猪骨髓干细胞体外分化和优化移植治疗急性心肌梗死的实验研究
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摘要
目的：探讨猪骨髓间充质干细胞(mesenchymal stem cells，MSCs)增殖和分化潜能的不均一性，以及自发分化和5-氮胞苷诱导分化为心肌细胞的潜能。
方法：抽取中华小型猪骨髓，密度梯度离心法[Percoll(1.077g／ml)]分离出单个核细胞，贴壁培养法筛选MSCs，原代(PO)培养后按1：3比例每3天传代一次。选取第1，4，8和10代(P1，P4，P8和P10)MSCs，研究其向心肌样细胞白发分化潜能，以及在5-氮胞苷诱导后4周能否分化为心肌细胞。连续镜下观察其形态变化；计数法分析MSCs增殖模式；免疫细胞化学染色法检测MSCs的表面标志(CD34、CD45、CD90和SH2)及心肌细胞特异性的蛋白表达，包括a-横纹肌肌动蛋白(a-sarcomeric actin，a-actin)、心肌肌钙蛋白-T(cardiactroponin-T，cTn-T)、肌球蛋白重链(myosin heavy chain，MHC)和连接蛋白43(connexin 43，Cx43)；以Real Time RT-PCR定量检测分化细胞是否表达心肌特异性基因及其表达量，包括GATA4、肌球蛋白轻链(myosin light chain，MLC)、MHC、肌浆网受磷蛋白(phospholamban)、a-actin、Cx43、结蛋白(desmin)和骨骼肌肌动蛋白(skeletal muscle actin，SM-actin)；透射电镜观察分化细胞超微结构。
结果：体外培养条件下，P1、P4、P8和大部分P10代MSCs形态一致，呈梭形成纤维细胞样形态，但部分P10代细胞多核、胞浆延长、和邻近细胞相互连接，转变为肌管样形态。5-氮胞苷诱导1周后，部分P1、P4和P8代MSCs表现为较长的胞浆形态和多核化，4周后更多细胞相互连接形成不规则的管样结构，而P10代MSCs则形成相对规则的肌管样结构。
计数法显示，各代MSCs培养3天后开始进入对数生长期直至平台期。P1、P4和P8代MSCs增殖速率相似(P=0.908)，倍增时间(population doubling time，PD)约48h。和上述早代MSCs相比，接种3天后，P10代MSCs增殖速率显著变慢(P＜0.05)，PD约为72h。
免疫细胞化学染色显示超过95％的贴壁细胞表达CD90、SH2，而不表达CD34、CD45，说明绝大多数贴壁细胞为MSCs。自然培养的P1、P4和P8代MSCs不表达心肌特异性结构蛋白，而部分P10代MSCs(10.3±1.8％)表达心肌特异蛋白：α-actin，MHC和cTn-T。各代MSCs经5-氮胞苷诱导后4周，均表达上述蛋白；其中，P1、P4和P8代MSCs向心肌细胞分化效率相似(18.3±8.2％vs 17.6±11.9％vs 16.5±5.0％，P＞0.05)，但均显著低于P10代MSCs的诱导成心肌细胞分化效率(44.5±4.2％，P=0.0001)。
RT-PCR结果表明，P1、P4和P8代MSCs在自然培养状态下无心肌特异性基因表达，而P10代MSCs则表达心肌特异性转录因子GATA4和结构基因desmin、phospholabman、MHC和MLC。5-氮胞苷诱导后4周，各代MSCs均表达上述基因。实时定量PCR结果表明，GATA4(P=0.018)和MLC(P＜0.0001)在P10代MSCs诱导后4周的表达丰度显著高于同期诱导的P4代诱导分化细胞。
透射电镜显示在自然培养状态下，P1、P4和P8代MSCs无任何肌细胞样结构，而部分P10代MSCs有不规则肌丝；5-氮胞苷诱导后4周，P1、P4和P8代MSCs分化的细胞有较多不规则肌丝、散乱肌节和横纹，而P10代MSCs分化的细胞可见丰富肌丝、规则肌节和横纹，尤其是闰盘结构。
结论：1．猪骨髓MSCs在体外自然培养条件下，形态学、增殖能力和分化潜能是不均一的，从P10代开始增殖变慢、部分细胞自发分化为心肌样细胞。2．5-氮胞苷可诱导MSCs分化为心肌样细胞，但是P10代MSCs分化的心肌细胞更趋成熟，具有相对成熟的心肌细胞结构，且分化效率更高。
目的：研究~(18)氟—脱氧葡萄糖(~(18)fluoro deoxy-glucose，~(18)F~-FDG)标记的骨髓单个核细胞(mononuclear cells，MNCs)经冠状动脉输注后在急性心肌梗死(acutemyocardial infarction，AMI)猪模型的早期全身分布、心肌内定位和对心功能的影响。
方法：选取10月龄中华小型猪14头(30±5kg)，分为对照组(经冠脉输注PBS，n=7)和移植组(经冠脉输注自体骨髓MNCs，n=7)。开胸后于紧靠第一对角支开口稍远处，胶管套扎左前降支(left anterior descending，LAD)冠状动脉90分钟制备AMI模型。AMI后1周，抽取自体骨髓分离出MNCs，以放射性同位素~(18)F-FDG(5mCi=185MBq)和荧光染料DAPI标记，调密度至1×10~9细胞／10ml。以over-the-wire balloon球囊导管在LAD结扎处阻断前向血流，同时输注标记的MNCs(1×10~9细胞／猪，n=6)，1小时后以双核素(~(18)氟／~(99)锝)—单光子发射计算机断层扫描(single photon emission computed tomography，SPECT)检测其全身分布和心脏内定位。分别以AMI后1周(移植当天)和移植后6周作为考察基线和终点，以~(99m)锝-甲氧基异丁基异腈(~(99m)Tc-MIBI)-SPECT检测心肌灌注缺损，以核磁共振成像(magnetic resonance imaging，MRI)检测心功能。终点指标检测结束后处死动物进行组织学检测，免疫组化法检测MNCs在梗死区和梗死周边区分化潜能以及毛细血管密度。
结果：AMI后24h内对照组和移植组分别有一只动物死于心力衰竭和心室颤动。~(18)F~-FDG标记后即刻，MNCs活力达97％以上，标记后1小时仍在93％以上，与非标记MNCs相似。放射活性检测表明，90.8±1.9％的放射性活性存留于MNCs中，上清液中放射性活性小于10％。DAPI对活细胞的标记效率为100％。
