熟地提取物通过调节SDF-1α/CXCR4信号途径活化内皮祖细胞保护梗死心肌的研究
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摘要
背景
内皮广泛存在于人体各级动、静脉及毛细血管内膜表层,对维持血管的正常功能起着重要的作用。从胚胎时期的血管发生形成原始血管网到成年后的生理状态下的血管内皮代谢及病理状态下的血管内皮修复,内皮细胞的作用贯穿于这一系列生理、病理过程的始终。内皮细胞的损伤及功能紊乱导致了心、脑、肾等重要脏器血管性疾病的发生、发展,并与其预后直接相关。
内皮祖细胞(endothelial progenitor cells EPCs)作为内皮细胞的前体,最早由Asahara等发现,并于2002年定义为一种阳性表达CD34、CD133、VEGFR2,且可与荆豆凝集素(UEA-1)和乙酰化低密度脂蛋白(acLDL)特异性结合的具有部分内皮功能和分化能力的单个核细胞群。内皮祖细胞(EPCs)来源于骨髓,存在于体循环中,它既能循环、增殖、迁移、分化为成熟的内皮细胞参与血管再生,也能够结合在血管内皮层,刺激邻近内皮细胞的增殖。许多内皮祖细胞激动剂,如粒细胞集落刺激因子,血管内皮生长因子和他汀类药物,已被证实可以激活内皮祖细胞,然而这些EPC激动剂的各种不良反应,如血管通透性增加、再狭窄率升高以及严重的肝功能损害等,严重限制了它们在临床治疗中的应用。
熟地黄是一种沿用千年的传统中药,味甜、甘,性温。归肝经、肾经,有补血滋润、益精填髓之功效。可用于治疗血虚萎黄、眩晕心悸、月经不调、崩不止,肝肾阴亏、潮热盗汗、遗精阳痿、不育不孕,腰膝酸软、耳鸣耳聋、头目昏花、须发早白,消渴、便秘、肾虚喘促等症。在现代医学研究中,熟地黄提取物(Rehmannia glutinosa extract RGE)被证实可以促进造血干细胞的增殖、分化,并促进骨髓DNA含量的增加。由此,我们假设：在生理状态下,熟地提取物RGE是否可作用于内皮祖细胞,从而达到其“补骨填髓”之功效。
目的
1掌握熟地提取物灌胃全身给药方法,观察不同浓度熟地提取物灌胃在不同时间点对生理状态下大鼠循环和骨髓内皮祖细胞作用变化
2筛选最佳给药浓度和作用时间,为后期实验提供基础。探索内皮祖细胞的体外分离、诱导培养、鉴定及功能检测方法。
方法
1地黄提取物的制备
熟地黄粗粉经反复水溶、醇提得到地黄粗多糖,并以Sevag法去除蛋白纯化为地黄多糖,并检测其浓度、纯度、提取率。
2实验分组
80只雄性WISTAR大鼠随机分为对照组(20只,RGE低浓度组(20只,0.38g/kg·d),RGE中浓度组(20只,0.75g/kg·d),RGE高浓度组(20只,1.5g/kg·d)。各组分别于3天、4周、8周、12周、16周取外周血,于4周、8周、12周、16周处死动物取骨髓。
3内皮祖细胞分离、培养
取大鼠外周血及骨髓,用密度梯度法以淋巴细胞分离液Ficoll分离出单个核细胞,以4～5×107的细胞密度将单个核细胞平铺于25cm2的细胞培养瓶中,用加入了诱导因子的培养基EBM-2诱导培养。
4内皮祖细胞鉴定
贴壁的细胞用DiL-acLDL和FITC-UEA-1标记,DAPI染核,荧光共聚焦显微镜下观察,DiL-acLDL和FITC-UEA-1双表达的细胞记作EPCs。
5内皮祖细胞计数
分离出的单个核细胞与CD34、CD133、VEFFR2一抗分别孵育,后再与之相对应的二抗：IgG-PerCP-Cy5.5、IgG-F(ab)2-PE、IgG-FITC孵育。流式细胞仪分析细胞群中三种标志物阳性表达的细胞数。
6内皮祖细胞功能检测
(1)增殖功能检测：MTT法检测EPCs增殖能力。
(2)迁移功能检测：Transwell小室法检测EPCs迁移能力。
(3)成管功能检测：Matrigel胶辅助下检测EPCs管腔结构的形成能力。
7统计学分析
统计学处理数量资料以均数±标准差(x±s)表示,所有数据用软件SPSS11.5进行统计分析。两组间数据的比较采用独立样本的t检验,多组之间的比较用单因素方差分析。多组间的两两比较用费希尔的LSD法。当P＜0.05,认为有统计学差异。
结果
1实验动物的一般情况
80只雄性WISTAR大鼠,5～6周龄,体重在160～180克,普通饮食,正常昼夜节律,适应性喂养3天,颈动脉取外周血。经口灌胃给药,分为RGE低、中、高三个浓度组和对照组。每日每只大鼠灌胃药物剂量为1m1,每周根据体重变化调整给药浓度,对照组每日生理盐水1ml灌胃。各组大鼠健康状况正常,试验中无自然死亡。
2流式细胞术分析内皮祖细胞数量
流式细胞仪分析结果显示,与对照组相比,各药物组外周血和骨髓EPCs数量随灌药时间的延长而增加。在外周血：8周末,中、高剂量组EPCs增加量较对照组有统计学意义(P＜0.01～0.05)；12周末和16周末,三种剂量的RGE灌胃均使EPCs较对照组显著增多(P＜0.01),但各剂量组12至16周的增长速率(8.53%、1.57%、1.79%)明显低于8至12周的增长率(10.43%、12.84%、16.50%)。在骨髓：8周末,中、高剂量组EPCs数量较对照组有增加(P＜0.05)；12周末和16周末,三种剂量的RGE灌胃均使EPCs较对照组显著增多(P＜0.01),各组8至12周增长速率为正(6.95%、12.10%、17.14%)然而12至16周增长速率明显下降(高浓度组3.34%)或增长停滞(低、中浓度组)。
3外周血内皮祖细胞功能检测
外周血分离单个核细胞,培养基EBM-2诱导培养48h换液,上清中的未贴壁细胞离心后重新加入,再经过一个48h后弃去不贴壁细胞,继续诱导培养48h。培养瓶内的EPCs以胰酶消化并计数,备用功能检测。
(1)增殖功能检测
体外诱导培养6天的外周血EPCs用MTT法检测细胞增殖能力。在16周末,中、高浓度组增殖能力与对照组相比有所升高(P＜0.05),与各组4周末数据相比亦有统计学差异(P＜0.05)。其余时间点各组增值能力无统计学差异。(2)迁移功能检测
Transwell小室法用于检测体外培养6天的外周血EPCs的迁移能力。可见12周末,中、高浓度组可使迁移能力相较对照组和各组的4周末数据有所增高(P＜0.05),但这种数量变化不明显,且从12周末到16周末这种增殖能力没有进一步的增长。(3)成管功能检测
体外培养6天的外周血EPCs平铺于Matrigel胶预孵育的96孔板上,检测EPCs管状结构形成能力。高浓度组在8周末相较于4周末城管能力有所增长(P＜0.05),12周末成管能力的增高与对照组相比有统计学意义(P＜0.05)。低、中、高浓度组在16周末成管能力相较于对照组均有显著增高(P＜0.05),与各组4周末数据相比较亦有统计学差异(P<0.05)。
4骨髓内皮祖细胞功能检测
骨髓PBS悬浊液中分离单个核细胞,培养基EBM-2诱导培养48h换液,传代,与上清液中分离出的未贴壁细胞共培养,48h后换液,弃上清,继续诱导培养4天。培养瓶内的EPCs以胰酶消化并计数,备用功能检测。
(1)增殖功能检测
体外诱导培养6天的骨髓EPCs用MTT法检测细胞增殖能力。在12周末,中、高浓度组增殖能力与对照组相比有所升高(P＜0.05),与各组4周、8周末数据相比亦有所增加(P＜0.05)。16周末,这种增值能力的增长进一步加剧(P＜0.01)。
(2)迁移功能检测
Transwell小室法用于检测体外培养6天的骨髓EPCs的迁移能力。12周末和16周末,三种浓度的药物刺激组迁移能力相较于对照组和各组4周末数据均有所增加(P＜0.05),然而这种增长并不明显。
(3)成管功能检测
体外培养6天的骨髓EPCs平铺于Matrigel胶预孵育的96孔板上,检测其管状结构形成能力。12周末,中、高浓度组EPCs成管功能的增高与对照组相比有统计学意义(P＜0.05)。在16周末,这种相比对照组成管功能的增长更加明显(P＜0.01～0.05),且相对于各组4周末数据已有所增长(P＜0.01～0.05)。这种变化在高浓度组更加显著(P＜0.01)。
结论
1在正常生理条件下,三种不同浓度的熟地提取物溶液灌胃喂养成年雄性大鼠,均可导致大鼠体内EPCs数目的增多。相较于外周血来源的EPCs,骨髓来源EPCs的这种效应更为明显。且三种药物浓度中高浓度组作用较为突出,增长速率在灌胃后的12周左右达到最大。
2在生理条件下,RGE灌胃可活化大鼠外周血和骨髓中EPCs的功能。但这种活化作用在外周血EPCs中表现多不显著。在骨髓中,EPCs的活化多发生于灌胃后的12周左右。
背景
心肌梗死(myocardial infarction)是指在冠状动脉病变的基础上,冠状动脉的血流中断,使相应的心肌出现严重而持久地急性缺血,最终导致心肌的缺血性坏死。因此,血管再通及建立有效的侧支循环是挽救缺血心肌,改善心肌梗死预后及降低死亡率的首要措施。越来越多的证据表明细胞移植和治疗性血管再生是治疗终末期冠状动脉疾病所致心肌梗死的最具潜力和研究意义的新方法。近年来的研究显示：骨髓来源的干细胞分化形成的内皮祖细胞(endothelial progenitor cells, EPC)可在心梗后动员、迁移、归巢到损伤部位,并促进缺血区血管的新生,从而有效保护梗死后的心肌。近年来对熟地的现代医学作用研究显示,熟地可有效促进血细胞和骨髓增殖,治疗心脑血管缺血性疾病。然而研究方法的陈旧、手段的单一、机制探讨的肤浅限制了熟地在临床上的推广,且目前为止尚未有研究将熟地的作用机制与内皮祖细胞相连。
血管发生和血管生成是新血管形成的两种基本形式。血管发生是指胚胎中的成血管细胞向内皮细胞系分化包绕形成原始血管网并最终分化成熟形成心血管系统；血管生成是指从在原有血管网基础上,通过内皮细胞迁移、增殖、伸展及管状结构形成等步骤最终长成新生毛细血管的复杂过程。成熟个体中的血管再生主要通过新毛细血管的生成得以实现。参与成熟个体中血管再生的细胞为内皮祖细胞。因此内皮祖细胞成为当今医疗领域中治疗缺血性疾病的研究焦点,尤其是在治疗心肌梗死中成为新的治疗靶点。生理状态下,外周血中的EPCs数量较少,而骨髓中的EPCs多处于相对静息状态；心梗发生后在急性期内,骨髓中的EPCs被动员到外周血中,活化并归巢到损伤部位,通过参与损伤部位血管的新生维持损伤部位的血供。
我们由实验Ⅰ证实了熟地提取物在生理状态下对内皮祖细胞的动员作用,由此我们做出推断：熟地提取物是否可以动员心梗条件下体内的EPC从而达到保护受损心肌,治疗心肌梗死的作用。本实验中我们针对这一假设展开实验。
目的
1.建立大鼠心肌梗死模型,探讨中药熟地黄在心梗的急性期和慢性期对大鼠缺血心肌的保护作用。
2.观察心梗后大鼠外周血和骨髓中内皮祖细胞的数量、功能变化,探讨内皮祖细胞在急性期和慢性期对心梗的治疗作用,并观察熟地对内皮祖细胞这一作用的影响。
方法：
1.实验动物
120只雄性WISTAR大鼠(8周龄,体重180～200g)随机分为两组(每组60只)：空白组生理盐水1ml／d灌胃,试验组熟地提取物生理盐水溶液1.5g/kg·d灌胃。
两组分别灌胃8周后,每组又随机分为对照组(16只,不手术),假手术组(16只,手术开胸穿线不结扎),心梗组(28只,行冠状动脉前降支结扎术)。至此实验大鼠分为6组：生理盐水对照组(N-b,16只)、生理盐水假手术组(N-m,16只)、生理盐水心梗组(N-MI,28只)、熟地对照组(RGE-b,16只)、熟地假手术组(RGE-m,16只)、熟地心梗组(RGE-MI,28只。
术后继续灌胃喂养4周,分别于术后第3天,1周末,2周末,4周末处死各组4～7只大鼠。
2.心梗模型的建立
法一：大鼠用2.5%的戊巴比妥钠(30mg/kg)腹腔注射麻醉。仰卧位将大鼠固定在手术台上,剪除胸部被毛。胸骨左侧3～4肋间心尖搏动最明显处横向剪开胸部皮肤约1.5～2cm,钝性分离皮下组织、胸大肌、前锯肌。顺肋间肌纹理钝性撑开3～4肋间,可见搏动的心尖,在心尖以上0.5cm处快速穿过可吸收缝合线,闭合式结扎。对合前锯肌、胸大肌,胸部皮肤缝合前轻挤压缝合口排出胸腔气体,缝合皮肤切口。皮下注射青霉素8万IU/kg常规抗感染。
法二：乙醚吸入式麻醉大鼠,仰卧位固定于手术台上,剪除颈部及胸部被毛。纵向剪开大鼠颈部皮肤约1.5～2cm,分离皮下组织及肌肉,钝性分离气管,于气管环之间剪开气管半周。行气管插管,连接呼吸机,调整呼吸频率、呼吸比和潮气量。胸骨左缘3mm处纵向剪开皮肤约4cm,钝性分离皮下组织、胸大肌、前锯肌。于第三肋间剪开肋间肌暴露胸膜。撑开肋骨,于呼气相刺破胸膜,暴露心脏。在肺动脉圆锥与左心耳交界下方2mm处以可吸收缝合线结扎冠状动脉前降支。逐层缝合肌肉和皮肤。大鼠苏醒后拔除气管插管,清理气道血块及分泌物。对合颈部肌肉及皮下组织,缝合皮肤切口。皮下注射青霉素8万IU／kg常规抗感染。
假手术组手术步骤同上,冠状动脉处穿过结扎线但不打结。
3.存活率分析
保留存活记录并绘制生存曲线。
4.心电图及超声心动图检测
所有大鼠于术前和术中进行心电图监测,显示即时心功能情况。大鼠于术前和心梗后3天、1周、2周、4周,处死之前行经壁超声心动图检测,B型超声记录心脏运动情况,M型超声测量同一个心动周期内左室收缩末期和舒张末期的左室内径,系统计算左室收缩末期容积(LV-s),左室舒张末期容积(LV-d),左室相对质量(LV-mass),左室射血分数(EF%),左室短轴缩短率(FS%),血流E峰、A峰比值(E／A)。
5.血清学指标检测
酶联免疫吸附法(ELISA)测定血清中心肌损伤相关指标Tn-T、BNP的含量,反应心肌损伤程度。
6.外周血、骨髓中内皮祖细胞量的测定
外周血／骨髓细胞悬液经红细胞裂解后与CD34、CD133、EFFR2抗体共孵育,后再与之相对应的二抗：IgG-PerCP-Cy5.5、IgG-F(ab)2-PE、IgG-FITC共孵育。流式细胞仪分析细胞群中三种标志物阳性表达的细胞数。
7.体外培养的骨髓内皮祖细胞功能测定
(1)增殖功能检测：MTT法检测EPCs增殖能力。
(2)迁移功能检测：Transwell小室法检测EPCs迁移能力。
(3)成管功能检测：Matrigel胶辅助下的EPCs管腔结构形成能力检测。
8.组织病理学染色
留取大鼠心脏标本,行苏木素-伊红染色(HE),POLEY缺血染色,观察心肌组织形状,及各组心肌缺血情况。
9.心肌细胞凋亡情况检测
TUNEL法检测各组梗死区及其周边区心肌细胞的凋亡情况
10.免疫组织化学染色
免疫组织化学检测梗死部分心肌血管新生情况,检测新生毛细血管内皮相关指标CD133、VEGFR2。
11.实时定量聚合酶链反应(Real-time PCR)检测组织mRNA水平Real-time PCR检测心肌组织内血管新生相关指标CD133、VEGFR2的表达水平。
12.统计学分析
统计学处理数量资料以均数±标准差(x±s)表示,所有数据用软件SPSS11.5进行统计分析。两组数据间比较用独立样本的t检验,多组之间的两两比较用单因素方差分析。多组间的两两比较用费希尔的LSD法。当P<0.05,认为有统计学差异。
结果：
1.实验动物的一般情况
本研究共涉及雄性WISTAR大鼠120只,假手术32只,心梗手术56只。行心梗手术的大鼠当天死亡5只,手术成功率为91%,心梗急性期3天内死亡4只,急性期存活率为84%,其后的试验中死于超声麻醉意外的2只,不明原因死亡2只,总存活率为77%。假手术组术后急性期死亡2只,存活率为94%。
2.心电图及心脏超声检测结果
大鼠于术前和术中进行心电图监测,显示结扎后ST段改变和病理性Q波的出现。大鼠术后超声结果与术前相比较：左室收缩期内径(LV-s)、左室舒张期内径(LV-d)、左室相对质量(LV-mass)升高,左室射血分数(EF)、左室短轴缩短率(FS)下降,E／A倒置(P<0.01＜0.05)。这些指标的变化标志着心梗模型的成功建立。