MNCs经冠脉输注后1小时，~(18)F-FDG-SPECT显示，心脏中的放射活性占全身的6.8±1.8％，90％以上的放射活性位于肝、脾和外周循环。双核素SPECT扫描相同层面的心肌并进行图像重叠，显示在所有移植动物心脏中，~(18)F-FDG放射活性全部聚集于灌注缺损区，相关性分析表明，心脏中放射活性强度与灌注缺损面积呈显著正相关(P=0.001，相关系数=0.973)。
基线时对照组和移植组心肌灌注缺损面积无显著差异(47.1±7.5％vs45.6±6.6％，P=0.713)，终点时，移植组灌注缺损面积显著小于对照组(23.9±5.1％vs 44.8±6.7％，P＜0.0001)。
MRI检测表明，基线时，对照组和移植组的各项心功能参数无显著差异(P＞0.05)。终点时，与对照组相比，移植组的心功能各项参数除左室舒张末期容积(end-diastolic volume，EDV)外均有显著改善，其中左室射血分数(leftventricular ejection fraction，LVEF)由43.9±8.5％增加到51.2±10.4％(差值d=7.3±4.8％)，而对照组由43.5±7.3％变为44.2±8.2％(d=0.7±2.0％，P=0.011)；左室收缩末期容积(end-systolic volume，ESV)由32.0±10.1ml变为31.5±10.3ml(d=-0.5±3.3 ml)，而对照组则从34.7±6.4 ml增加到38.5±7.3ml(d=3.8±3.1ml，P=0.039)；运动障碍节段数由8.2±1.2个减少至4.8±0.8个(d=-3.3±0.5)，对照组由7.8±1.0个减少为7.2±0.8个(d=-0.7±0.5，P＜0.0001)；室壁增厚率从-18.2±7.3％增加至45.7±11.1％(d=63.8±4.1％)，对照组从-17.5±6.3％减至-22.3±5.7％(d=-4.8±3.9％，P＜0.0001)；梗死面积从6.9±1.1 cm~2减小为3.7±0.7 cm~2(d=-3.1±0.8cm~2)，而对照组从6.6±0.8cm~2变为6.9±0.4cm~2(d=-0.3+0.5 cm~2，P＜0.0001)；左室质量指数从59.8±8.9g／m~2增加到64.2±7.9g／m~2(d=-4.3±1.5g／m~2)，而对照组从61.7±7.2g／m~2变为72.7±4.6g／m~2(d=-11.0±4.4 g／m~2，P=0.007)。移植组EDV由56.2±8.4ml变为63.3±7.8ml(d=7.2±4.1ml)，而对照组则从58.8±6.6ml增加到68.7±5.3ml(d=9.8±2.6ml，P=0.211)。
H&E染色和Masson’s三色染色结果显示，对照组心肌梗死区有严重纤维化和炎症细胞浸润，存活心肌较少，而移植组纤维化和炎症细胞浸润较轻，且伴有存活心肌。免疫荧光显示DAPI标记的细胞表达心肌和血管特异性的结构蛋白，包括α-横纹肌肌动蛋白、心肌肌钙蛋白T、连接蛋白43、Von Willebrand因子和血管平滑肌肌动蛋白。与对照组比较，移植组梗死区(1.8±0.5 vs 3.7±1.1／HPF，P＜0.003)和梗死周边区(8.7±2.0 vs 4.9±1.3／HPF，P＜0.003)毛细血管密度均显著增加。
结论：本研究表明：1．~(18)F-FDG标记和双核素SPECT显像可以用于监测骨髓MNCs经冠脉输注后全身分布和心肌内定位。2．经冠脉输注骨髓MNCs可显著减小灌注缺损面积、改善心功能和阻止心室重塑。
目的：探索性研究以通心络超微粉和他汀类药物改良急性心肌梗死(acutemyocardial infarction，AMI)猪模型的心肌微环境，对移植骨髓间充质干细胞(mesenchymal stem cells，MSCs)在体存活、分化和生物学活性的影响。
方法：56只中华小型猪(30±5kg)分为8组，每组7只：对照组、MSCs移植组、单纯通心络超微粉、辛伐他汀和阿托伐他汀干预组、三种药物改良+MSCs移植组。开胸结扎左前降支冠状动脉90min制备AMI模型，再灌注后随即于梗死区和梗死周边区心肌内注射自体骨髓MSCs(3×10~7细胞／500μl)，对照组以相同方法注射500μl低糖DMEM培养基。在6个药物干预组，分别于AMI前3天开始给予口服小剂量通心络超微粉(0.05g／d．kg)、辛伐他汀(0.25mg／d．kg)和阿托伐他汀(0.25mg／d．kg)，AMI当天和其后继续使用4天。
在移植后1周(基线)和6周(终点)，以单光子发射计算机断层显象(singlephoton emission computed tomography，SPECT)和核磁共振成像(magneticresonance imaging，MRI)检测心肌灌注缺损和心功能。免疫荧光检测移植细胞在体存活和分化；TUNEL法检测梗死周边细胞凋亡；分别以黄嘌呤氧化酶法和硫代巴比妥酸法测定梗死区超氧化物歧化酶(superoxide dismutase，SOD)活性和丙二醛(malondialdehyde，MDA)含量；以Western blot免疫印迹法检测梗死区白细胞介素-1β(interleukin--1β，IL-1β)、IL-6和肿瘤坏死因子-α(tumor necrosisfactor，TNF-α)的表达水平，以及梗死周边区促凋亡蛋白Bax和抑凋亡蛋白Bcl-2表达水平。
结果：SPECT扫描显示，基线时各组心肌灌注缺损面积无显著差异(51.8±16.5％，46.5±9.2％，47.9±12.2％，52.7±15.5％，49.7±16.8％，53.7±14.9％，48.9±15.9％vs 50.7±14.5％，P＞0.05)。终点时，MSCs组、通心络超微粉组和对照组无显著差异(47.3±13.2％，44.8±13.9％vs 47.8±11.1％，P＞0.05)，而辛伐他汀组和阿托伐他汀组灌注缺损面积均小于对照组(39.3±12.4％，38.7±13.1％vs47.8±11.1％，P＜0.05)；尤其是通心络超微粉+MSCs组、辛伐他汀+MSCs组、阿托伐他汀+MSCs组的灌注缺损面积进一步显著减小(27.6±7.4％，28.3±7.7％，29.3±9.0％vs 47.8±11.1％，P＜0.05)。
MRI检测显示，各组心功能参数在基线时无显著差异(P＞0.05)。终点时，与对照组相比，MSCs组和通心络超微粉组的各项心功能参数均无显著差异(P＞0.05)，而辛伐他汀组运动障碍节段数(P=0.039)和梗死面积(P=0.0061)、阿托伐他汀组的运动障碍节段数(P=0.04)均小于对照组。和对照组相比，在通心络超微粉、辛伐他汀和阿托伐他汀改良的MSCs移植组，除左室舒张末期容积(end-diastolic volume，EDV)外，各项心功能参数均有显著改善(P＜0.05)。
H&E染色和Masson’s Trichrome染色显示，在对照组，梗死区呈连续性大片状纤维化，有较多炎性细胞浸润，未见存活心肌细胞。MSCs组和通心络超微粉组心肌梗死区和对照组相似，而辛伐他汀组和阿托伐他汀组梗死区纤维化和炎性细胞浸润稍轻，有少量存活心肌。在通心络超微粉、辛伐他汀和阿托伐他汀改良的MSCs移植组，炎症细胞浸润显著减轻，存活心肌较多，纤维化呈散在分布。
在通心络超微粉、辛伐他汀和阿托伐他汀改良的MSCs移植组，移植细胞在体存活显著多于单纯MSCs移植组(223.1±26.9，310.6±83.8，373.9±90.3 vs73.2±21.3／HPF，P＜0.0001)。在各MSCs移植组，均有部分移植细胞表达心肌细胞特异性蛋白和血管内皮细胞特异性的因子Ⅷ；但三种药物改良的MSCs移植组，移植细胞向心肌细胞分化效率均显著高于单纯MSCs移植组(55.6±12.1％，46.0±5.1％，44.4±12.5％vs 8.7±2.4％，P＜0.0001)。各移植组均有部分MSCs表达心肌细胞特异性的连接蛋白Connexin43，但上述三种药物改良的移植组Connexin43表达显著强于单纯MSCs移植组(22.4±6.6，17.0±1.6，14.2±2.6 vs4.7±1.8，P＜0.0001)。
与对照组相比，MSCs组、通心络超微粉组、辛伐他汀组和阿托伐他汀组梗死区(1.8±0.8，1.9±0.5，2.2±0.7，1.9±0.6 vs 1.8±0.5／HPF，P＞0.05)和梗死周边毛细血管密度(5.2±1.4，5.4±0.8，5.4±1.2，5.4±1.0 vs 4.9±1.3／HPF，P＞0.05)均无显著性差异；而三种药物改良的MSCs移植组梗死区(4.1±0.7，3.9±1.0，3.9±0.9vs 1.8±0.5／HPF，P＜0.0001)和梗死周边毛细血管密度(8.9±1.7，8.5±1.6，8.6±1.2vs 4.9±1.3／HPF，P＜0.0001)均显著高于对照组。
终点时，对照组凋亡指数显著高于Sham组(10.1±1.8 vs 0.9±0.3，P＜0.0001)，MSCs组(9.2±1.4，P＞0.05)和对照组无显著差异，而通心络超微粉组(5.6±1.7)、辛伐他汀组(5.7±1.5)和阿托伐他汀组(5.2±1.9)凋亡指数均显著小于对照组(P＜0.0001)，和单纯药物干预组相比，各药物改良的MSCs移植组凋亡指数进一步减小(2.4±0.5，2.3±0.3，1.9±0.3，P＜0.0001)。Western blot检测表明，对照组促凋亡蛋白Bax表达显著增加(P＜0.0001)，抑凋亡蛋白表达显著降低(P＜0.0001)；和对照组相比，MSCs组、三种药物组和药物改良的MSCs移植组均使Bax表达降低(P＜0.0001)和Bcl-2增加(P＜0.0001)；其中，各药物改良的MSCs移植组在药物组的基础上使Bax表达进一步降低(P＜0.05)。
终点时，对照组梗死区SOD活力较Sham组显著降低(82.5±8.2 vs 122.5±12.2U／mg蛋白，P＜0.0001)，MSCs组(88.6±8.3 U／mg蛋白)和对照组相比无显著性差异(P＞0.05)；但通心络超微粉组(95.5±9.6U／mg蛋白)、辛伐他汀组(100.8±12.1U／mg蛋白)、阿托伐他汀组(98.8±13.4U／mg蛋白)、通心络超微粉+MSCs(101.7±9.1U／mg蛋白)、辛伐他汀+MSCs(108.5±10.2U／mg蛋白)和阿托伐他汀+MSCs组(109.0±13.8U／mg蛋白)均显著高于对照组(P＜0.0001)。对照组梗死区MDA含量较Sham组显著升高(9.0±0.8 vs 5.5±0.9nmol／mg蛋白，P＜0.0001)，MSCs组(8.5±0.6nmol／mg蛋白)和对照组相比无显著差异(P＞0.05)；而通心络超微粉组(5.9±0.7nmol／mg蛋白)、辛伐他汀组(5.8±0.7nmol／mg蛋白)、阿托伐他汀组(5.7±1.3nmol／mg蛋白)、通心络超微粉+MSCs(5.7±0.7nmol／mg蛋白)、辛伐他汀+MSCs(5.6±0.9nmol／mg蛋白)和阿托伐他汀+MSCs组(5.5±1.1nmol／mg蛋白)均显著低于对照组(P＜0.0001)。
Western blot检测显示，对照组梗死区IL-1β，IL-6和TNF-α表达较Sham组显著升高(P＜0.0001)，和对照组相比，MSCs组、三种药物干预组和药物改良的MSCs移植组梗死区上述因子表达均显著降低(P＜0.0001)。
结论：本实验表明：1．在AMI再灌注后即刻心肌内移植自体骨髓MSCs，其存活和分化能力有限，尽管能抑制促凋亡蛋白和部分炎症因子的表达，但并未产生显著的心功能获益；2．围移植期使用通心络超微粉、辛伐他汀和阿托伐他汀可显著改良梗死心肌局部微环境，增强移植的骨髓MSCs在体存活和分化，产生显著的心功能获益。
Objectives: Mesenchymal stem cells (MSCs) are a group of heterogenous stem cells with multipotency in growth and differentiation. This study was to explore the proliferative potential and cardiomyogenic development of porcine bone marrow-derived MSCs stimulated with or without 5-azacytidine.