超声结果显示：在大鼠心梗急性期(术后3天至术后1周),熟地组和生理盐水组大鼠的左室射血分数几乎没有差异；而在心梗的慢性期(术后2周至术后4周),与生理盐水组相比熟地组可明显改善心梗造成的EF、FS的降低(P＜0.05)。
3.血清学检测
心梗术后血清中Tn-T和BNP含量与对照组和假手术组相比显著升高(P＜0.01)亦证实了心梗模型的成功建立。
心梗后,熟地组血清Tn-T含量在术后1周末开始下降(P＜0.01-0.05),而生理盐水组则在术后4周末开始下降(P＜0.05),证实熟地可在心梗后降低心肌损伤。
心梗后,生理盐水组血清中的BNP含量随着时间延长呈上升趋势(P＜0.01-0.05),而在熟地组这种上升趋势则不明显。提示熟地干预可降低心梗后发生心肌细胞相关延时性损伤的风险。
4.外周血和骨髓中EPCs含量的测定
心梗术后与术前相比,熟地组和生理盐水外周血的EPCs含量均升高而骨髓中EPCs含量则降低(P＜0.01～0.05),说明心梗发生后,心肌损伤导致骨髓中的EPCs被动员到了外周血中。在心肌梗死发生后的慢性期(2周末和4周末),熟地组外周血EPCs的增长幅度大于生理盐水组(P＜0.01),而熟地组骨髓EPCs的降低不如生理盐水组明显(P＜0.01),可见熟地可以增加动员到外周血的EPCs数量同时保持骨髓中EPCs的储备量。由此亦可判断,熟地作用于心梗大鼠,除了增加了EPCs动员量以外,体内EPCs总量也有所增加。
5.骨髓EPCs功能的测定
(1)增殖功能检测：心肌的梗死使生理盐水组和熟地组的EPCs增殖能力均有所上升(P＜0.01),这种上升在生理盐水组于术后2周开始下降(P＜0.05),而在熟地组这种增殖能力则一直保持在较高水平。可见熟地可促进心梗后EPCs的增值能力。
(2)迁移功能检测：心梗后生理盐水组和熟地组的EPCs迁移能力均有所增加(P＜0.01),而在心梗术后4周,熟地组EPCs的迁移能力比生理盐水组更加活跃(P＜0.05)。
(3)成管功能检测：从心梗术后2周熟地组EPCs参与成管的数量明显多于生理盐水组(P＜0.05)。在空白对照组,也有类似的情况发生。说明,熟地可强化心梗后EPCs的成管功能,亦能在生理状态下激活这种功能。
6.组织病理学染色检测
(1)HE染色显示：术后1周开始,梗死心肌组织发生明显的组织形态学变化：梗死区肌纤维溶解、断裂,排列紊乱。
(2)POLEY缺血染色：缺血心肌染为红色,而正常心肌组织为蓝色。在心梗的急性期(术后3天到1周)熟地组和生理盐水组差异不明显；到心梗慢性期(术后2周到4周)熟地组缺血心肌的面积较之生理盐水组下降明显(P＜0.05)。
7.心肌细胞凋亡检测
TUNEL染色法用于检测心肌细胞凋亡情况。在心梗术后2周和4周,熟地组心肌细胞的凋亡量明显少于生理盐水组。
8.免疫组织化学检测
(1)心肌梗死后,在两种干预(生理盐水和熟地)下,VEGFR2在心肌组织中的表达均随时间变化有所增加(P＜0.05-0.01),而在心梗发生1周以后(熟地灌胃9周)开始,VEGFR2在熟地组中的表达逐渐高于在生理盐水组中的表达(P＜0.05～0.01)。
(2)心肌梗死后,在两种干预(生理盐水和熟地)下,CD133在心肌组织中的表达均随时间变化有所增加(P＜0.05-0.01),而在心梗的慢性期(心梗术后2周到4周,CD133在熟地组中表达的增多明显高于生理盐水组(P＜0.01)。
9.Real-time PCR检测
为了验证免疫组织化学中对VEGFR2、CD133表达量变化的检测,我们用Real-time PCR在mRNA水平验证VEGFR2和CD133的表达量。结果显示VEGFR2和CD133在rnRNA水平的变化趋势与免疫组织化学染色法中检测到的它们在蛋白水平的变化趋势一致。结论：
(1)熟地灌胃可降低心梗后心肌细胞的缺血坏死和凋亡,通过增加血管新生改善梗死心肌的血供,保护梗死后的心室功能,改善预后。
(2)熟地灌胃可增加心肌梗死后骨髓动员到外周血的EPCs数量,并保持EPCs的较高活性,促进其参与管腔形成；于此同时,又可保持骨髓EPCs数量的相对稳定。
(3)与生理盐水组相比,熟地的治疗作用大多发生在心肌梗死的慢性恢复期,而非急性发作期。
背景：
生理状态下,外周血和组织中内皮祖细胞(Endothelial progenitor cells, EPCs)的数量极低,骨髓中有一定数量的EPCs,但多处于静息状态。局部损伤或在一些激动剂的作用下,骨髓EPCs可动员迁移到外周血,定植于损伤局部。在EPCs的动员迁移过程中,基质细胞衍生因子(Stromal-derived factor, SDF)发挥了重要作用。
基质细胞衍生因子1(SDF-1或称CXCL12)是目前已知的唯一能够迁移造血干细胞的趋化因子,SDF-1表达量的变化促使EPCs改变其原有的静息状态并进一步活化、迁移、定植、分化。SDF-1与其受体CXCR4结合,使CXCR4由低能量构象状态进入过渡状态,CXCR4内部盐桥断裂暴露结合位点,与SDF-1α进一步结合使CXCR4进入激活状态,启动下游的一系列信号转导,一系列级联过程最终导致了EPCs的活化和迁移。更有研究指出,在EPCs介导的心梗后的损伤修复过程中,SDF-1α/CXCR4的激活,损伤局部SDF-1α表达的上调最终指导了骨髓EPCs迁移到外周血并归巢到损伤部位。
在实验Ⅰ和实验Ⅱ中,我们已证实熟地在生理状态及心梗模型造成的病理状态下均可促进骨髓和外周血EPCs的数量和功能,且在心梗的慢性期通过活化EPCs参与心肌梗死区及周边区血管的新生达到治疗心梗的效果。我们推测,熟地对EPCs的这种动员、迁移、归巢、血管新生等功能的促进作用是否通过激活SDF-1α/CXCR4系统实现。
目的
3.观察熟地提取物在体外刺激内皮祖细胞的情况。
4.探讨熟地提取物激活内皮祖细胞的作用机制。
方法：
13.细胞来源
取雄性WISTAR大鼠长骨(肱骨、股骨、胫骨)髓腔中的骨髓,分离单个核细胞,诱导培养为元代内皮祖细胞。
14.细胞的培养鉴定
EGM-2培养基诱导培养骨髓来源的单个核细胞,96小时后收集贴壁细胞,流式细胞仪分析,DiL-acLDL及FITC-UEA-1荧光双染,荧光共聚焦显微镜下观察,鉴定其为内皮祖细胞。
15.细胞刺激
(1)浓度梯度筛选：无血清培养基培养内皮祖细胞24小时后,熟地黄提取物分别以10、25、50、100、500及1000μg/ml的浓度加入植有内皮祖细胞的6孔板各孔的培养基中,对照组仅以培养基培养。72小时后收集各孔细胞和上清液,待检。
(2)最佳作用浓度筛选：无血清培养基培养内皮祖细胞24小时后,以适宜浓度梯度(10,25,50,100μg/ml)刺激6孔板内的内皮祖细胞,对照组仅以培养基培养,抑制剂组用CXCR4的特异性抑制剂AMD3100在5μg/ml的浓度下预刺激1小时后加熟地提取物刺激(100μg/ml)。72小时后收集各孔细胞和上清液,待检。
(3)最佳作用时间筛选：无血清培养基培养内皮祖细胞24小时后,以最佳浓度刺激6孔板内的内皮祖细胞(SDF-1α为50μg/ml, CXCR4为25μg/ml),对照组仅以培养基培养,分别于刺激后的6、12、24、48、72小时收集各孔细胞和上清液,待检。
(4)机制探讨分析：无血清培养基培养内皮祖细胞24小时后,分别以SDF-1α的最佳作用浓度和时间(50μg/ml,24h)及CXCR4的最佳作用浓度和时间(25μg/ml,48h)刺激6孔板内的细胞,对照组仅以培养基培养,抑制剂组用CXCR4的特异性抑制剂AMD3100在5μg/ml的浓度下预刺激1小时后加熟地提取物刺激(50μg/ml,24h及25μg/ml,48h),去除含RGE的培养基,加普通培养基进行细胞功能学检测。
16.增殖功能检测
MTT法检测各组细胞增殖能力。17.Western Blot检测
(1)提取心肌组织的蛋白,测定蛋白浓度,并通过凝胶电泳、转膜、一抗及相应二抗的标记、曝光等步骤检测CXCR4、SDF-1α、β-actin等的蛋白表达水平。
(2)提取各组细胞蛋白,同上方法检测CXCR4、SDF-1α、β-actin等的蛋白表达水平。18.Real-time RT-PCR检测
(1)提取心肌组织的RNA,逆转录成cDNA,通过荧光实时定量聚合酶链反应,检测CXCR4、SDF-1α、β-actin等的RNA表达水平。
(2)提取各组细胞RNA,同上方法检测CXCR4、SDF-1α、β-actin等的RNA表达水平
19.统计学分析
统计学处理数量资料以均数±标准差(x±s)表示,所得数据用软件SPSS11.5进行统计学分析。当P＜0.05,认为所比较的两组有统计学差异。
结果：
1.心肌免疫组化染色
免疫组化染色检测心肌中内皮祖细胞动员相关因子CXCR4的表达情况。可见与空白对照组及假手术组相比,心梗阻大鼠心肌CXCR4表达增高(P＜0.01)；在梗死组中,与生理盐水灌胃相比,熟地灌胃可在梗死后的慢性期(2周到4周)增加心肌中CXCR4的表达(P＜0.01)；在空白对照组和假手术组,熟地灌胃与生理盐水灌胃相比亦可增加心肌的CXCR4表达量(P＜0.05-0.01)。
2.心肌组织蛋白表达量检测
提取各组大鼠心肌组织的蛋白,WesternBlot法检测SDF-1α/CXCR4的表达情况。通过Image Pro-Plus软件对蛋白印迹进行定量分析发现：与对照组和假手术组相比,心梗组CXCR4表达升高(P＜0.01),SDF-1α亦升高(P＜0.01),但SDF-1α的升高在心梗的急性期(3天到1周)内表现不明显；在心梗大鼠中,与生理盐水灌胃相比,熟地灌胃使CXCR4表达在心梗的慢性期升高(P＜0.01),熟地对SDF-1α表达的上调作用表现在心梗1周后(P＜0.01)而在之后的慢性期内熟地对SDF-1α作用不明显；在正常对照组,熟地亦可上调CXCR4的表达(P＜0.05～0.01),但熟地的这种上调作用对SDF-1α的表达影响不大。
3.心肌组织基因表达量检测
提取各组大鼠心肌组织RNA,逆转录为cDNA, Real-time PCR法定量检测SDF-1α/CXCR4的基因表达情况。可见与对照组和假手术组相比,心梗组CXCR4表达升高(P＜0.01),SDF-1α的表达亦有所升高(P＜0.01),且SDF-1α及CXCR4的表达增加随时间的发展呈递进关系(P＜0.05～0.01)；在心梗大鼠中,与生理盐水灌胃相比,熟地灌胃使CXCR4的表达在心梗的慢性期上调(P＜0.05～0.01),熟地对SDF-1α表达的上调作用直至心梗后4周方开始显现(P<0.05)。
4.RGE刺激细胞浓度的筛选
在体外实验中,用不同浓度的RGE(浓度分别为10μg/ml,25μg/ml,50μg/ml,100μg/ml,500μg/ml,1000μg/ml)刺激元代培养的内皮祖细胞,72小时后收集细胞进行增殖活性检测,发现元代细胞用500μg/ml和1000μg/ml浓度的RGE刺激后,其增殖能力与对照组相比明显降低(P<0.05)。提示高浓度(500μg/ml,1000μg/ml) RGE因其对细胞增殖能力抑制作用而不适合用于接下来的体外细胞实验中。
5.RGE刺激细胞的最佳浓度选择
用筛选出的4种浓度RGE(10μg/ml,25μg/ml,50μg/ml)刺激细胞,与对照组和其它各浓度组相比,25μg/ml组CXCR4的蛋白以及RNA表达水平均升高(P＜0.01)；50μg/ml组SDF-1α的蛋白及RNA表达水平升高(P＜0.01)。抑制剂组与其它各组相比,CXCR4的蛋白以及RNA表达水平均降低(P＜0.01),SDF-1α的蛋白和RNA表达水平升高(P＜0.01)。由此我们推出：25μg/ml是RGE刺激CXCR4表达上调的较适宜浓度；50μg/ml是RGE刺激SDF-1α表达上调的较适宜浓度；在AMD3100的作用下,CXCR4的翻译和转录下调,而SDF-1α翻译和转录上调。
6.RGE刺激细胞的最佳时间选择
以25μg/ml的RGE浓度刺激元代细胞,分别于刺激后的6小时、12小时、24小时、48小时、72小时收集细胞,检测CXCR4的表达,发现与对照组和其它各组相比,刺激48小时的细胞CXCR4的蛋白和RNA表达量增加(P<0.05),可见48小时是RGE刺激CXCR4表达上调的较适宜时间。
以50μg/ml的RGE浓度刺激元代细胞,分别于刺激后的6小时、12小时、24小时、48小时、72小时收集细胞,检测SDF-1α的表达,发现与对照组和其它各组相比,刺激24小时的细胞SDF-1α的蛋白和RNA表达量增加(P＜0.01),可见24小时是RGE刺激SDF-1α表达上调的较适宜时间。
7.细胞功能检测
用RGE对CXCR4和SDF-1α的最佳作用浓度和时间(25μg/ml,48h以及50μg/ml,24h)分别刺激细胞后,去除含有RGE的培养基,换正常培养基进行细胞功能检测,与对照组和抑制剂组相比,元代EPC的成管能力增强,尤其RGE25μg/ml刺激48小时组,成管能力明显高于对照组和抑制剂组(P＜0.05-0.01)；抑制剂组细胞基本不参与新的管腔形成,其功能明显低于其他各组(P＜0.01)。
结论：
1.熟地提取物灌胃可上调生理状态下和心梗状态下心肌组织中SDF-1α及CXCR4的蛋白和RNA表达。
2.体外试验中,熟地提取物刺激元代培养的EPCs,可使细胞和上清中SDF-1α及CXCR4的蛋白和RNA表达升高,并且CXCR4的抑制剂AMD3100可抵消熟地提取物的这一作用。
3.SDF-1α及CXCR4升高的EPCs功能明显增强,AMD3100竞争性抑制CXCR4表达使细胞功能减弱甚至丧失。
4.熟地通过上调SDF-1α/CXCR4的表达激活EPCs,熟地的这种作用在体外可被AMD3100抵消。
Background
Endothelial progenitor cells (EPCs) were firstly found by Pro. Asahara, and were defined as precursors of endothelial cells which positive expressed CD34, CD133, VEGFR2. EPCs derived from the bone marrow, existed in the systemic circulation. It can proliferate, migrate, and differentiate into mature endothelial cells and participate in angiogenesis, besides it is capable of binding in the blood vessels and stimulating the proliferation of neighboring endothelial cells. Many EPCs agonists such as granulocyte-colony stimulating factor, vascular endothelial growth factor (VEGF) and statins, can mobilize EPCs in bone marrow. However adverse reactions, such as increased vascular permeability and high ratio of restenosis and liver damage, limit their use in clinical.
Prepared Rehmannia glutinosa (PRG), belongs to the family of Scrophulariaceae, is a widely used traditional Chinese medicinal herb. It has been used to treat hypodynamia caused by many kinds of diseases. It has been effective and safe, but the involved mechanism has not been verified. Recently, Rehmannia glutinosa extract (RGE) has been used in modern medicine studies. RGE can stimulate the proliferation and differentiation of hematopoietic stem cells in bone marrow and increase the DNA content of bone marrow. So we supposed that Rehmannia glutinosa extract (RGE) could activate EPCs.
Objectives
Grasp the method of RGE systemic administration. Investigate the changed effect of RGE on EPCs in bone marrow and circulation blood of normal rats along with the differ in concentration and duration of RGE. Screen the optimal concentration and duration for RGE, and provide basis for the subsequent experiments. Explore the separation, induced cultivation, identification and functional measurements for EPCs in vitro.
Method
1. Preparations of Rehmannia glutinosa extract (RGE)
Rude polyoses was extracted form Rehmannia glutinosa powder by water dissolving and alcohol extracting repeatedly. And then RGE was extracted and purified in Sevag way. The concentration, purification and extraction ratio were tested.
2. Animal group
80male Wistar rats were random divided into four groups:in the control group (n=20) rats were oral-treated with normal saline, in the RGE-L group (n=20) rats were oral-treated with RGE at0.38g·kg-1·day-1, in the RGE-M group (n=20) rats were oral-treated with RGE at0.75g·kg-1·day-1, in the RGE-H group rats were oral-treated with RGE at1.5g·kg-1·day-1.5rats in each group were sacrificed at4,8,12,16weeks after systemic delivery.
3Isolation and cultivation of EPCs
10ml peripheral blood was obtained from rats by aspiration of the heart. Bone-marrow cells were obtained by flushing the cavity of femurs, tibias, and humerus with growth medium EBM-2. Peripheral blood and bone marrow mononuclear cells were isolated by Ficoll density-gradient centrifugation.10million isolated cells were resuspended in growth medium EBM-2and plated in25-cm2culture flasks. After48h, non-adherent cells were discarded and growth medium was changed every2days.
4. Identification of EPCs
Direct fluorescent staining was used to detect dual binding of FITC-UEA-land Dil-acLDL and the nuclei were stained with DAPI. Cells double stained for Dil-Ac-LDL and FITC-UEA-1were considered EPCs. Immunocytochemistry followed standard protocols.
5. Determination of EPC number
Fluorescence-activated cell sorting (FACS) was used to determine the EPC population in blood and bone marrow of rats. Briefly, fresh anticoagulation blood or bone-marrow PBS suspension (200μl) was incubated with the monoclonal antibodies: anti-VEGFR2, anti-CD133and anti-CD34-PerCP-Cy5.5for20min at room temperature, then with2ml Lysing solution for10min and washed with PBS twice by centrifugation. The cells were resuspended with200μl PBS, then incubated with the secondary antibodies:goat polyclonal rabbit IgG-FITC and goat polyclonal mouse IgG-F(ab)2fragment PE for30min at room temperature. Cells were washed with PBS and resuspended in400μl PBS.
6. function test of EPCs
(1) MTT assay was used to evaluate EPC proliferation.
(2) EPC migration was evaluated by use of a Transwell chamber.
(3) Capillary-like tube formation was analyzed by use of Matrigel Matrix.
7. Statistical analysis
Data are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05.
Results
1. General situation of rats
The rats (6weeks old, body weight at160-180g) were fed a regular rat chow and housed in normal night-day conditions under standard temperature and humidity. The rats in each group were normally health and no natural mortality during the test.
2. The number of EPCs was evaluated by FACS
The FASC showed that the number of EPCs increased with RGE systemic delivery both in blood and in bone marrow, and this tendency intensified with the time went by. In peripheral blood, at the end of8weeks, the EPCs number in RGE-M and RGE-H groups was statistically increased compared to control group (P<0.01-0.05); at the end of12and16weeks, the EPCs number in all the three RGE groups was increased significantly (P<0.01), however the ratios from12to16weeks (8.53%,1.57%,1.79%) were much lower than that from8to12weeks (10.43%,12.84%,16.50%). In bone marrow, at the end of8weeks, the EPCs number in RGE-M and RGE-H groups was increased compared with control group (P<0.05); at the end of12and16weeks, the EPCs number in all the three RGE groups was increased significantly (P<0.01), however the ratios were positive from8to12weeks (6.95%、12.10%.17.14%) and the ratios decreased (RGE-H3.34%) or negative (RGE-L RGE-M groups) from8to12weeks.
3. The functional test of EPCs in peripheral blood
Mononuclear cells were isolated from peripheral blood, induced cultured by EVM-2for48h, the nonadherented cells were centrifuged and co-cultured for another48h, and then discarded the cells still not adherent culture for the third48h. These cells were digested by trypsin and counted for use.
(1) Proliferation test
MTT showed the proliferation of EPCs from peripheral blood. At the end of16weeks, the proliferation of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05) and the same group at the end of4weeks (P<0.05). The other concentration at other time points had no statistical different.
(2) Migration test
Migration of EPCs in peripheral blood was evaluated by use of Transwell chamber. At the end of12weeks, it increased in RGE-M and RGE-H group compared to control group and the same group at the end of4weeks (P<0.05), however these changes seemed shyly. And as from12to16week, it had no further growth.
(3) Tube-formation test
Tube-formation capacity of peripheral blood EPCs was evaluated by Matrigel Matrix. In RGE-H group, at the end of8weeks it had increased compared to the end of4weeks (P<0.05), and at the end of12weeks it was more than control group (P<0.05). At the end of16weeks, the tube-formation capacity of EPCs in all the RGE groups significantly increased compared with control group and the same group at the end of4weeks as well.
4. The functional test of EPCs in bone marrow
Mononuclear cells were isolated from bone marrow PBS solution, induced cultured by EVM-2for48h, the nonadherented cells were centrifuged and co-cultured for another48h, then transfer of culture and discarded the cells still not adherent. After culture for6days, cells were digested by trypsin and counted for use.
(1) Proliferation test
MTT showed the proliferation of EPCs from bone marrow. At the end of12weeks, the proliferation of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05) and the same group at the end of4and8weeks (P<0.05). At the end of16weeks, it increased further more in all RGE groups than control group.
(2) Migration test
Migration of EPCs in bone marrow was evaluated by use of Transwell chamber. At the end of12and16weeks, it increased in all the RGE groups compared to control group and the same group at the end of4,8weeks (P<0.05), however the extent didn't significant.
(3) Tube-formation test
Tube-formation capacity of bone marrow EPCs was evaluated by Matrigel Matrix. At the end of12weeks, the tube-formation capacity of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05). At the end of16weeks, it significantly increased in all the RGE groups compared with control group and the same group at the end of4weeks (P<0.01-0.05). The RGE-H group was more significant than other groups (P<0.01).
Conclusion
1. Under physiological condition, rats systemic delivery with three different concentration of RGE, and lead to the number of EPCs in peripheral blood and bone marrow increased. This increase was much more significant in bone marrow than peripheral blood. The RGE-H group had the most extrusive effect among the three concentrations. The increment speed was maximum at about12weeks after oral-feed.
2. At the condition of physiology, RGE systemic delivery was able to active the EPCs in peripheral blood and bone marrow. This activation in peripheral blood EPCs was not so obvious as that in bone marrow EPCs. And in bone marrow, the activation of EPCs often appeared12weeks after oral-feed.
Background
Myocardial infarction (MI) occurs with the deprivation of coronary blood and is usually caused by stenosis or occlusion of the coronary artery. The culminating event is necrosis of myocardial tissue and dysfunction of the left ventricle. Therefore, recanalization and establishment of collateral circulation are primary measures for saving ischemic myocardium, improving the prognosis of myocardial infarction and reducing mortality. Many studies proved that cell transplantation and therapeutic angiogenesis is a most potential and significant method for the treatment of end-stage coronary artery disease. Recent studies showed that endothelial progenitor cells (EPCs) which were differentiated from bone marrow-derived stem cells can be activated after myocardial infarction, and mobilization, migration, homing to the site of injury and participated in angiogenesis, so that protect the infarcted myocardium. The modern medical studies about Prepared Rehmannia Glutinosa showed that itcould effectively promote the proliferation of blood cells and bone marrow, it could also be used for the treatment of ischemic cardiovascular and cerebrovascular diseases. Obsolete research methods, relatively single mechanism, superficial studies about mechanism, however superficial limits the promotion of Rehmannia in clinical research. And so far no research connected the mechanism of Rehmannia Glutinosa Extra (RGE) with the action of endothelial progenitor cells yet.
Objectives
1. Establish a rat model of myocardial infarction, and investigate the protective effect of Chinese medicine Prepared Rehmannia Glutinosa on infarcted rat myocardium in acute and chronic phase.
2. Observed the change of endothelial progenitor cells in rat peripheral blood and bone marrow after myocardial infarction, including number and function. Explore the therapeutic effect of endothelial progenitor cells in the acute and chronic phase of myocardial infarction. Discuss the role of RGE on endothelial progenitor cells' therapeutic effect.
Methods
1. Animal group
A total of120male Wistar rats (8weeks old; body weight180-200g) were randomized to2groups (n=60each) for treatment:high-dose RGE (1.5g·kg-1·day-1orally) for8weeks, then left anterior descending coronary artery ligation (n=28), mock surgery (n=16) or no treatment (n=16), then RGE orally for4weeks; or normal saline (NS) as the above protocol. Then rats were sacrificed,4～7each on the3rd day and the end of1,2,4weeks, recording as day3, week1, week2and week4respectively.
2. The establishment of a rat myocardial infarction model.
Method1:The rats were narcotized by2.5%napental (30mg/kg), and fixed the rat on the operating table in supine position, cut off its chest hair. Horizontal cut the chest shin about1.5～2cm at the3to4left intercostal sternal where the apex beat most obvious and blunt dissection of the subcutaneous tissue, pectoralis major, serratus anterior muscle. Blunt softened3to4intercostal shun intercostal muscle texture, seen the beating apex, fast through the myocardium0.5cm above the apex with the absorbable suture, and closed ligation. Light squeeze together serratus anterior muscle, the pectoralis major, chest skin and discharge pleural gas before suture the skin incision. Subcutaneous injection of penicillin80,000IU/kg of conventional anti-infective.
Method2:The rats were narcotized by ethylether, and fixed the rat on the operating table in supine position, cut off its neck and chest hair. Longitudinal cut open the rat neck skin about1.5～2cm, separate subcutaneous tissue and muscle, blunt separation trachea, cut open tracheal half cycle between trachea ring. Do endotracheal intubations, connection breathing machine, adjust the frequency of breathing, breathing ratio and tidal volume. Longitudinal cut open chest skin about4cm at the left edge of the sternal3mm place, blunt separation subcutaneous tissue, and the pectoralis major, former saw muscle. Cut open the intercostal muscle on the third intercostals space to exposure pleura. Open the frame, punctured pleura at the breathe out phase, exposing the heart. Absorpted suture ligation at the anterior descending coronary artery in place2mm below the common border of the pulmonary artery cone and the left atrium. Layered suture muscle and skin. Remove the endotracheal intubation after rats were awake, and clearing the airway blood clot and secretion.
The rats in mock groups underwent mock surgery with a silk suture across the coronary artery without ligation.
3. Mortality analysis:
Reserved survival record and draw the survival curve.
4. Electrocardiography and echocardiography analysis
Before and after surgery, rats underwent electrocardiography (ECG) by use of a Micromaxx P04224system and ultrasonic cardiography (UCG) by a high-frequency duplex ultrasonic cardiogram system and a transducer. Rats underwent ultrasonic cardiography at day3, weeks1,2and4before sacrificed. The transducer for ultrasonic cardiography was placed at the left thoraces between the3rd and4th ribs to obtain B-mode tracings of the heart from just below the level of the papillary muscles of the mitral valve. We obtained left-ventricular end-diastolic diameters (LVD-d) and end-systolic diameters (LVD-s) with M-mode tracings between the anterior and posterior walls. The time of end-diastole and end-systole was defined as time of maximum and minimum diameter of the left ventricle, respectively, in one heart cycle. Following the American Society of Echocardiology leading-edge method, we obtained3images, on average, in each view, which were averaged over three consecutive cycles. The system calculated the left-ventricular end-diastolic volume (LV-d), left-ventricular end-systolic volume (LV-s), mass of the left ventricle (LV-mass), left-ventricular fractional shortening (LVFS) and left-ventricular ejection fraction (LVEF)
5. Serological markers detection
ELISA was used to measure cardiac troponin T (Tn-T) and brain natriuretic peptide (BNP) concentration in serum for left ventricular function evaluation, by use of a BNP kit (Rat-45, Abcam, USA) and Tn-T kit (TSZ ELISA, USA). Briefly, standards and diluted serum of rats were added into the pre-coated96-well plates and incubated for30min in37℃. After a washing with PBS, the horseradish peroxidase-conjugated anti-body was added for30min incubation at37℃. After a washing by PBS, the tetramethylbenzibine substrate was added. After reaching the desired color density, the reaction was terminated by stop solution. OD450was determined by use of an ELISA plate reader (Varioskan Flash, Thermo Fisher, Germany). Each samples repeated in3wells.