Methods: In this study, the bone marrow was aspirated from Chinese mini-swine. The mononuclear cells were isolated by density gradient centrifugation through 1.077g/ml Percoll and then cultured in low-glucose DMEM containing 10% fetal calf serum. After primary culture, relatively purified MSCs were obtained by using the method of adherence screening. The cultured MSCs in vitro were passaged every 3 days after primary culture, and MSCs at passage 1, 4, 8 and 10 (P1, P4, P8 and P 10) were examined for their proliferation and differentiation stimulated with or without 5-azacytidine (10μM) in vitro by cell counting, immunocytochemistry, transmission electrical microscopy and RT-PCR/real time qPCR.
Results: In normal growth medium, approximately 10.34±1.8% of porcine bone marrow-derived MSCs at passage 10 in vitro spontaneously take on cardiomyocyte-like phenotype and structure, and express cardimyocyte-specific genes and proteins, however, earlier passages of MSCs don't. Four weeks after induction by 5-azacytidine, MSCs at passage 1, 4 and 8 all express cardiomyocyte-specific genes and proteins and have cardiomyocyte-like ultrastructure such as irregular myofilaments in vitro. However only MSCs at passage 10 express connexin 43 at the level of gene and protein, and take on relatively mature cardiomyocyte ultrastructure, including myofilaments, sarcomeres and transverse striations, especially intercalated discs in vitro. The MSC-derived myogenic cells showed cardiac myogenic but not skeletal myogenic markers at both mRNA and protein levels. In addition, the cardiomyogenic differentiation efficiency of MSCs at passage 10 are significantly higher than that of earlier passages (44.5±4.2% vs. 18.3±8.2%, 17.6±11.9%,16.5±5.0%, respectively, all P＜0.0001).
Conclusion: The potency of growth and differentiation of porcine bone marrow-derived MSCs spontaneously or in response to stimulation of 5-azacytidine is dependent upon the endogenous gene expression and passage condition of MSCs. The myogenic development from the subset of MSCs at higher passages provides further evidence supporting the potential application of MSCs in cardiac stem cell therapy for treatment of heart failure and myocardial infarction.
Objectives: This study was to investigate: (1) the biodistribution and myocardial localization of intracoronarily delivered bone marrow-derived mononuclear cells (MNCs) after acute myocardial infarction (AMI) in vivo, and (2) the beneficial effects of intracoronary infusion of autologous MNCs on post-infarction hearts of swine.
Methods: Fourteen Chinese swine were divided into two groups, including group control (n=7) and group 2 (intracoronary delivery of MNCs, n=7), and AMI models were made by occlusion of left anterior descending coronary artery for 90 minutes. Bone marrow-derived MNCs (1×10~9 cells per animal) were radiolabeled with 185 MBq ~(18)F-fluoro-deoxy-glucose (~(18)F-FDG, specific activity, 18.5 MBq/ml=SmCi/ml) and infused into the infarct-related coronary artery (n=6) 1 week after AMI. MNC biodistribution after intracoronary infusion was determined by dual-nuclide single photon emission computed tomography (SPECT), including ~(99m)Tc-sestamibi imaging for localization and size of perfusion defect and ~(18)F-FDG imaging for anatomic localization and distribution of radiolabeled MNCs. The potentials of differentiation of implanted cells in vivo and capillary density in both infarcted and peri-infarct region were detected by immunofluorescent analysis. Cardiac function and area of perfusion defect were detected at baseline (1 week after myocardial infarction) and endpoint (6 weeks after transplantation) by magnetic resonance imaging (MRI) and ~(99m)Tc-sestamibi SPECT.
Results: More than 90% of the total radioactivity was cell bound. Cell viability of radiolabeled MNCs was more than 97%. One hour after cell infusion, all animals underwent dual-nuclide SPECT imaging. After intracoronary transfer, 6.8±1.8% of ~(18)F-FDG-labeled MNCs was detected in the infarcted myocardium; the remaining activity was found primarily in liver and spleen. Dual-nuclide SPECT showed that MNCs predominantly engrafted in the under-perfused region. The retention rate of infused cells in hearts has a significantly positive correlation with under-perfused area (P=0.001, Pearson correlation=0.973). At endpoint, there were severe fibrosis and inflammatory cell infiltration observed in infarcted zone with seldom surviving myocardium in group control, in contrast, there were less fibrosis and inflammatory cell infiltration in group 2 with more surviving myocardium. In group 2, the capillary density was significantly more than that in group control in both infarcted zone (P＜0.003) and peri-infarct zone (P＜0.003). MRI showed that all parameters at baseline were not significantly different between 2 groups (all P＞0.05). At endpoint, regional wall thickening, left ventricular ejection fraction were increased, while left ventricular mass index, dyskinetic segments, end-systolic volume and infarcted size were decreased in group 2 compared with control group (all P＜0.05). SPECT also showed that the area of perfusion defect significantly decreased in group 2 at endpoint compared with control group (P＜0.0001).
Conclusion: The present study demonstrate: (1) ~(18)F-FDG labeling and dual-nuclide SPECT imaging can be used to monitor biodistribution and myocardial homing of MNCs, and (2) intracoronary delivery of MNCs can decrease infarcted area, improve cardiac function and prevent ventricular remodeling after AMI.
Objectives: To study whether drug-facilitation in the perioperative period of mesenchymal stem cells (MSCs) transplantation can improve survival, differentiation and subsequent activities of implanted cells in swine hearts with acute myocardial infarction (AMI).
Methods: Fifty-six Chinese mini-pigs were divided into 8 groups (n=7 in every group), including group 1 (control), group 2 (MSCs transplantation alone), group 3 (Tongxinluo administration, TXL), group 4 (TXL + MSCs), group 5 (simvastatin),group 6 (simvastatin + MSCs), group 7 (atorvastatin), group 8 (atorvastatin + MSCs), and AMI models were made by occlusion of left anterior descending coronary artery for 90 minutes. Then autologous bone marrow MSCs (3×10~7 cells per animal) were injected into infarcted and peri-infarct myocardium immediately after AMI and reperfusion. The survival and differentiation of implanted cells in vivo were detected with immunofluorescent staining. The data of cardiac function were obtained at baseline (1 week after transplantation) and endpoint (6 weeks after transplantation) with single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). The cellular apoptosis in the peri-infarction region was detected with TUNEL assay and oxidative stress level was investigated in the post-infarct myocardium. Inflammatory cytokines within the post-infarcted myocardium, including interleukin-1β(IL-1β), IL-6 and tumor necrosis factor (TNF-α), were detected with Western blotting at the protein level.