6. Determination of EPC number in peripheral blood and bone marrow
Fluorescence-activated cell sorting (FACS) was used to determine the EPC population in blood and bone marrow of rats. Briefly, fresh anticoagulation blood or bone-marrow PBS suspension (200μl) was incubated with the monoclonal antibodies: anti-VEGFR2(Abeam, USA,1mg/ml,1:100), anti-CD133(Abeam, USA,0.5mg/ml,1:100) and anti-CD34-PerCP-Cy5.5(Santa Cruz Biotechnology, Santa Cruz, CA;0.2mg/ml,1:10) for20min at room temperature, then with2ml Lysing solution (BD, USA) for10min and washed with PBS twice by centrifugation. The cells were resuspended with200μl PBS, then incubated with the secondary antibodies:goat polyclonal rabbit IgG-FITC (Abeam, USA,2mg/ml,1:80) and goat polyclonal mouse IgG-F(ab)2fragment PE (Abeam, USA,0.5mg/ml,1:40) for30min at room temperature. Cells were washed with PBS and resuspended in400μl PBS. Flow cytometry involved use of a FACS calibur flow cytometer and Cell-Quest software (BD Biosciences, USA). Each analysis included at least10,000cells.
7. Function detection of EPCs from bone marrow.
(4) MTT assay was used to evaluate EPC proliferation.
(5) EPC migration was evaluated by use of a Transwell chamber.
(6) Capillary-like tube formation was analyzed by use of Matrigel Matrix.
8. Histological analysis
Myocardial tissues (approximately2mm thick) in the left ventricle of rats were removed and fixed in4%pre-cooled paraformaldehyde for72h, then embedded in paraffin, and sectioned into slices5μm thick. Haematoxylin eosin (HE) and Poley's stain was used to observe the form of the myocardium and assess the ischemic myocardial area. Images were visualized under an optical microscope at×200magnification.
9. Apoptosis of myocardium detection.
Myocardial tissue sections underwent the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using an in situ detection kit (Roche, Germany) following the manufacturer's instructions. The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total myocardial cells.
10. Immunohistochemical analysis
Immunohistochemical staining involved standard techniques as described. Briefly, endogenous peroxidase activity was inhibited by incubation with3%H2O2. Sections were blocked with5%calf serum in PBS and incubated overnight at4℃with the monoclonal antibodies:anti-VEGFR2(Abcam, USA,1mg/ml,1:100), anti-CD133(Abcam, USA,0.5mg/ml,1:50) and anti-CXCR4(Abcam, USA,1mg/ml,1:100). After a washing with PBS, sections were incubated with secondary antibody at37℃for30min. Immunohistochemical staining was visualized by use of a diaminobenzidine kit (Zhongshan Goldenbridge Biotechnology, Beijing). Samples were counter stained with hematoxylin for nuclei.
11. Real-time quantitative polymerase chain reaction (RT-PCR)
Tissue samples were frozen with the use of liquid nitrogen. Total RNA was extracted by use of TRIZOL reagent (Invitrogen, USA), quantified by spectrophotometry and reverse transcribed by use of the M-MLV Reverse Transcriptase System (Osaka, Japan) with oligo-dT primers. The mRNA expression of VEGFR2, CD133, and CXCR4in myocardium was examined by real-time RT-PCR with SYBR Green Real-time PCR Master Mix (TOYOBO, Life Science Department, Japan) and an MYIQTM Single Color Real-Time PCR Detection System (Bio-Rad, Germany). The mRNA sequences were obtained from Gene-bank (NCBI, Bethesda, MD; Tablel). Actin level was an internal control. Experiments were performed in triplicate, and data were analyzed by the2-△△CT method.
12. Statistical analysis
Data are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05
Results
1. General situation of rats
The rats (6weeks old, body weight at160-180g) were fed a regular rat chow and housed in normal night-day conditions under standard temperature and humidity. The rats in each group were normally health and no natural mortality during the test.
2. Electrocardiography and echocardiography analysis
After surgery induction, the ECG revealed elevated ST segment and pathologic waveforms, the UCG revealed changes in left ventricular wall mobility, blood flow at the mitral valve and the increased LV-d, LV-s, LV-mass, the decreased LVEF, LVFS, the reversed E/A ratio (P<0.01-0.05). These revealed the successful establishment of the MI model. After MI, the function of left ventricular was reflected by LVEF of UCG. In acute stage (day3to week1), the MI groups with both treatment showed almost no difference in LVEF, while as to the chronic stage (week2and week4), the recovery of LVEF was greater with RGE than NS (P<0.05). These revealed that RGE systemic delivery protected the function of left ventricular in chronic stage after MI.
3. Serological markers detection
The significantly up-regulated serum levels of Tn-T and BNP in NS-MI and GRE-MI groups also showed the successful establishment of mouse MI model (P<0.01). After MI, the high level of Tn-T decreased in RGE group (from week1, P<0.01～0.05) earlier than that in NS group (from week4, P<0.01). As for BNP, the level increased from day3to week4with NS (P<0.01～0.05), while had no changes with RGE treatment after MI. These revealed that RGE systemic delivery decreased myocardial damage and protected them from further inflammatory reaction after MI.
4. Determination of EPC number in peripheral blood and bone marrow
FACS was used to analyze the quantity of EPCs marked by CD34, VEGFR2and CD133in blood and bone marrow. After MI, the quantity of EPCs in peripheral blood increased (P<0.01) and it decreased in bone marrow (P<0.05) with both RGE and NS. These suggested that the EPCs in bone marrow were mobilized to peripheral blood as the injury of myocardium. In chronic stage after MI, the increase of EPCs in peripheral blood was more significant with RGE compared to NS (P<0.01), and the decrease of EPCs in bone was not so much with RGE as it with NS. These suggested that in chronic stage after MI, RGE was able to increase EPCs mobilizing to ischemic myocardium and maintain the quantity of EPCs stored in bone marrow. With the increased EPC population in both bone marrow and peripheral blood, the total number of EPCs in vivo was much more with RGE than NS.
5. Function detection of EPCs from bone marrow
By MTT assay, we tested the proliferation of EPCs in each group. The MI surgery made the proliferated activity of EPCs up-regulated with both GRE and NS (P<0.01). However, it maintained at a high level with RGE compared to it decreased in week2and4with NS (P<0.05). These showed that RGE was able to up-regulated the proliferation of EPCs after MI, especially in chronic stage.
At week4after MI, the migration of EPCs was more active with RGE than NS (P<0.05). From week2after MI, EPCs participated in capillary-like tube formation were increased with RGE compared to NS (P<0.05), and a similar increase occurred in normal rats with RGE relative to NS (P<0.05). These suggested REG's function on motivating EPCs tube-formation capacity after MI as well as in normal physiological status
6. Histological analysis
HE staining showed that at weekl after MI, myocardial tissue infarction occurred obvious histomorphology changes:infarction area muscle fiber dissolving, fracture, arrangement disorder.
Poley's stain showed the ischemic myocardial zone (stained red) in normal myocardium (stained blue). In chronic stage after MI, the relative ischemic area was lower with RGE than NS (P<0.05).
7. Apoptosis of myocardium detection
TUNEL stain and quantitative analysis showed that the apoptotic myocardium was less with RGE than NS in chronic stage after MI (P<0.01～0.05). These showed RGE's function on improving ischemic myocardium and decreasing myocardial apoptosis.
8. Immunohistochemical analysis
Immunohistochemistry showed the fluctuant expression of VEGFR2and CD133, which were signals of new-born capillary. After MI, the expression of VEGFR2increased from week1until week4with RGE (P<0.05-0.01), and was much more significant than that with NS (P<0.05～0.01). In chronic stage after MI, the expression of CD133also increased more with RGE than NS (P<0.01). The expression fluctuation at the level of mRNA was almost the same. These revealed that RGE was able to promote the newborn of capillary at the chronic stage of MI.
9. Real-time quantitative polymerase chain reaction(RT-PCR)
Real-time PCR showed the fluctuant expression at the level of mRNA on VEGFR2and CD133, which were signals of new-born capillary. The expression of VEGFR2increased from week1until week4with RGE (P<0.05～0.01) after MI, and was more significant than that with NS (P<0.05～0.01). In chronic stage of MI, the expression of CD133mRNA also increased with RGE compared to NS (P<0.01).
Conclusions
1. RGE systermic delivery after myocardial infarction can reduce necrosis and apoptosis of the myocardial ischemic cells, protect the infarction ventricular function through increasing angiogenesis and improving blood supply in infarction region, thus improve the prognosis of myocardial infarction.
2. RGE systermic delivery can increase the quantity of EPCs mobilized from bone marrow to peripheral blood after myocardial infarction, and maintain a highly active of EPCs, promote their participated into tube cavity formation. At the same time, RGE could also keep the relative stability number of EPCs in bone marrow.
3. Compared with the NS group, the effects of RGE in the treatment of myocardial infarction almost occurred in the chronic stage rather than acute stage.
Background
In physiological state, the number of endothelial progenitor cells (EPCs) in peripheral blood and tissue is very low, there are a certain number of EPCs in the bone marrow, however, most of them were under the resting state. Local damage or some EPCs agonist was able to mobilize the EPCs from bone marrow to the peripheral blood, and plante in the damage localization. Stromal cell-derived factor (SDF) played an important role in the process of EPCs' mobilization and migration.
Activated EPCs first migrate to the ischemic tissue for their roles. Stromal-derived factor-1(SDF-1, or CXCL12) is the only known chemokine capable of migration of hematopoietic stem cells (HSCs), as the fluctuations in SDF-1expression controlled the fluctuated steady-state of HSCs and their progenitors in peripheral blood. Among these, the SDF-1α and its receptor4(CXCR4) play a key role in mobilization and migration of EPCs. After MI, SDF-1α/CXCR4interaction plays a crucial role in recruiting EPCs to the ischemic myocardium, the increased CXCR4expression lead to increased EPCs homing to the ischemic zone and participated in therapeutic angiogenesis. These suggest that the SDF-1α/CXCR4cascade is critical for the regulation of EPCs, and it might be an important therapeutic target for cardiovascular diseases especially in MI.
In experiment Ⅰ and experiment Ⅱ, we had confirmed that Rehmannia Glutinosa Extract (RGE) could up-regulated the number and function of EPCs in bone marrow and peripheral blood, under the state of pathologic and physiologic caused by myocardial infarction model. In the chronic phase of myocardial infarction, RGE treated the myocardial infarction through activating EPCs to participate in angiogenesis in ischemic myocardium and the surrounding areas as well. We suggested that the effects of RGE on EPCs mobilization, migration, homing and angiogenesis function was through activating SDF-la/CXCR4cascade.
Objectives
1. Observe the situation of RGE stimulating EPCs in vitro.
2. Investigated the mechanism of RGE activating EPCs.
Materials and methods
1. Cell resource
Get from long bones (humerus, femur, tibia) of male WISTAR rat, isolated the mononuclear cells from bone marrow, induced and cultured primary endothelial progenitor cells.
2. Cultivation and identification of EPCs
Induced and cultured the bone marrow-derived mononuclear cells by EGM-2medium. After96hours, collected the adherented cells, analyzed these cells by Fluorescence-activated cell sorting (FACS) and Direct fluorescent staining. The EPCs were double stained by DiL-acLDL and FITC-UEA-1, the nuclei were stained with DAPI, and viewed by laser scanning confocal microscope.
3. Cell stimulation
(1) Concentration gradient screening:the EPCs were cultured with a serum-free medium for24hours, RGE were added to the6-well plates with endothelial progenitor cells at the concentration of10,25,50,100,500and1000μg/ml, the control group EPCs were cultured only with the medium. Collected the cells and supernatant of each well after72hours, waiting to be tested.
(2) The optimal concentration screening:the EPCs were cultured with a serum-free medium for24hours, RGE were added to the6-well plates with endothelial progenitor cells at the concentration of10,25,50and100μg/ml, the control group EPCs were cultured only with the medium. In the inhibitor group, EPCs were pre-stimulated with AMD3100(specific inhibitors of the CXCR4,5μg/ml) for one hour and then with RGE (100μg/ml). Each group of cells and supernatant were collected after72hours stimulation, waiting to be tested.
(3) The optimal duration screening:the EPCs were cultured with a serum-free medium for24hours, RGE stimulated EPCs at its optimal concentration (50μg/ml for SDF-1α,25μg/ml for CXCR4). The control group EPCs were cultured only with the medium. Collected the cells and supernatant at6,12,24,48,72hours after stimulation respectively, waiting to be tested.
(4) Mechanism analysis:the EPCs were cultured with a serum-free medium for24hours, stimulated the EPCs with RGE at the optimal concentration and duration of SDF-1α (50μg/ml,24h) and CXCR4(25μg/ml,48h), in the control group EPCs were cultured only with the medium. In the inhibitor group, EPCs were pre-stimulated with AMD3100(5μg/ml) for one hour and then with RGE (50μg/ml for24h,25μg/ml for48h), remove the RGE-containing medium, plus normal medium for functional test.
4. Proliferation function detection
5. Western blot analysis
(1) Extract the myocardial tissue protein, measure the protein concentration, and exam the protein expression levels of CXCR4, SDF-1α and β-actin by gel electrophoresis, membrane transform, incubating with antibody and exposure etc.
(2) Extract the protein from EPCs, exam the protein expression levels of CXCR4, SDF-1α and p-actin as above.
6. RT-PCR analysis
(1) Extract the myocardial tissue mRNA, reverse transcribed into cDNA, exam the mRNA levels of CXCR4, SDF-1α and β-actin by quantitative real-time polymerase chain reaction.
(2) Extract the mRNA from EPCs, exam the mRNA levels of CXCR4, SDF-1α and β-actin as above.
7. Statistical analysis
Data are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05.
Results
1. Myocardial immunohistochemical staining
Immunohistochemical staining was used to detect the expression of CXCR4in myocardium. We can see that, compared with the control group and the sham group, the CXCR4expression increased in ischemic myocardium (P<0.01); for the infarction groups, compared with normal saline (NS), RGE could increase myocardial expression of CXCR4in the chronic phase of MI (2weeks to4weeks)(P<0.01); RGE could also increase myocardial CXCR4expression in the control group and the sham group (P<0.05～0.01).
2. The protein expression in myocardial tissue
Extract the myocardial tissue protein, test the expression of SDF-1α/CXCR4cascade by western blot. Image Pro-Plus software was used for quantitative analysis and then
内皮广泛存在于人体各级动、静脉及毛细血管内膜表层,对维持血管的正常功能起着重要的作用。从胚胎时期的血管发生形成原始血管网到成年后的生理状态下的血管内皮代谢及病理状态下的血管内皮修复,内皮细胞的作用贯穿于这一系列生理、病理过程的始终。内皮细胞的损伤及功能紊乱导致了心、脑、肾等重要脏器血管性疾病的发生、发展,并与其预后直接相关。
内皮祖细胞(endothelial progenitor cells EPCs)作为内皮细胞的前体,最早由Asahara等发现,并于2002年定义为一种阳性表达CD34、CD133、VEGFR2,且可与荆豆凝集素(UEA-1)和乙酰化低密度脂蛋白(acLDL)特异性结合的具有部分内皮功能和分化能力的单个核细胞群。内皮祖细胞(EPCs)来源于骨髓,存在于体循环中,它既能循环、增殖、迁移、分化为成熟的内皮细胞参与血管再生,也能够结合在血管内皮层,刺激邻近内皮细胞的增殖。许多内皮祖细胞激动剂,如粒细胞集落刺激因子,血管内皮生长因子和他汀类药物,已被证实可以激活内皮祖细胞,然而这些EPC激动剂的各种不良反应,如血管通透性增加、再狭窄率升高以及严重的肝功能损害等,严重限制了它们在临床治疗中的应用。
熟地黄是一种沿用千年的传统中药,味甜、甘,性温。归肝经、肾经,有补血滋润、益精填髓之功效。可用于治疗血虚萎黄、眩晕心悸、月经不调、崩不止,肝肾阴亏、潮热盗汗、遗精阳痿、不育不孕,腰膝酸软、耳鸣耳聋、头目昏花、须发早白,消渴、便秘、肾虚喘促等症。在现代医学研究中,熟地黄提取物(Rehmannia glutinosa extract RGE)被证实可以促进造血干细胞的增殖、分化,并促进骨髓DNA含量的增加。由此,我们假设：在生理状态下,熟地提取物RGE是否可作用于内皮祖细胞,从而达到其“补骨填髓”之功效。
目的
1掌握熟地提取物灌胃全身给药方法,观察不同浓度熟地提取物灌胃在不同时间点对生理状态下大鼠循环和骨髓内皮祖细胞作用变化
2筛选最佳给药浓度和作用时间,为后期实验提供基础。探索内皮祖细胞的体外分离、诱导培养、鉴定及功能检测方法。
方法
1地黄提取物的制备
熟地黄粗粉经反复水溶、醇提得到地黄粗多糖,并以Sevag法去除蛋白纯化为地黄多糖,并检测其浓度、纯度、提取率。
2实验分组
80只雄性WISTAR大鼠随机分为对照组(20只,RGE低浓度组(20只,0.38g/kg·d),RGE中浓度组(20只,0.75g/kg·d),RGE高浓度组(20只,1.5g/kg·d)。各组分别于3天、4周、8周、12周、16周取外周血,于4周、8周、12周、16周处死动物取骨髓。
3内皮祖细胞分离、培养
取大鼠外周血及骨髓,用密度梯度法以淋巴细胞分离液Ficoll分离出单个核细胞,以4～5×107的细胞密度将单个核细胞平铺于25cm2的细胞培养瓶中,用加入了诱导因子的培养基EBM-2诱导培养。
4内皮祖细胞鉴定
贴壁的细胞用DiL-acLDL和FITC-UEA-1标记,DAPI染核,荧光共聚焦显微镜下观察,DiL-acLDL和FITC-UEA-1双表达的细胞记作EPCs。
5内皮祖细胞计数
分离出的单个核细胞与CD34、CD133、VEFFR2一抗分别孵育,后再与之相对应的二抗：IgG-PerCP-Cy5.5、IgG-F(ab)2-PE、IgG-FITC孵育。流式细胞仪分析细胞群中三种标志物阳性表达的细胞数。
6内皮祖细胞功能检测
(1)增殖功能检测：MTT法检测EPCs增殖能力。
(2)迁移功能检测：Transwell小室法检测EPCs迁移能力。
(3)成管功能检测：Matrigel胶辅助下检测EPCs管腔结构的形成能力。
7统计学分析
统计学处理数量资料以均数±标准差(x±s)表示,所有数据用软件SPSS11.5进行统计分析。两组间数据的比较采用独立样本的t检验,多组之间的比较用单因素方差分析。多组间的两两比较用费希尔的LSD法。当P＜0.05,认为有统计学差异。
结果
1实验动物的一般情况
80只雄性WISTAR大鼠,5～6周龄,体重在160～180克,普通饮食,正常昼夜节律,适应性喂养3天,颈动脉取外周血。经口灌胃给药,分为RGE低、中、高三个浓度组和对照组。每日每只大鼠灌胃药物剂量为1m1,每周根据体重变化调整给药浓度,对照组每日生理盐水1ml灌胃。各组大鼠健康状况正常,试验中无自然死亡。
2流式细胞术分析内皮祖细胞数量
流式细胞仪分析结果显示,与对照组相比,各药物组外周血和骨髓EPCs数量随灌药时间的延长而增加。在外周血：8周末,中、高剂量组EPCs增加量较对照组有统计学意义(P＜0.01～0.05)；12周末和16周末,三种剂量的RGE灌胃均使EPCs较对照组显著增多(P＜0.01),但各剂量组12至16周的增长速率(8.53%、1.57%、1.79%)明显低于8至12周的增长率(10.43%、12.84%、16.50%)。在骨髓：8周末,中、高剂量组EPCs数量较对照组有增加(P＜0.05)；12周末和16周末,三种剂量的RGE灌胃均使EPCs较对照组显著增多(P＜0.01),各组8至12周增长速率为正(6.95%、12.10%、17.14%)然而12至16周增长速率明显下降(高浓度组3.34%)或增长停滞(低、中浓度组)。
3外周血内皮祖细胞功能检测
外周血分离单个核细胞,培养基EBM-2诱导培养48h换液,上清中的未贴壁细胞离心后重新加入,再经过一个48h后弃去不贴壁细胞,继续诱导培养48h。培养瓶内的EPCs以胰酶消化并计数,备用功能检测。
(1)增殖功能检测
体外诱导培养6天的外周血EPCs用MTT法检测细胞增殖能力。在16周末,中、高浓度组增殖能力与对照组相比有所升高(P＜0.05),与各组4周末数据相比亦有统计学差异(P＜0.05)。其余时间点各组增值能力无统计学差异。(2)迁移功能检测
Transwell小室法用于检测体外培养6天的外周血EPCs的迁移能力。可见12周末,中、高浓度组可使迁移能力相较对照组和各组的4周末数据有所增高(P＜0.05),但这种数量变化不明显,且从12周末到16周末这种增殖能力没有进一步的增长。(3)成管功能检测
体外培养6天的外周血EPCs平铺于Matrigel胶预孵育的96孔板上,检测EPCs管状结构形成能力。高浓度组在8周末相较于4周末城管能力有所增长(P＜0.05),12周末成管能力的增高与对照组相比有统计学意义(P＜0.05)。低、中、高浓度组在16周末成管能力相较于对照组均有显著增高(P＜0.05),与各组4周末数据相比较亦有统计学差异(P<0.05)。
4骨髓内皮祖细胞功能检测
骨髓PBS悬浊液中分离单个核细胞,培养基EBM-2诱导培养48h换液,传代,与上清液中分离出的未贴壁细胞共培养,48h后换液,弃上清,继续诱导培养4天。培养瓶内的EPCs以胰酶消化并计数,备用功能检测。
(1)增殖功能检测
体外诱导培养6天的骨髓EPCs用MTT法检测细胞增殖能力。在12周末,中、高浓度组增殖能力与对照组相比有所升高(P＜0.05),与各组4周、8周末数据相比亦有所增加(P＜0.05)。16周末,这种增值能力的增长进一步加剧(P＜0.01)。
(2)迁移功能检测
Transwell小室法用于检测体外培养6天的骨髓EPCs的迁移能力。12周末和16周末,三种浓度的药物刺激组迁移能力相较于对照组和各组4周末数据均有所增加(P＜0.05),然而这种增长并不明显。
(3)成管功能检测
体外培养6天的骨髓EPCs平铺于Matrigel胶预孵育的96孔板上,检测其管状结构形成能力。12周末,中、高浓度组EPCs成管功能的增高与对照组相比有统计学意义(P＜0.05)。在16周末,这种相比对照组成管功能的增长更加明显(P＜0.01～0.05),且相对于各组4周末数据已有所增长(P＜0.01～0.05)。这种变化在高浓度组更加显著(P＜0.01)。
结论
1在正常生理条件下,三种不同浓度的熟地提取物溶液灌胃喂养成年雄性大鼠,均可导致大鼠体内EPCs数目的增多。相较于外周血来源的EPCs,骨髓来源EPCs的这种效应更为明显。且三种药物浓度中高浓度组作用较为突出,增长速率在灌胃后的12周左右达到最大。
2在生理条件下,RGE灌胃可活化大鼠外周血和骨髓中EPCs的功能。但这种活化作用在外周血EPCs中表现多不显著。在骨髓中,EPCs的活化多发生于灌胃后的12周左右。
背景
心肌梗死(myocardial infarction)是指在冠状动脉病变的基础上,冠状动脉的血流中断,使相应的心肌出现严重而持久地急性缺血,最终导致心肌的缺血性坏死。因此,血管再通及建立有效的侧支循环是挽救缺血心肌,改善心肌梗死预后及降低死亡率的首要措施。越来越多的证据表明细胞移植和治疗性血管再生是治疗终末期冠状动脉疾病所致心肌梗死的最具潜力和研究意义的新方法。近年来的研究显示：骨髓来源的干细胞分化形成的内皮祖细胞(endothelial progenitor cells, EPC)可在心梗后动员、迁移、归巢到损伤部位,并促进缺血区血管的新生,从而有效保护梗死后的心肌。近年来对熟地的现代医学作用研究显示,熟地可有效促进血细胞和骨髓增殖,治疗心脑血管缺血性疾病。然而研究方法的陈旧、手段的单一、机制探讨的肤浅限制了熟地在临床上的推广,且目前为止尚未有研究将熟地的作用机制与内皮祖细胞相连。
血管发生和血管生成是新血管形成的两种基本形式。血管发生是指胚胎中的成血管细胞向内皮细胞系分化包绕形成原始血管网并最终分化成熟形成心血管系统；血管生成是指从在原有血管网基础上,通过内皮细胞迁移、增殖、伸展及管状结构形成等步骤最终长成新生毛细血管的复杂过程。成熟个体中的血管再生主要通过新毛细血管的生成得以实现。参与成熟个体中血管再生的细胞为内皮祖细胞。因此内皮祖细胞成为当今医疗领域中治疗缺血性疾病的研究焦点,尤其是在治疗心肌梗死中成为新的治疗靶点。生理状态下,外周血中的EPCs数量较少,而骨髓中的EPCs多处于相对静息状态；心梗发生后在急性期内,骨髓中的EPCs被动员到外周血中,活化并归巢到损伤部位,通过参与损伤部位血管的新生维持损伤部位的血供。
我们由实验Ⅰ证实了熟地提取物在生理状态下对内皮祖细胞的动员作用,由此我们做出推断：熟地提取物是否可以动员心梗条件下体内的EPC从而达到保护受损心肌,治疗心肌梗死的作用。本实验中我们针对这一假设展开实验。
目的
1.建立大鼠心肌梗死模型,探讨中药熟地黄在心梗的急性期和慢性期对大鼠缺血心肌的保护作用。
2.观察心梗后大鼠外周血和骨髓中内皮祖细胞的数量、功能变化,探讨内皮祖细胞在急性期和慢性期对心梗的治疗作用,并观察熟地对内皮祖细胞这一作用的影响。
方法：
1.实验动物
120只雄性WISTAR大鼠(8周龄,体重180～200g)随机分为两组(每组60只)：空白组生理盐水1ml／d灌胃,试验组熟地提取物生理盐水溶液1.5g/kg·d灌胃。
两组分别灌胃8周后,每组又随机分为对照组(16只,不手术),假手术组(16只,手术开胸穿线不结扎),心梗组(28只,行冠状动脉前降支结扎术)。至此实验大鼠分为6组：生理盐水对照组(N-b,16只)、生理盐水假手术组(N-m,16只)、生理盐水心梗组(N-MI,28只)、熟地对照组(RGE-b,16只)、熟地假手术组(RGE-m,16只)、熟地心梗组(RGE-MI,28只。
术后继续灌胃喂养4周,分别于术后第3天,1周末,2周末,4周末处死各组4～7只大鼠。
2.心梗模型的建立
法一：大鼠用2.5%的戊巴比妥钠(30mg/kg)腹腔注射麻醉。仰卧位将大鼠固定在手术台上,剪除胸部被毛。胸骨左侧3～4肋间心尖搏动最明显处横向剪开胸部皮肤约1.5～2cm,钝性分离皮下组织、胸大肌、前锯肌。顺肋间肌纹理钝性撑开3～4肋间,可见搏动的心尖,在心尖以上0.5cm处快速穿过可吸收缝合线,闭合式结扎。对合前锯肌、胸大肌,胸部皮肤缝合前轻挤压缝合口排出胸腔气体,缝合皮肤切口。皮下注射青霉素8万IU/kg常规抗感染。
法二：乙醚吸入式麻醉大鼠,仰卧位固定于手术台上,剪除颈部及胸部被毛。纵向剪开大鼠颈部皮肤约1.5～2cm,分离皮下组织及肌肉,钝性分离气管,于气管环之间剪开气管半周。行气管插管,连接呼吸机,调整呼吸频率、呼吸比和潮气量。胸骨左缘3mm处纵向剪开皮肤约4cm,钝性分离皮下组织、胸大肌、前锯肌。于第三肋间剪开肋间肌暴露胸膜。撑开肋骨,于呼气相刺破胸膜,暴露心脏。在肺动脉圆锥与左心耳交界下方2mm处以可吸收缝合线结扎冠状动脉前降支。逐层缝合肌肉和皮肤。大鼠苏醒后拔除气管插管,清理气道血块及分泌物。对合颈部肌肉及皮下组织,缝合皮肤切口。皮下注射青霉素8万IU／kg常规抗感染。
假手术组手术步骤同上,冠状动脉处穿过结扎线但不打结。
3.存活率分析
保留存活记录并绘制生存曲线。
4.心电图及超声心动图检测
所有大鼠于术前和术中进行心电图监测,显示即时心功能情况。大鼠于术前和心梗后3天、1周、2周、4周,处死之前行经壁超声心动图检测,B型超声记录心脏运动情况,M型超声测量同一个心动周期内左室收缩末期和舒张末期的左室内径,系统计算左室收缩末期容积(LV-s),左室舒张末期容积(LV-d),左室相对质量(LV-mass),左室射血分数(EF%),左室短轴缩短率(FS%),血流E峰、A峰比值(E／A)。
5.血清学指标检测
酶联免疫吸附法(ELISA)测定血清中心肌损伤相关指标Tn-T、BNP的含量,反应心肌损伤程度。
6.外周血、骨髓中内皮祖细胞量的测定
外周血／骨髓细胞悬液经红细胞裂解后与CD34、CD133、EFFR2抗体共孵育,后再与之相对应的二抗：IgG-PerCP-Cy5.5、IgG-F(ab)2-PE、IgG-FITC共孵育。流式细胞仪分析细胞群中三种标志物阳性表达的细胞数。
7.体外培养的骨髓内皮祖细胞功能测定
(1)增殖功能检测：MTT法检测EPCs增殖能力。
(2)迁移功能检测：Transwell小室法检测EPCs迁移能力。
(3)成管功能检测：Matrigel胶辅助下的EPCs管腔结构形成能力检测。
8.组织病理学染色
留取大鼠心脏标本,行苏木素-伊红染色(HE),POLEY缺血染色,观察心肌组织形状,及各组心肌缺血情况。
9.心肌细胞凋亡情况检测
TUNEL法检测各组梗死区及其周边区心肌细胞的凋亡情况
10.免疫组织化学染色
免疫组织化学检测梗死部分心肌血管新生情况,检测新生毛细血管内皮相关指标CD133、VEGFR2。
11.实时定量聚合酶链反应(Real-time PCR)检测组织mRNA水平Real-time PCR检测心肌组织内血管新生相关指标CD133、VEGFR2的表达水平。
12.统计学分析
统计学处理数量资料以均数±标准差(x±s)表示,所有数据用软件SPSS11.5进行统计分析。两组数据间比较用独立样本的t检验,多组之间的两两比较用单因素方差分析。多组间的两两比较用费希尔的LSD法。当P<0.05,认为有统计学差异。
结果：
1.实验动物的一般情况
本研究共涉及雄性WISTAR大鼠120只,假手术32只,心梗手术56只。行心梗手术的大鼠当天死亡5只,手术成功率为91%,心梗急性期3天内死亡4只,急性期存活率为84%,其后的试验中死于超声麻醉意外的2只,不明原因死亡2只,总存活率为77%。假手术组术后急性期死亡2只,存活率为94%。
2.心电图及心脏超声检测结果