Results: Baseline SPECT showed that the area of perfusion defect was not significantly different among all groups (51.8±16.5%, 46.5±9.2%, 47.9±12.2%, 52.7±15.5%, 49.7±16.8%, 53.7±14.9%3 48.9±15.9% vs 50.7±14.5%, all P＞0.05). At endpoint, perfusion defect in group MSCs and TXL was not different compared with group control (47.3±13.2%, 44.8±13.9% vs 47.8±11.1%, both P＞0.05). However, it was significantly decreased in group simvastatin, atorvastatin and three drug-facilitating MSCs groups compared with group control (39.3±12.4%, 38.7±13.1%, 27.6±7.4%, 28.3±7.7%, 29.3±9.0% vs 47.8±11.1%, all P＜0.05).
Baseline MRI indicated that there were not significant differences in cardiac function among all groups (all P＞0.05). At endpoint, every cardiac function parameter in group MSCs and TXL was not significantly different compared with group control (all P＞0.05), however, the number of dyskinetic segments (P=0.039) and infarcted size (P=0.0061) in group simvastatin, and the number of dyskinetic segments (P=0.04) in group atorvastatin, were remarkably decreased compared with group control. Except end-diastolic volume (EDV), other parameters of cardiac function in the three drug-facilitating MSCs transplantation groups were significantly improved compared with group control (all P＜0.05).
H&E and Masson's Trichrome staining showed that there were serious and extensive fibrosis and inflammatory cell infiltration with seldom surviving myocardium within infarcted regions in group control, MSCs and TXL. However, fibrosis and inflammation were slighter in group simvastatin and atorvastatin than that in group control. Furthermore, inflammation and fibrosis were significantly decreased in the three drug-facilitating MSCs transplantation groups. The implanted MSC survivals in the three drug-facilitating MSCs transplantation groups were significantly more that that in group MSCs (223.1±26.9, 310.6±83.8, 373.9±90.3 vs 73.2±21.3/HPF, all P＜0.0001), and were the same as the cardiomyogenic differentiation efficiencies (55.6±12.1%, 46.0±5.1%, 44.4±12.5% vs 8.7±2.4%, all P＜0.0001) and connexin 43 expression (22.4±6.6, 17.0±1.6, 14.2±2.6 vs 4.7±1.8, all P＜0.0001). In addition, the capillary densities within infarcted and peri-infarction regions in group MSCs, TXL, simvastatin and atorvastatin were not significantly different from that in group control (all P＞0.05). However, they were significantly more in the three drug-facilitating MSCs transplantation groups than that in group control (all P＜0.0001).
At endpoint, the apoptotic index in group control was significantly increased compared with that in group sham (10.1±1.8 vs 0.9±0.3, P＜0.0001). There was not significant difference in the apoptotic index between group control and MSCs (9.2±1.4, P＞0.05), however, it was remarkably decreased in group TXL (5.6±1.7), simvastatin (5.7±1.5) and atorvastatin (5.2±1.9) compared with group control (all P＜0.0001). Based on the drug application, it was less in the three drug-facilitating MSCs transplantation groups than that in group control (2.4±0.5, 2.3±0.3, 1.9±0.3, P＜0.0001). In addition, Western blotting indicated that Bax increased and Bcl-2 decreased significantly in group control compared with group sham (both P＜0.0001). However, Bax was decreased and Bcl-2 increased in group MSCs, three drug application and drug-facilitating MSCs transplantation groups (all P＜0.0001).
In addition, SOD activity in group control was significantly decreased compared with group sham (82.5±8.2 vs 122.5±12.2 U/mg protein, P＜0.0001) and MDA contents decreased (9.0±0.8 vs 5.5±0.9nmol/mg protein, P＜0.0001). They were not remarkably different between group control and MSCs (P＞0.05). However, the three drug application and three drug-facilitating MSCs transplantation significantly increased SOD activity (all P＜0.0001) and decreased MDA contents (all P＜0.0001).
Furthermore, the expressions of IL-1β, IL-6 and TNF-αin group control were significantly higher than that in group sham (all P＜0.0001), however, they were remarkably decreased by the three drug application and three drug-facilitating MSCs transplantation (all P＜0.0001).
Conclusion: Our study demonstrated (1) immediately intramyocardial injection of MSCs after AMI and reperfusion resulted in limited survival and differentiation potential of implanted cells in vivo, without significant benefits in cardiac function; (2) TXL-, simvastatin- and atorvastatin-facilitation resulted in significantly powerful survival and differentiation potential of implanted cells in vivo via inhibition of apoptosis, oxidative stress and inflammatory responses, accompanying by significant benefits in cardiac function.