大鼠于术前和术中进行心电图监测,显示结扎后ST段改变和病理性Q波的出现。大鼠术后超声结果与术前相比较：左室收缩期内径(LV-s)、左室舒张期内径(LV-d)、左室相对质量(LV-mass)升高,左室射血分数(EF)、左室短轴缩短率(FS)下降,E／A倒置(P<0.01＜0.05)。这些指标的变化标志着心梗模型的成功建立。
超声结果显示：在大鼠心梗急性期(术后3天至术后1周),熟地组和生理盐水组大鼠的左室射血分数几乎没有差异；而在心梗的慢性期(术后2周至术后4周),与生理盐水组相比熟地组可明显改善心梗造成的EF、FS的降低(P＜0.05)。
3.血清学检测
心梗术后血清中Tn-T和BNP含量与对照组和假手术组相比显著升高(P＜0.01)亦证实了心梗模型的成功建立。
心梗后,熟地组血清Tn-T含量在术后1周末开始下降(P＜0.01-0.05),而生理盐水组则在术后4周末开始下降(P＜0.05),证实熟地可在心梗后降低心肌损伤。
心梗后,生理盐水组血清中的BNP含量随着时间延长呈上升趋势(P＜0.01-0.05),而在熟地组这种上升趋势则不明显。提示熟地干预可降低心梗后发生心肌细胞相关延时性损伤的风险。
4.外周血和骨髓中EPCs含量的测定
心梗术后与术前相比,熟地组和生理盐水外周血的EPCs含量均升高而骨髓中EPCs含量则降低(P＜0.01～0.05),说明心梗发生后,心肌损伤导致骨髓中的EPCs被动员到了外周血中。在心肌梗死发生后的慢性期(2周末和4周末),熟地组外周血EPCs的增长幅度大于生理盐水组(P＜0.01),而熟地组骨髓EPCs的降低不如生理盐水组明显(P＜0.01),可见熟地可以增加动员到外周血的EPCs数量同时保持骨髓中EPCs的储备量。由此亦可判断,熟地作用于心梗大鼠,除了增加了EPCs动员量以外,体内EPCs总量也有所增加。
5.骨髓EPCs功能的测定
(1)增殖功能检测：心肌的梗死使生理盐水组和熟地组的EPCs增殖能力均有所上升(P＜0.01),这种上升在生理盐水组于术后2周开始下降(P＜0.05),而在熟地组这种增殖能力则一直保持在较高水平。可见熟地可促进心梗后EPCs的增值能力。
(2)迁移功能检测：心梗后生理盐水组和熟地组的EPCs迁移能力均有所增加(P＜0.01),而在心梗术后4周,熟地组EPCs的迁移能力比生理盐水组更加活跃(P＜0.05)。
(3)成管功能检测：从心梗术后2周熟地组EPCs参与成管的数量明显多于生理盐水组(P＜0.05)。在空白对照组,也有类似的情况发生。说明,熟地可强化心梗后EPCs的成管功能,亦能在生理状态下激活这种功能。
6.组织病理学染色检测
(1)HE染色显示：术后1周开始,梗死心肌组织发生明显的组织形态学变化：梗死区肌纤维溶解、断裂,排列紊乱。
(2)POLEY缺血染色：缺血心肌染为红色,而正常心肌组织为蓝色。在心梗的急性期(术后3天到1周)熟地组和生理盐水组差异不明显；到心梗慢性期(术后2周到4周)熟地组缺血心肌的面积较之生理盐水组下降明显(P＜0.05)。
7.心肌细胞凋亡检测
TUNEL染色法用于检测心肌细胞凋亡情况。在心梗术后2周和4周,熟地组心肌细胞的凋亡量明显少于生理盐水组。
8.免疫组织化学检测
(1)心肌梗死后,在两种干预(生理盐水和熟地)下,VEGFR2在心肌组织中的表达均随时间变化有所增加(P＜0.05-0.01),而在心梗发生1周以后(熟地灌胃9周)开始,VEGFR2在熟地组中的表达逐渐高于在生理盐水组中的表达(P＜0.05～0.01)。
(2)心肌梗死后,在两种干预(生理盐水和熟地)下,CD133在心肌组织中的表达均随时间变化有所增加(P＜0.05-0.01),而在心梗的慢性期(心梗术后2周到4周,CD133在熟地组中表达的增多明显高于生理盐水组(P＜0.01)。
9.Real-time PCR检测
为了验证免疫组织化学中对VEGFR2、CD133表达量变化的检测,我们用Real-time PCR在mRNA水平验证VEGFR2和CD133的表达量。结果显示VEGFR2和CD133在rnRNA水平的变化趋势与免疫组织化学染色法中检测到的它们在蛋白水平的变化趋势一致。结论：
(1)熟地灌胃可降低心梗后心肌细胞的缺血坏死和凋亡,通过增加血管新生改善梗死心肌的血供,保护梗死后的心室功能,改善预后。
(2)熟地灌胃可增加心肌梗死后骨髓动员到外周血的EPCs数量,并保持EPCs的较高活性,促进其参与管腔形成；于此同时,又可保持骨髓EPCs数量的相对稳定。
(3)与生理盐水组相比,熟地的治疗作用大多发生在心肌梗死的慢性恢复期,而非急性发作期。
背景：
生理状态下,外周血和组织中内皮祖细胞(Endothelial progenitor cells, EPCs)的数量极低,骨髓中有一定数量的EPCs,但多处于静息状态。局部损伤或在一些激动剂的作用下,骨髓EPCs可动员迁移到外周血,定植于损伤局部。在EPCs的动员迁移过程中,基质细胞衍生因子(Stromal-derived factor, SDF)发挥了重要作用。
基质细胞衍生因子1(SDF-1或称CXCL12)是目前已知的唯一能够迁移造血干细胞的趋化因子,SDF-1表达量的变化促使EPCs改变其原有的静息状态并进一步活化、迁移、定植、分化。SDF-1与其受体CXCR4结合,使CXCR4由低能量构象状态进入过渡状态,CXCR4内部盐桥断裂暴露结合位点,与SDF-1α进一步结合使CXCR4进入激活状态,启动下游的一系列信号转导,一系列级联过程最终导致了EPCs的活化和迁移。更有研究指出,在EPCs介导的心梗后的损伤修复过程中,SDF-1α/CXCR4的激活,损伤局部SDF-1α表达的上调最终指导了骨髓EPCs迁移到外周血并归巢到损伤部位。
在实验Ⅰ和实验Ⅱ中,我们已证实熟地在生理状态及心梗模型造成的病理状态下均可促进骨髓和外周血EPCs的数量和功能,且在心梗的慢性期通过活化EPCs参与心肌梗死区及周边区血管的新生达到治疗心梗的效果。我们推测,熟地对EPCs的这种动员、迁移、归巢、血管新生等功能的促进作用是否通过激活SDF-1α/CXCR4系统实现。
目的
3.观察熟地提取物在体外刺激内皮祖细胞的情况。
4.探讨熟地提取物激活内皮祖细胞的作用机制。
方法：
13.细胞来源
取雄性WISTAR大鼠长骨(肱骨、股骨、胫骨)髓腔中的骨髓,分离单个核细胞,诱导培养为元代内皮祖细胞。
14.细胞的培养鉴定
EGM-2培养基诱导培养骨髓来源的单个核细胞,96小时后收集贴壁细胞,流式细胞仪分析,DiL-acLDL及FITC-UEA-1荧光双染,荧光共聚焦显微镜下观察,鉴定其为内皮祖细胞。
15.细胞刺激
(1)浓度梯度筛选：无血清培养基培养内皮祖细胞24小时后,熟地黄提取物分别以10、25、50、100、500及1000μg/ml的浓度加入植有内皮祖细胞的6孔板各孔的培养基中,对照组仅以培养基培养。72小时后收集各孔细胞和上清液,待检。
(2)最佳作用浓度筛选：无血清培养基培养内皮祖细胞24小时后,以适宜浓度梯度(10,25,50,100μg/ml)刺激6孔板内的内皮祖细胞,对照组仅以培养基培养,抑制剂组用CXCR4的特异性抑制剂AMD3100在5μg/ml的浓度下预刺激1小时后加熟地提取物刺激(100μg/ml)。72小时后收集各孔细胞和上清液,待检。
(3)最佳作用时间筛选：无血清培养基培养内皮祖细胞24小时后,以最佳浓度刺激6孔板内的内皮祖细胞(SDF-1α为50μg/ml, CXCR4为25μg/ml),对照组仅以培养基培养,分别于刺激后的6、12、24、48、72小时收集各孔细胞和上清液,待检。
(4)机制探讨分析：无血清培养基培养内皮祖细胞24小时后,分别以SDF-1α的最佳作用浓度和时间(50μg/ml,24h)及CXCR4的最佳作用浓度和时间(25μg/ml,48h)刺激6孔板内的细胞,对照组仅以培养基培养,抑制剂组用CXCR4的特异性抑制剂AMD3100在5μg/ml的浓度下预刺激1小时后加熟地提取物刺激(50μg/ml,24h及25μg/ml,48h),去除含RGE的培养基,加普通培养基进行细胞功能学检测。
16.增殖功能检测
MTT法检测各组细胞增殖能力。17.Western Blot检测
(1)提取心肌组织的蛋白,测定蛋白浓度,并通过凝胶电泳、转膜、一抗及相应二抗的标记、曝光等步骤检测CXCR4、SDF-1α、β-actin等的蛋白表达水平。
(2)提取各组细胞蛋白,同上方法检测CXCR4、SDF-1α、β-actin等的蛋白表达水平。18.Real-time RT-PCR检测
(1)提取心肌组织的RNA,逆转录成cDNA,通过荧光实时定量聚合酶链反应,检测CXCR4、SDF-1α、β-actin等的RNA表达水平。
(2)提取各组细胞RNA,同上方法检测CXCR4、SDF-1α、β-actin等的RNA表达水平
19.统计学分析
统计学处理数量资料以均数±标准差(x±s)表示,所得数据用软件SPSS11.5进行统计学分析。当P＜0.05,认为所比较的两组有统计学差异。
结果：
1.心肌免疫组化染色
免疫组化染色检测心肌中内皮祖细胞动员相关因子CXCR4的表达情况。可见与空白对照组及假手术组相比,心梗阻大鼠心肌CXCR4表达增高(P＜0.01)；在梗死组中,与生理盐水灌胃相比,熟地灌胃可在梗死后的慢性期(2周到4周)增加心肌中CXCR4的表达(P＜0.01)；在空白对照组和假手术组,熟地灌胃与生理盐水灌胃相比亦可增加心肌的CXCR4表达量(P＜0.05-0.01)。
2.心肌组织蛋白表达量检测
提取各组大鼠心肌组织的蛋白,WesternBlot法检测SDF-1α/CXCR4的表达情况。通过Image Pro-Plus软件对蛋白印迹进行定量分析发现：与对照组和假手术组相比,心梗组CXCR4表达升高(P＜0.01),SDF-1α亦升高(P＜0.01),但SDF-1α的升高在心梗的急性期(3天到1周)内表现不明显；在心梗大鼠中,与生理盐水灌胃相比,熟地灌胃使CXCR4表达在心梗的慢性期升高(P＜0.01),熟地对SDF-1α表达的上调作用表现在心梗1周后(P＜0.01)而在之后的慢性期内熟地对SDF-1α作用不明显；在正常对照组,熟地亦可上调CXCR4的表达(P＜0.05～0.01),但熟地的这种上调作用对SDF-1α的表达影响不大。
3.心肌组织基因表达量检测
提取各组大鼠心肌组织RNA,逆转录为cDNA, Real-time PCR法定量检测SDF-1α/CXCR4的基因表达情况。可见与对照组和假手术组相比,心梗组CXCR4表达升高(P＜0.01),SDF-1α的表达亦有所升高(P＜0.01),且SDF-1α及CXCR4的表达增加随时间的发展呈递进关系(P＜0.05～0.01)；在心梗大鼠中,与生理盐水灌胃相比,熟地灌胃使CXCR4的表达在心梗的慢性期上调(P＜0.05～0.01),熟地对SDF-1α表达的上调作用直至心梗后4周方开始显现(P<0.05)。
4.RGE刺激细胞浓度的筛选
在体外实验中,用不同浓度的RGE(浓度分别为10μg/ml,25μg/ml,50μg/ml,100μg/ml,500μg/ml,1000μg/ml)刺激元代培养的内皮祖细胞,72小时后收集细胞进行增殖活性检测,发现元代细胞用500μg/ml和1000μg/ml浓度的RGE刺激后,其增殖能力与对照组相比明显降低(P<0.05)。提示高浓度(500μg/ml,1000μg/ml) RGE因其对细胞增殖能力抑制作用而不适合用于接下来的体外细胞实验中。
5.RGE刺激细胞的最佳浓度选择
用筛选出的4种浓度RGE(10μg/ml,25μg/ml,50μg/ml)刺激细胞,与对照组和其它各浓度组相比,25μg/ml组CXCR4的蛋白以及RNA表达水平均升高(P＜0.01)；50μg/ml组SDF-1α的蛋白及RNA表达水平升高(P＜0.01)。抑制剂组与其它各组相比,CXCR4的蛋白以及RNA表达水平均降低(P＜0.01),SDF-1α的蛋白和RNA表达水平升高(P＜0.01)。由此我们推出：25μg/ml是RGE刺激CXCR4表达上调的较适宜浓度；50μg/ml是RGE刺激SDF-1α表达上调的较适宜浓度；在AMD3100的作用下,CXCR4的翻译和转录下调,而SDF-1α翻译和转录上调。
6.RGE刺激细胞的最佳时间选择
以25μg/ml的RGE浓度刺激元代细胞,分别于刺激后的6小时、12小时、24小时、48小时、72小时收集细胞,检测CXCR4的表达,发现与对照组和其它各组相比,刺激48小时的细胞CXCR4的蛋白和RNA表达量增加(P<0.05),可见48小时是RGE刺激CXCR4表达上调的较适宜时间。
以50μg/ml的RGE浓度刺激元代细胞,分别于刺激后的6小时、12小时、24小时、48小时、72小时收集细胞,检测SDF-1α的表达,发现与对照组和其它各组相比,刺激24小时的细胞SDF-1α的蛋白和RNA表达量增加(P＜0.01),可见24小时是RGE刺激SDF-1α表达上调的较适宜时间。
7.细胞功能检测
用RGE对CXCR4和SDF-1α的最佳作用浓度和时间(25μg/ml,48h以及50μg/ml,24h)分别刺激细胞后,去除含有RGE的培养基,换正常培养基进行细胞功能检测,与对照组和抑制剂组相比,元代EPC的成管能力增强,尤其RGE25μg/ml刺激48小时组,成管能力明显高于对照组和抑制剂组(P＜0.05-0.01)；抑制剂组细胞基本不参与新的管腔形成,其功能明显低于其他各组(P＜0.01)。
结论：
1.熟地提取物灌胃可上调生理状态下和心梗状态下心肌组织中SDF-1α及CXCR4的蛋白和RNA表达。
2.体外试验中,熟地提取物刺激元代培养的EPCs,可使细胞和上清中SDF-1α及CXCR4的蛋白和RNA表达升高,并且CXCR4的抑制剂AMD3100可抵消熟地提取物的这一作用。
3.SDF-1α及CXCR4升高的EPCs功能明显增强,AMD3100竞争性抑制CXCR4表达使细胞功能减弱甚至丧失。
4.熟地通过上调SDF-1α/CXCR4的表达激活EPCs,熟地的这种作用在体外可被AMD3100抵消。
Background
Endothelial progenitor cells (EPCs) were firstly found by Pro. Asahara, and were defined as precursors of endothelial cells which positive expressed CD34, CD133, VEGFR2. EPCs derived from the bone marrow, existed in the systemic circulation. It can proliferate, migrate, and differentiate into mature endothelial cells and participate in angiogenesis, besides it is capable of binding in the blood vessels and stimulating the proliferation of neighboring endothelial cells. Many EPCs agonists such as granulocyte-colony stimulating factor, vascular endothelial growth factor (VEGF) and statins, can mobilize EPCs in bone marrow. However adverse reactions, such as increased vascular permeability and high ratio of restenosis and liver damage, limit their use in clinical.