方法：抽取中华小型猪骨髓，密度梯度离心法[Percoll(1.077g／ml)]分离出单个核细胞，贴壁培养法筛选MSCs，原代(PO)培养后按1：3比例每3天传代一次。选取第1，4，8和10代(P1，P4，P8和P10)MSCs，研究其向心肌样细胞白发分化潜能，以及在5-氮胞苷诱导后4周能否分化为心肌细胞。连续镜下观察其形态变化；计数法分析MSCs增殖模式；免疫细胞化学染色法检测MSCs的表面标志(CD34、CD45、CD90和SH2)及心肌细胞特异性的蛋白表达，包括a-横纹肌肌动蛋白(a-sarcomeric actin，a-actin)、心肌肌钙蛋白-T(cardiactroponin-T，cTn-T)、肌球蛋白重链(myosin heavy chain，MHC)和连接蛋白43(connexin 43，Cx43)；以Real Time RT-PCR定量检测分化细胞是否表达心肌特异性基因及其表达量，包括GATA4、肌球蛋白轻链(myosin light chain，MLC)、MHC、肌浆网受磷蛋白(phospholamban)、a-actin、Cx43、结蛋白(desmin)和骨骼肌肌动蛋白(skeletal muscle actin，SM-actin)；透射电镜观察分化细胞超微结构。
结果：体外培养条件下，P1、P4、P8和大部分P10代MSCs形态一致，呈梭形成纤维细胞样形态，但部分P10代细胞多核、胞浆延长、和邻近细胞相互连接，转变为肌管样形态。5-氮胞苷诱导1周后，部分P1、P4和P8代MSCs表现为较长的胞浆形态和多核化，4周后更多细胞相互连接形成不规则的管样结构，而P10代MSCs则形成相对规则的肌管样结构。
计数法显示，各代MSCs培养3天后开始进入对数生长期直至平台期。P1、P4和P8代MSCs增殖速率相似(P=0.908)，倍增时间(population doubling time，PD)约48h。和上述早代MSCs相比，接种3天后，P10代MSCs增殖速率显著变慢(P＜0.05)，PD约为72h。
免疫细胞化学染色显示超过95％的贴壁细胞表达CD90、SH2，而不表达CD34、CD45，说明绝大多数贴壁细胞为MSCs。自然培养的P1、P4和P8代MSCs不表达心肌特异性结构蛋白，而部分P10代MSCs(10.3±1.8％)表达心肌特异蛋白：α-actin，MHC和cTn-T。各代MSCs经5-氮胞苷诱导后4周，均表达上述蛋白；其中，P1、P4和P8代MSCs向心肌细胞分化效率相似(18.3±8.2％vs 17.6±11.9％vs 16.5±5.0％，P＞0.05)，但均显著低于P10代MSCs的诱导成心肌细胞分化效率(44.5±4.2％，P=0.0001)。
RT-PCR结果表明，P1、P4和P8代MSCs在自然培养状态下无心肌特异性基因表达，而P10代MSCs则表达心肌特异性转录因子GATA4和结构基因desmin、phospholabman、MHC和MLC。5-氮胞苷诱导后4周，各代MSCs均表达上述基因。实时定量PCR结果表明，GATA4(P=0.018)和MLC(P＜0.0001)在P10代MSCs诱导后4周的表达丰度显著高于同期诱导的P4代诱导分化细胞。
透射电镜显示在自然培养状态下，P1、P4和P8代MSCs无任何肌细胞样结构，而部分P10代MSCs有不规则肌丝；5-氮胞苷诱导后4周，P1、P4和P8代MSCs分化的细胞有较多不规则肌丝、散乱肌节和横纹，而P10代MSCs分化的细胞可见丰富肌丝、规则肌节和横纹，尤其是闰盘结构。
结论：1．猪骨髓MSCs在体外自然培养条件下，形态学、增殖能力和分化潜能是不均一的，从P10代开始增殖变慢、部分细胞自发分化为心肌样细胞。2．5-氮胞苷可诱导MSCs分化为心肌样细胞，但是P10代MSCs分化的心肌细胞更趋成熟，具有相对成熟的心肌细胞结构，且分化效率更高。
目的：研究~(18)氟—脱氧葡萄糖(~(18)fluoro deoxy-glucose，~(18)F~-FDG)标记的骨髓单个核细胞(mononuclear cells，MNCs)经冠状动脉输注后在急性心肌梗死(acutemyocardial infarction，AMI)猪模型的早期全身分布、心肌内定位和对心功能的影响。
方法：选取10月龄中华小型猪14头(30±5kg)，分为对照组(经冠脉输注PBS，n=7)和移植组(经冠脉输注自体骨髓MNCs，n=7)。开胸后于紧靠第一对角支开口稍远处，胶管套扎左前降支(left anterior descending，LAD)冠状动脉90分钟制备AMI模型。AMI后1周，抽取自体骨髓分离出MNCs，以放射性同位素~(18)F-FDG(5mCi=185MBq)和荧光染料DAPI标记，调密度至1×10~9细胞／10ml。以over-the-wire balloon球囊导管在LAD结扎处阻断前向血流，同时输注标记的MNCs(1×10~9细胞／猪，n=6)，1小时后以双核素(~(18)氟／~(99)锝)—单光子发射计算机断层扫描(single photon emission computed tomography，SPECT)检测其全身分布和心脏内定位。分别以AMI后1周(移植当天)和移植后6周作为考察基线和终点，以~(99m)锝-甲氧基异丁基异腈(~(99m)Tc-MIBI)-SPECT检测心肌灌注缺损，以核磁共振成像(magnetic resonance imaging，MRI)检测心功能。终点指标检测结束后处死动物进行组织学检测，免疫组化法检测MNCs在梗死区和梗死周边区分化潜能以及毛细血管密度。
结果：AMI后24h内对照组和移植组分别有一只动物死于心力衰竭和心室颤动。~(18)F~-FDG标记后即刻，MNCs活力达97％以上，标记后1小时仍在93％以上，与非标记MNCs相似。放射活性检测表明，90.8±1.9％的放射性活性存留于MNCs中，上清液中放射性活性小于10％。DAPI对活细胞的标记效率为100％。
MNCs经冠脉输注后1小时，~(18)F-FDG-SPECT显示，心脏中的放射活性占全身的6.8±1.8％，90％以上的放射活性位于肝、脾和外周循环。双核素SPECT扫描相同层面的心肌并进行图像重叠，显示在所有移植动物心脏中，~(18)F-FDG放射活性全部聚集于灌注缺损区，相关性分析表明，心脏中放射活性强度与灌注缺损面积呈显著正相关(P=0.001，相关系数=0.973)。
基线时对照组和移植组心肌灌注缺损面积无显著差异(47.1±7.5％vs45.6±6.6％，P=0.713)，终点时，移植组灌注缺损面积显著小于对照组(23.9±5.1％vs 44.8±6.7％，P＜0.0001)。
MRI检测表明，基线时，对照组和移植组的各项心功能参数无显著差异(P＞0.05)。终点时，与对照组相比，移植组的心功能各项参数除左室舒张末期容积(end-diastolic volume，EDV)外均有显著改善，其中左室射血分数(leftventricular ejection fraction，LVEF)由43.9±8.5％增加到51.2±10.4％(差值d=7.3±4.8％)，而对照组由43.5±7.3％变为44.2±8.2％(d=0.7±2.0％，P=0.011)；左室收缩末期容积(end-systolic volume，ESV)由32.0±10.1ml变为31.5±10.3ml(d=-0.5±3.3 ml)，而对照组则从34.7±6.4 ml增加到38.5±7.3ml(d=3.8±3.1ml，P=0.039)；运动障碍节段数由8.2±1.2个减少至4.8±0.8个(d=-3.3±0.5)，对照组由7.8±1.0个减少为7.2±0.8个(d=-0.7±0.5，P＜0.0001)；室壁增厚率从-18.2±7.3％增加至45.7±11.1％(d=63.8±4.1％)，对照组从-17.5±6.3％减至-22.3±5.7％(d=-4.8±3.9％，P＜0.0001)；梗死面积从6.9±1.1 cm~2减小为3.7±0.7 cm~2(d=-3.1±0.8cm~2)，而对照组从6.6±0.8cm~2变为6.9±0.4cm~2(d=-0.3+0.5 cm~2，P＜0.0001)；左室质量指数从59.8±8.9g／m~2增加到64.2±7.9g／m~2(d=-4.3±1.5g／m~2)，而对照组从61.7±7.2g／m~2变为72.7±4.6g／m~2(d=-11.0±4.4 g／m~2，P=0.007)。移植组EDV由56.2±8.4ml变为63.3±7.8ml(d=7.2±4.1ml)，而对照组则从58.8±6.6ml增加到68.7±5.3ml(d=9.8±2.6ml，P=0.211)。