Prepared Rehmannia glutinosa (PRG), belongs to the family of Scrophulariaceae, is a widely used traditional Chinese medicinal herb. It has been used to treat hypodynamia caused by many kinds of diseases. It has been effective and safe, but the involved mechanism has not been verified. Recently, Rehmannia glutinosa extract (RGE) has been used in modern medicine studies. RGE can stimulate the proliferation and differentiation of hematopoietic stem cells in bone marrow and increase the DNA content of bone marrow. So we supposed that Rehmannia glutinosa extract (RGE) could activate EPCs.
Objectives
Grasp the method of RGE systemic administration. Investigate the changed effect of RGE on EPCs in bone marrow and circulation blood of normal rats along with the differ in concentration and duration of RGE. Screen the optimal concentration and duration for RGE, and provide basis for the subsequent experiments. Explore the separation, induced cultivation, identification and functional measurements for EPCs in vitro.
Method
1. Preparations of Rehmannia glutinosa extract (RGE)
Rude polyoses was extracted form Rehmannia glutinosa powder by water dissolving and alcohol extracting repeatedly. And then RGE was extracted and purified in Sevag way. The concentration, purification and extraction ratio were tested.
2. Animal group
80male Wistar rats were random divided into four groups:in the control group (n=20) rats were oral-treated with normal saline, in the RGE-L group (n=20) rats were oral-treated with RGE at0.38g·kg-1·day-1, in the RGE-M group (n=20) rats were oral-treated with RGE at0.75g·kg-1·day-1, in the RGE-H group rats were oral-treated with RGE at1.5g·kg-1·day-1.5rats in each group were sacrificed at4,8,12,16weeks after systemic delivery.
3Isolation and cultivation of EPCs
10ml peripheral blood was obtained from rats by aspiration of the heart. Bone-marrow cells were obtained by flushing the cavity of femurs, tibias, and humerus with growth medium EBM-2. Peripheral blood and bone marrow mononuclear cells were isolated by Ficoll density-gradient centrifugation.10million isolated cells were resuspended in growth medium EBM-2and plated in25-cm2culture flasks. After48h, non-adherent cells were discarded and growth medium was changed every2days.
4. Identification of EPCs
Direct fluorescent staining was used to detect dual binding of FITC-UEA-land Dil-acLDL and the nuclei were stained with DAPI. Cells double stained for Dil-Ac-LDL and FITC-UEA-1were considered EPCs. Immunocytochemistry followed standard protocols.
5. Determination of EPC number
Fluorescence-activated cell sorting (FACS) was used to determine the EPC population in blood and bone marrow of rats. Briefly, fresh anticoagulation blood or bone-marrow PBS suspension (200μl) was incubated with the monoclonal antibodies: anti-VEGFR2, anti-CD133and anti-CD34-PerCP-Cy5.5for20min at room temperature, then with2ml Lysing solution for10min and washed with PBS twice by centrifugation. The cells were resuspended with200μl PBS, then incubated with the secondary antibodies:goat polyclonal rabbit IgG-FITC and goat polyclonal mouse IgG-F(ab)2fragment PE for30min at room temperature. Cells were washed with PBS and resuspended in400μl PBS.
6. function test of EPCs
(1) MTT assay was used to evaluate EPC proliferation.
(2) EPC migration was evaluated by use of a Transwell chamber.
(3) Capillary-like tube formation was analyzed by use of Matrigel Matrix.
7. Statistical analysis
Data are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05.
Results
1. General situation of rats
The rats (6weeks old, body weight at160-180g) were fed a regular rat chow and housed in normal night-day conditions under standard temperature and humidity. The rats in each group were normally health and no natural mortality during the test.
2. The number of EPCs was evaluated by FACS
The FASC showed that the number of EPCs increased with RGE systemic delivery both in blood and in bone marrow, and this tendency intensified with the time went by. In peripheral blood, at the end of8weeks, the EPCs number in RGE-M and RGE-H groups was statistically increased compared to control group (P<0.01-0.05); at the end of12and16weeks, the EPCs number in all the three RGE groups was increased significantly (P<0.01), however the ratios from12to16weeks (8.53%,1.57%,1.79%) were much lower than that from8to12weeks (10.43%,12.84%,16.50%). In bone marrow, at the end of8weeks, the EPCs number in RGE-M and RGE-H groups was increased compared with control group (P<0.05); at the end of12and16weeks, the EPCs number in all the three RGE groups was increased significantly (P<0.01), however the ratios were positive from8to12weeks (6.95%、12.10%.17.14%) and the ratios decreased (RGE-H3.34%) or negative (RGE-L RGE-M groups) from8to12weeks.
3. The functional test of EPCs in peripheral blood
Mononuclear cells were isolated from peripheral blood, induced cultured by EVM-2for48h, the nonadherented cells were centrifuged and co-cultured for another48h, and then discarded the cells still not adherent culture for the third48h. These cells were digested by trypsin and counted for use.
(1) Proliferation test
MTT showed the proliferation of EPCs from peripheral blood. At the end of16weeks, the proliferation of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05) and the same group at the end of4weeks (P<0.05). The other concentration at other time points had no statistical different.
(2) Migration test
Migration of EPCs in peripheral blood was evaluated by use of Transwell chamber. At the end of12weeks, it increased in RGE-M and RGE-H group compared to control group and the same group at the end of4weeks (P<0.05), however these changes seemed shyly. And as from12to16week, it had no further growth.
(3) Tube-formation test
Tube-formation capacity of peripheral blood EPCs was evaluated by Matrigel Matrix. In RGE-H group, at the end of8weeks it had increased compared to the end of4weeks (P<0.05), and at the end of12weeks it was more than control group (P<0.05). At the end of16weeks, the tube-formation capacity of EPCs in all the RGE groups significantly increased compared with control group and the same group at the end of4weeks as well.
4. The functional test of EPCs in bone marrow
Mononuclear cells were isolated from bone marrow PBS solution, induced cultured by EVM-2for48h, the nonadherented cells were centrifuged and co-cultured for another48h, then transfer of culture and discarded the cells still not adherent. After culture for6days, cells were digested by trypsin and counted for use.
(1) Proliferation test
MTT showed the proliferation of EPCs from bone marrow. At the end of12weeks, the proliferation of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05) and the same group at the end of4and8weeks (P<0.05). At the end of16weeks, it increased further more in all RGE groups than control group.
(2) Migration test
Migration of EPCs in bone marrow was evaluated by use of Transwell chamber. At the end of12and16weeks, it increased in all the RGE groups compared to control group and the same group at the end of4,8weeks (P<0.05), however the extent didn't significant.
(3) Tube-formation test
Tube-formation capacity of bone marrow EPCs was evaluated by Matrigel Matrix. At the end of12weeks, the tube-formation capacity of EPCs increased in RGE-M and RGE-H group compared with control group (P<0.05). At the end of16weeks, it significantly increased in all the RGE groups compared with control group and the same group at the end of4weeks (P<0.01-0.05). The RGE-H group was more significant than other groups (P<0.01).
Conclusion
1. Under physiological condition, rats systemic delivery with three different concentration of RGE, and lead to the number of EPCs in peripheral blood and bone marrow increased. This increase was much more significant in bone marrow than peripheral blood. The RGE-H group had the most extrusive effect among the three concentrations. The increment speed was maximum at about12weeks after oral-feed.
2. At the condition of physiology, RGE systemic delivery was able to active the EPCs in peripheral blood and bone marrow. This activation in peripheral blood EPCs was not so obvious as that in bone marrow EPCs. And in bone marrow, the activation of EPCs often appeared12weeks after oral-feed.
Background
Myocardial infarction (MI) occurs with the deprivation of coronary blood and is usually caused by stenosis or occlusion of the coronary artery. The culminating event is necrosis of myocardial tissue and dysfunction of the left ventricle. Therefore, recanalization and establishment of collateral circulation are primary measures for saving ischemic myocardium, improving the prognosis of myocardial infarction and reducing mortality. Many studies proved that cell transplantation and therapeutic angiogenesis is a most potential and significant method for the treatment of end-stage coronary artery disease. Recent studies showed that endothelial progenitor cells (EPCs) which were differentiated from bone marrow-derived stem cells can be activated after myocardial infarction, and mobilization, migration, homing to the site of injury and participated in angiogenesis, so that protect the infarcted myocardium. The modern medical studies about Prepared Rehmannia Glutinosa showed that itcould effectively promote the proliferation of blood cells and bone marrow, it could also be used for the treatment of ischemic cardiovascular and cerebrovascular diseases. Obsolete research methods, relatively single mechanism, superficial studies about mechanism, however superficial limits the promotion of Rehmannia in clinical research. And so far no research connected the mechanism of Rehmannia Glutinosa Extra (RGE) with the action of endothelial progenitor cells yet.
Objectives
1. Establish a rat model of myocardial infarction, and investigate the protective effect of Chinese medicine Prepared Rehmannia Glutinosa on infarcted rat myocardium in acute and chronic phase.
2. Observed the change of endothelial progenitor cells in rat peripheral blood and bone marrow after myocardial infarction, including number and function. Explore the therapeutic effect of endothelial progenitor cells in the acute and chronic phase of myocardial infarction. Discuss the role of RGE on endothelial progenitor cells' therapeutic effect.
Methods
1. Animal group
A total of120male Wistar rats (8weeks old; body weight180-200g) were randomized to2groups (n=60each) for treatment:high-dose RGE (1.5g·kg-1·day-1orally) for8weeks, then left anterior descending coronary artery ligation (n=28), mock surgery (n=16) or no treatment (n=16), then RGE orally for4weeks; or normal saline (NS) as the above protocol. Then rats were sacrificed,4～7each on the3rd day and the end of1,2,4weeks, recording as day3, week1, week2and week4respectively.
2. The establishment of a rat myocardial infarction model.
Method1:The rats were narcotized by2.5%napental (30mg/kg), and fixed the rat on the operating table in supine position, cut off its chest hair. Horizontal cut the chest shin about1.5～2cm at the3to4left intercostal sternal where the apex beat most obvious and blunt dissection of the subcutaneous tissue, pectoralis major, serratus anterior muscle. Blunt softened3to4intercostal shun intercostal muscle texture, seen the beating apex, fast through the myocardium0.5cm above the apex with the absorbable suture, and closed ligation. Light squeeze together serratus anterior muscle, the pectoralis major, chest skin and discharge pleural gas before suture the skin incision. Subcutaneous injection of penicillin80,000IU/kg of conventional anti-infective.
Method2:The rats were narcotized by ethylether, and fixed the rat on the operating table in supine position, cut off its neck and chest hair. Longitudinal cut open the rat neck skin about1.5～2cm, separate subcutaneous tissue and muscle, blunt separation trachea, cut open tracheal half cycle between trachea ring. Do endotracheal intubations, connection breathing machine, adjust the frequency of breathing, breathing ratio and tidal volume. Longitudinal cut open chest skin about4cm at the left edge of the sternal3mm place, blunt separation subcutaneous tissue, and the pectoralis major, former saw muscle. Cut open the intercostal muscle on the third intercostals space to exposure pleura. Open the frame, punctured pleura at the breathe out phase, exposing the heart. Absorpted suture ligation at the anterior descending coronary artery in place2mm below the common border of the pulmonary artery cone and the left atrium. Layered suture muscle and skin. Remove the endotracheal intubation after rats were awake, and clearing the airway blood clot and secretion.
The rats in mock groups underwent mock surgery with a silk suture across the coronary artery without ligation.
3. Mortality analysis:
Reserved survival record and draw the survival curve.
4. Electrocardiography and echocardiography analysis
Before and after surgery, rats underwent electrocardiography (ECG) by use of a Micromaxx P04224system and ultrasonic cardiography (UCG) by a high-frequency duplex ultrasonic cardiogram system and a transducer. Rats underwent ultrasonic cardiography at day3, weeks1,2and4before sacrificed. The transducer for ultrasonic cardiography was placed at the left thoraces between the3rd and4th ribs to obtain B-mode tracings of the heart from just below the level of the papillary muscles of the mitral valve. We obtained left-ventricular end-diastolic diameters (LVD-d) and end-systolic diameters (LVD-s) with M-mode tracings between the anterior and posterior walls. The time of end-diastole and end-systole was defined as time of maximum and minimum diameter of the left ventricle, respectively, in one heart cycle. Following the American Society of Echocardiology leading-edge method, we obtained3images, on average, in each view, which were averaged over three consecutive cycles. The system calculated the left-ventricular end-diastolic volume (LV-d), left-ventricular end-systolic volume (LV-s), mass of the left ventricle (LV-mass), left-ventricular fractional shortening (LVFS) and left-ventricular ejection fraction (LVEF)
5. Serological markers detection
ELISA was used to measure cardiac troponin T (Tn-T) and brain natriuretic peptide (BNP) concentration in serum for left ventricular function evaluation, by use of a BNP kit (Rat-45, Abcam, USA) and Tn-T kit (TSZ ELISA, USA). Briefly, standards and diluted serum of rats were added into the pre-coated96-well plates and incubated for30min in37℃. After a washing with PBS, the horseradish peroxidase-conjugated anti-body was added for30min incubation at37℃. After a washing by PBS, the tetramethylbenzibine substrate was added. After reaching the desired color density, the reaction was terminated by stop solution. OD450was determined by use of an ELISA plate reader (Varioskan Flash, Thermo Fisher, Germany). Each samples repeated in3wells.
6. Determination of EPC number in peripheral blood and bone marrow
Fluorescence-activated cell sorting (FACS) was used to determine the EPC population in blood and bone marrow of rats. Briefly, fresh anticoagulation blood or bone-marrow PBS suspension (200μl) was incubated with the monoclonal antibodies: anti-VEGFR2(Abeam, USA,1mg/ml,1:100), anti-CD133(Abeam, USA,0.5mg/ml,1:100) and anti-CD34-PerCP-Cy5.5(Santa Cruz Biotechnology, Santa Cruz, CA;0.2mg/ml,1:10) for20min at room temperature, then with2ml Lysing solution (BD, USA) for10min and washed with PBS twice by centrifugation. The cells were resuspended with200μl PBS, then incubated with the secondary antibodies:goat polyclonal rabbit IgG-FITC (Abeam, USA,2mg/ml,1:80) and goat polyclonal mouse IgG-F(ab)2fragment PE (Abeam, USA,0.5mg/ml,1:40) for30min at room temperature. Cells were washed with PBS and resuspended in400μl PBS. Flow cytometry involved use of a FACS calibur flow cytometer and Cell-Quest software (BD Biosciences, USA). Each analysis included at least10,000cells.
7. Function detection of EPCs from bone marrow.
(4) MTT assay was used to evaluate EPC proliferation.
(5) EPC migration was evaluated by use of a Transwell chamber.
(6) Capillary-like tube formation was analyzed by use of Matrigel Matrix.
8. Histological analysis
Myocardial tissues (approximately2mm thick) in the left ventricle of rats were removed and fixed in4%pre-cooled paraformaldehyde for72h, then embedded in paraffin, and sectioned into slices5μm thick. Haematoxylin eosin (HE) and Poley's stain was used to observe the form of the myocardium and assess the ischemic myocardial area. Images were visualized under an optical microscope at×200magnification.
9. Apoptosis of myocardium detection.
Myocardial tissue sections underwent the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using an in situ detection kit (Roche, Germany) following the manufacturer's instructions. The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total myocardial cells.
10. Immunohistochemical analysis
Immunohistochemical staining involved standard techniques as described. Briefly, endogenous peroxidase activity was inhibited by incubation with3%H2O2. Sections were blocked with5%calf serum in PBS and incubated overnight at4℃with the monoclonal antibodies:anti-VEGFR2(Abcam, USA,1mg/ml,1:100), anti-CD133(Abcam, USA,0.5mg/ml,1:50) and anti-CXCR4(Abcam, USA,1mg/ml,1:100). After a washing with PBS, sections were incubated with secondary antibody at37℃for30min. Immunohistochemical staining was visualized by use of a diaminobenzidine kit (Zhongshan Goldenbridge Biotechnology, Beijing). Samples were counter stained with hematoxylin for nuclei.
11. Real-time quantitative polymerase chain reaction (RT-PCR)
Tissue samples were frozen with the use of liquid nitrogen. Total RNA was extracted by use of TRIZOL reagent (Invitrogen, USA), quantified by spectrophotometry and reverse transcribed by use of the M-MLV Reverse Transcriptase System (Osaka, Japan) with oligo-dT primers. The mRNA expression of VEGFR2, CD133, and CXCR4in myocardium was examined by real-time RT-PCR with SYBR Green Real-time PCR Master Mix (TOYOBO, Life Science Department, Japan) and an MYIQTM Single Color Real-Time PCR Detection System (Bio-Rad, Germany). The mRNA sequences were obtained from Gene-bank (NCBI, Bethesda, MD; Tablel). Actin level was an internal control. Experiments were performed in triplicate, and data were analyzed by the2-△△CT method.
12. Statistical analysis
Data are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05
Results
1. General situation of rats
The rats (6weeks old, body weight at160-180g) were fed a regular rat chow and housed in normal night-day conditions under standard temperature and humidity. The rats in each group were normally health and no natural mortality during the test.
2. Electrocardiography and echocardiography analysis
After surgery induction, the ECG revealed elevated ST segment and pathologic waveforms, the UCG revealed changes in left ventricular wall mobility, blood flow at the mitral valve and the increased LV-d, LV-s, LV-mass, the decreased LVEF, LVFS, the reversed E/A ratio (P<0.01-0.05). These revealed the successful establishment of the MI model. After MI, the function of left ventricular was reflected by LVEF of UCG. In acute stage (day3to week1), the MI groups with both treatment showed almost no difference in LVEF, while as to the chronic stage (week2and week4), the recovery of LVEF was greater with RGE than NS (P<0.05). These revealed that RGE systemic delivery protected the function of left ventricular in chronic stage after MI.
3. Serological markers detection
The significantly up-regulated serum levels of Tn-T and BNP in NS-MI and GRE-MI groups also showed the successful establishment of mouse MI model (P<0.01). After MI, the high level of Tn-T decreased in RGE group (from week1, P<0.01～0.05) earlier than that in NS group (from week4, P<0.01). As for BNP, the level increased from day3to week4with NS (P<0.01～0.05), while had no changes with RGE treatment after MI. These revealed that RGE systemic delivery decreased myocardial damage and protected them from further inflammatory reaction after MI.
4. Determination of EPC number in peripheral blood and bone marrow
FACS was used to analyze the quantity of EPCs marked by CD34, VEGFR2and CD133in blood and bone marrow. After MI, the quantity of EPCs in peripheral blood increased (P<0.01) and it decreased in bone marrow (P<0.05) with both RGE and NS. These suggested that the EPCs in bone marrow were mobilized to peripheral blood as the injury of myocardium. In chronic stage after MI, the increase of EPCs in peripheral blood was more significant with RGE compared to NS (P<0.01), and the decrease of EPCs in bone was not so much with RGE as it with NS. These suggested that in chronic stage after MI, RGE was able to increase EPCs mobilizing to ischemic myocardium and maintain the quantity of EPCs stored in bone marrow. With the increased EPC population in both bone marrow and peripheral blood, the total number of EPCs in vivo was much more with RGE than NS.
5. Function detection of EPCs from bone marrow
By MTT assay, we tested the proliferation of EPCs in each group. The MI surgery made the proliferated activity of EPCs up-regulated with both GRE and NS (P<0.01). However, it maintained at a high level with RGE compared to it decreased in week2and4with NS (P<0.05). These showed that RGE was able to up-regulated the proliferation of EPCs after MI, especially in chronic stage.
At week4after MI, the migration of EPCs was more active with RGE than NS (P<0.05). From week2after MI, EPCs participated in capillary-like tube formation were increased with RGE compared to NS (P<0.05), and a similar increase occurred in normal rats with RGE relative to NS (P<0.05). These suggested REG's function on motivating EPCs tube-formation capacity after MI as well as in normal physiological status
6. Histological analysis
HE staining showed that at weekl after MI, myocardial tissue infarction occurred obvious histomorphology changes:infarction area muscle fiber dissolving, fracture, arrangement disorder.
Poley's stain showed the ischemic myocardial zone (stained red) in normal myocardium (stained blue). In chronic stage after MI, the relative ischemic area was lower with RGE than NS (P<0.05).
7. Apoptosis of myocardium detection
TUNEL stain and quantitative analysis showed that the apoptotic myocardium was less with RGE than NS in chronic stage after MI (P<0.01～0.05). These showed RGE's function on improving ischemic myocardium and decreasing myocardial apoptosis.
8. Immunohistochemical analysis
Immunohistochemistry showed the fluctuant expression of VEGFR2and CD133, which were signals of new-born capillary. After MI, the expression of VEGFR2increased from week1until week4with RGE (P<0.05-0.01), and was much more significant than that with NS (P<0.05～0.01). In chronic stage after MI, the expression of CD133also increased more with RGE than NS (P<0.01). The expression fluctuation at the level of mRNA was almost the same. These revealed that RGE was able to promote the newborn of capillary at the chronic stage of MI.
9. Real-time quantitative polymerase chain reaction(RT-PCR)
Real-time PCR showed the fluctuant expression at the level of mRNA on VEGFR2and CD133, which were signals of new-born capillary. The expression of VEGFR2increased from week1until week4with RGE (P<0.05～0.01) after MI, and was more significant than that with NS (P<0.05～0.01). In chronic stage of MI, the expression of CD133mRNA also increased with RGE compared to NS (P<0.01).
Conclusions
1. RGE systermic delivery after myocardial infarction can reduce necrosis and apoptosis of the myocardial ischemic cells, protect the infarction ventricular function through increasing angiogenesis and improving blood supply in infarction region, thus improve the prognosis of myocardial infarction.
2. RGE systermic delivery can increase the quantity of EPCs mobilized from bone marrow to peripheral blood after myocardial infarction, and maintain a highly active of EPCs, promote their participated into tube cavity formation. At the same time, RGE could also keep the relative stability number of EPCs in bone marrow.
3. Compared with the NS group, the effects of RGE in the treatment of myocardial infarction almost occurred in the chronic stage rather than acute stage.
Background
In physiological state, the number of endothelial progenitor cells (EPCs) in peripheral blood and tissue is very low, there are a certain number of EPCs in the bone marrow, however, most of them were under the resting state. Local damage or some EPCs agonist was able to mobilize the EPCs from bone marrow to the peripheral blood, and plante in the damage localization. Stromal cell-derived factor (SDF) played an important role in the process of EPCs' mobilization and migration.
Activated EPCs first migrate to the ischemic tissue for their roles. Stromal-derived factor-1(SDF-1, or CXCL12) is the only known chemokine capable of migration of hematopoietic stem cells (HSCs), as the fluctuations in SDF-1expression controlled the fluctuated steady-state of HSCs and their progenitors in peripheral blood. Among these, the SDF-1α and its receptor4(CXCR4) play a key role in mobilization and migration of EPCs. After MI, SDF-1α/CXCR4interaction plays a crucial role in recruiting EPCs to the ischemic myocardium, the increased CXCR4expression lead to increased EPCs homing to the ischemic zone and participated in therapeutic angiogenesis. These suggest that the SDF-1α/CXCR4cascade is critical for the regulation of EPCs, and it might be an important therapeutic target for cardiovascular diseases especially in MI.
In experiment Ⅰ and experiment Ⅱ, we had confirmed that Rehmannia Glutinosa Extract (RGE) could up-regulated the number and function of EPCs in bone marrow and peripheral blood, under the state of pathologic and physiologic caused by myocardial infarction model. In the chronic phase of myocardial infarction, RGE treated the myocardial infarction through activating EPCs to participate in angiogenesis in ischemic myocardium and the surrounding areas as well. We suggested that the effects of RGE on EPCs mobilization, migration, homing and angiogenesis function was through activating SDF-la/CXCR4cascade.
Objectives
1. Observe the situation of RGE stimulating EPCs in vitro.
2. Investigated the mechanism of RGE activating EPCs.
Materials and methods
1. Cell resource
Get from long bones (humerus, femur, tibia) of male WISTAR rat, isolated the mononuclear cells from bone marrow, induced and cultured primary endothelial progenitor cells.
2. Cultivation and identification of EPCs
Induced and cultured the bone marrow-derived mononuclear cells by EGM-2medium. After96hours, collected the adherented cells, analyzed these cells by Fluorescence-activated cell sorting (FACS) and Direct fluorescent staining. The EPCs were double stained by DiL-acLDL and FITC-UEA-1, the nuclei were stained with DAPI, and viewed by laser scanning confocal microscope.
3. Cell stimulation
(1) Concentration gradient screening:the EPCs were cultured with a serum-free medium for24hours, RGE were added to the6-well plates with endothelial progenitor cells at the concentration of10,25,50,100,500and1000μg/ml, the control group EPCs were cultured only with the medium. Collected the cells and supernatant of each well after72hours, waiting to be tested.
(2) The optimal concentration screening:the EPCs were cultured with a serum-free medium for24hours, RGE were added to the6-well plates with endothelial progenitor cells at the concentration of10,25,50and100μg/ml, the control group EPCs were cultured only with the medium. In the inhibitor group, EPCs were pre-stimulated with AMD3100(specific inhibitors of the CXCR4,5μg/ml) for one hour and then with RGE (100μg/ml). Each group of cells and supernatant were collected after72hours stimulation, waiting to be tested.
(3) The optimal duration screening:the EPCs were cultured with a serum-free medium for24hours, RGE stimulated EPCs at its optimal concentration (50μg/ml for SDF-1α,25μg/ml for CXCR4). The control group EPCs were cultured only with the medium. Collected the cells and supernatant at6,12,24,48,72hours after stimulation respectively, waiting to be tested.
(4) Mechanism analysis:the EPCs were cultured with a serum-free medium for24hours, stimulated the EPCs with RGE at the optimal concentration and duration of SDF-1α (50μg/ml,24h) and CXCR4(25μg/ml,48h), in the control group EPCs were cultured only with the medium. In the inhibitor group, EPCs were pre-stimulated with AMD3100(5μg/ml) for one hour and then with RGE (50μg/ml for24h,25μg/ml for48h), remove the RGE-containing medium, plus normal medium for functional test.
4. Proliferation function detection
5. Western blot analysis
(1) Extract the myocardial tissue protein, measure the protein concentration, and exam the protein expression levels of CXCR4, SDF-1α and β-actin by gel electrophoresis, membrane transform, incubating with antibody and exposure etc.
(2) Extract the protein from EPCs, exam the protein expression levels of CXCR4, SDF-1α and p-actin as above.
6. RT-PCR analysis
(1) Extract the myocardial tissue mRNA, reverse transcribed into cDNA, exam the mRNA levels of CXCR4, SDF-1α and β-actin by quantitative real-time polymerase chain reaction.
(2) Extract the mRNA from EPCs, exam the mRNA levels of CXCR4, SDF-1α and β-actin as above.
7. Statistical analysis
Data are expressed as mean±SD and were assessed by one-sample Kolmogorov-Smirnov test to check for normal distribution. Differences between2groups were assessed by unpaired t-test and among multiple groups by ANOVA followed by post-hoc two-tailed Newman-Keuls test. Data analysis involved use of SPSS11.5(SPSS Inc., Chicago, IL). Statistical significance was set at P<0.05.
Results
1. Myocardial immunohistochemical staining
Immunohistochemical staining was used to detect the expression of CXCR4in myocardium. We can see that, compared with the control group and the sham group, the CXCR4expression increased in ischemic myocardium (P<0.01); for the infarction groups, compared with normal saline (NS), RGE could increase myocardial expression of CXCR4in the chronic phase of MI (2weeks to4weeks)(P<0.01); RGE could also increase myocardial CXCR4expression in the control group and the sham group (P<0.05～0.01).
2. The protein expression in myocardial tissue
Extract the myocardial tissue protein, test the expression of SDF-1α/CXCR4cascade by western blot. Image Pro-Plus software was used for quantitative analysis and then
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