H&E染色和Masson’s三色染色结果显示，对照组心肌梗死区有严重纤维化和炎症细胞浸润，存活心肌较少，而移植组纤维化和炎症细胞浸润较轻，且伴有存活心肌。免疫荧光显示DAPI标记的细胞表达心肌和血管特异性的结构蛋白，包括α-横纹肌肌动蛋白、心肌肌钙蛋白T、连接蛋白43、Von Willebrand因子和血管平滑肌肌动蛋白。与对照组比较，移植组梗死区(1.8±0.5 vs 3.7±1.1／HPF，P＜0.003)和梗死周边区(8.7±2.0 vs 4.9±1.3／HPF，P＜0.003)毛细血管密度均显著增加。
结论：本研究表明：1．~(18)F-FDG标记和双核素SPECT显像可以用于监测骨髓MNCs经冠脉输注后全身分布和心肌内定位。2．经冠脉输注骨髓MNCs可显著减小灌注缺损面积、改善心功能和阻止心室重塑。
目的：探索性研究以通心络超微粉和他汀类药物改良急性心肌梗死(acutemyocardial infarction，AMI)猪模型的心肌微环境，对移植骨髓间充质干细胞(mesenchymal stem cells，MSCs)在体存活、分化和生物学活性的影响。
方法：56只中华小型猪(30±5kg)分为8组，每组7只：对照组、MSCs移植组、单纯通心络超微粉、辛伐他汀和阿托伐他汀干预组、三种药物改良+MSCs移植组。开胸结扎左前降支冠状动脉90min制备AMI模型，再灌注后随即于梗死区和梗死周边区心肌内注射自体骨髓MSCs(3×10~7细胞／500μl)，对照组以相同方法注射500μl低糖DMEM培养基。在6个药物干预组，分别于AMI前3天开始给予口服小剂量通心络超微粉(0.05g／d．kg)、辛伐他汀(0.25mg／d．kg)和阿托伐他汀(0.25mg／d．kg)，AMI当天和其后继续使用4天。
在移植后1周(基线)和6周(终点)，以单光子发射计算机断层显象(singlephoton emission computed tomography，SPECT)和核磁共振成像(magneticresonance imaging，MRI)检测心肌灌注缺损和心功能。免疫荧光检测移植细胞在体存活和分化；TUNEL法检测梗死周边细胞凋亡；分别以黄嘌呤氧化酶法和硫代巴比妥酸法测定梗死区超氧化物歧化酶(superoxide dismutase，SOD)活性和丙二醛(malondialdehyde，MDA)含量；以Western blot免疫印迹法检测梗死区白细胞介素-1β(interleukin--1β，IL-1β)、IL-6和肿瘤坏死因子-α(tumor necrosisfactor，TNF-α)的表达水平，以及梗死周边区促凋亡蛋白Bax和抑凋亡蛋白Bcl-2表达水平。
结果：SPECT扫描显示，基线时各组心肌灌注缺损面积无显著差异(51.8±16.5％，46.5±9.2％，47.9±12.2％，52.7±15.5％，49.7±16.8％，53.7±14.9％，48.9±15.9％vs 50.7±14.5％，P＞0.05)。终点时，MSCs组、通心络超微粉组和对照组无显著差异(47.3±13.2％，44.8±13.9％vs 47.8±11.1％，P＞0.05)，而辛伐他汀组和阿托伐他汀组灌注缺损面积均小于对照组(39.3±12.4％，38.7±13.1％vs47.8±11.1％，P＜0.05)；尤其是通心络超微粉+MSCs组、辛伐他汀+MSCs组、阿托伐他汀+MSCs组的灌注缺损面积进一步显著减小(27.6±7.4％，28.3±7.7％，29.3±9.0％vs 47.8±11.1％，P＜0.05)。
MRI检测显示，各组心功能参数在基线时无显著差异(P＞0.05)。终点时，与对照组相比，MSCs组和通心络超微粉组的各项心功能参数均无显著差异(P＞0.05)，而辛伐他汀组运动障碍节段数(P=0.039)和梗死面积(P=0.0061)、阿托伐他汀组的运动障碍节段数(P=0.04)均小于对照组。和对照组相比，在通心络超微粉、辛伐他汀和阿托伐他汀改良的MSCs移植组，除左室舒张末期容积(end-diastolic volume，EDV)外，各项心功能参数均有显著改善(P＜0.05)。
H&E染色和Masson’s Trichrome染色显示，在对照组，梗死区呈连续性大片状纤维化，有较多炎性细胞浸润，未见存活心肌细胞。MSCs组和通心络超微粉组心肌梗死区和对照组相似，而辛伐他汀组和阿托伐他汀组梗死区纤维化和炎性细胞浸润稍轻，有少量存活心肌。在通心络超微粉、辛伐他汀和阿托伐他汀改良的MSCs移植组，炎症细胞浸润显著减轻，存活心肌较多，纤维化呈散在分布。
在通心络超微粉、辛伐他汀和阿托伐他汀改良的MSCs移植组，移植细胞在体存活显著多于单纯MSCs移植组(223.1±26.9，310.6±83.8，373.9±90.3 vs73.2±21.3／HPF，P＜0.0001)。在各MSCs移植组，均有部分移植细胞表达心肌细胞特异性蛋白和血管内皮细胞特异性的因子Ⅷ；但三种药物改良的MSCs移植组，移植细胞向心肌细胞分化效率均显著高于单纯MSCs移植组(55.6±12.1％，46.0±5.1％，44.4±12.5％vs 8.7±2.4％，P＜0.0001)。各移植组均有部分MSCs表达心肌细胞特异性的连接蛋白Connexin43，但上述三种药物改良的移植组Connexin43表达显著强于单纯MSCs移植组(22.4±6.6，17.0±1.6，14.2±2.6 vs4.7±1.8，P＜0.0001)。
与对照组相比，MSCs组、通心络超微粉组、辛伐他汀组和阿托伐他汀组梗死区(1.8±0.8，1.9±0.5，2.2±0.7，1.9±0.6 vs 1.8±0.5／HPF，P＞0.05)和梗死周边毛细血管密度(5.2±1.4，5.4±0.8，5.4±1.2，5.4±1.0 vs 4.9±1.3／HPF，P＞0.05)均无显著性差异；而三种药物改良的MSCs移植组梗死区(4.1±0.7，3.9±1.0，3.9±0.9vs 1.8±0.5／HPF，P＜0.0001)和梗死周边毛细血管密度(8.9±1.7，8.5±1.6，8.6±1.2vs 4.9±1.3／HPF，P＜0.0001)均显著高于对照组。
终点时，对照组凋亡指数显著高于Sham组(10.1±1.8 vs 0.9±0.3，P＜0.0001)，MSCs组(9.2±1.4，P＞0.05)和对照组无显著差异，而通心络超微粉组(5.6±1.7)、辛伐他汀组(5.7±1.5)和阿托伐他汀组(5.2±1.9)凋亡指数均显著小于对照组(P＜0.0001)，和单纯药物干预组相比，各药物改良的MSCs移植组凋亡指数进一步减小(2.4±0.5，2.3±0.3，1.9±0.3，P＜0.0001)。Western blot检测表明，对照组促凋亡蛋白Bax表达显著增加(P＜0.0001)，抑凋亡蛋白表达显著降低(P＜0.0001)；和对照组相比，MSCs组、三种药物组和药物改良的MSCs移植组均使Bax表达降低(P＜0.0001)和Bcl-2增加(P＜0.0001)；其中，各药物改良的MSCs移植组在药物组的基础上使Bax表达进一步降低(P＜0.05)。
终点时，对照组梗死区SOD活力较Sham组显著降低(82.5±8.2 vs 122.5±12.2U／mg蛋白，P＜0.0001)，MSCs组(88.6±8.3 U／mg蛋白)和对照组相比无显著性差异(P＞0.05)；但通心络超微粉组(95.5±9.6U／mg蛋白)、辛伐他汀组(100.8±12.1U／mg蛋白)、阿托伐他汀组(98.8±13.4U／mg蛋白)、通心络超微粉+MSCs(101.7±9.1U／mg蛋白)、辛伐他汀+MSCs(108.5±10.2U／mg蛋白)和阿托伐他汀+MSCs组(109.0±13.8U／mg蛋白)均显著高于对照组(P＜0.0001)。对照组梗死区MDA含量较Sham组显著升高(9.0±0.8 vs 5.5±0.9nmol／mg蛋白，P＜0.0001)，MSCs组(8.5±0.6nmol／mg蛋白)和对照组相比无显著差异(P＞0.05)；而通心络超微粉组(5.9±0.7nmol／mg蛋白)、辛伐他汀组(5.8±0.7nmol／mg蛋白)、阿托伐他汀组(5.7±1.3nmol／mg蛋白)、通心络超微粉+MSCs(5.7±0.7nmol／mg蛋白)、辛伐他汀+MSCs(5.6±0.9nmol／mg蛋白)和阿托伐他汀+MSCs组(5.5±1.1nmol／mg蛋白)均显著低于对照组(P＜0.0001)。
Western blot检测显示，对照组梗死区IL-1β，IL-6和TNF-α表达较Sham组显著升高(P＜0.0001)，和对照组相比，MSCs组、三种药物干预组和药物改良的MSCs移植组梗死区上述因子表达均显著降低(P＜0.0001)。
结论：本实验表明：1．在AMI再灌注后即刻心肌内移植自体骨髓MSCs，其存活和分化能力有限，尽管能抑制促凋亡蛋白和部分炎症因子的表达，但并未产生显著的心功能获益；2．围移植期使用通心络超微粉、辛伐他汀和阿托伐他汀可显著改良梗死心肌局部微环境，增强移植的骨髓MSCs在体存活和分化，产生显著的心功能获益。
Objectives: Mesenchymal stem cells (MSCs) are a group of heterogenous stem cells with multipotency in growth and differentiation. This study was to explore the proliferative potential and cardiomyogenic development of porcine bone marrow-derived MSCs stimulated with or without 5-azacytidine.
Methods: In this study, the bone marrow was aspirated from Chinese mini-swine. The mononuclear cells were isolated by density gradient centrifugation through 1.077g/ml Percoll and then cultured in low-glucose DMEM containing 10% fetal calf serum. After primary culture, relatively purified MSCs were obtained by using the method of adherence screening. The cultured MSCs in vitro were passaged every 3 days after primary culture, and MSCs at passage 1, 4, 8 and 10 (P1, P4, P8 and P 10) were examined for their proliferation and differentiation stimulated with or without 5-azacytidine (10μM) in vitro by cell counting, immunocytochemistry, transmission electrical microscopy and RT-PCR/real time qPCR.
Results: In normal growth medium, approximately 10.34±1.8% of porcine bone marrow-derived MSCs at passage 10 in vitro spontaneously take on cardiomyocyte-like phenotype and structure, and express cardimyocyte-specific genes and proteins, however, earlier passages of MSCs don't. Four weeks after induction by 5-azacytidine, MSCs at passage 1, 4 and 8 all express cardiomyocyte-specific genes and proteins and have cardiomyocyte-like ultrastructure such as irregular myofilaments in vitro. However only MSCs at passage 10 express connexin 43 at the level of gene and protein, and take on relatively mature cardiomyocyte ultrastructure, including myofilaments, sarcomeres and transverse striations, especially intercalated discs in vitro. The MSC-derived myogenic cells showed cardiac myogenic but not skeletal myogenic markers at both mRNA and protein levels. In addition, the cardiomyogenic differentiation efficiency of MSCs at passage 10 are significantly higher than that of earlier passages (44.5±4.2% vs. 18.3±8.2%, 17.6±11.9%,16.5±5.0%, respectively, all P＜0.0001).
Conclusion: The potency of growth and differentiation of porcine bone marrow-derived MSCs spontaneously or in response to stimulation of 5-azacytidine is dependent upon the endogenous gene expression and passage condition of MSCs. The myogenic development from the subset of MSCs at higher passages provides further evidence supporting the potential application of MSCs in cardiac stem cell therapy for treatment of heart failure and myocardial infarction.
Objectives: This study was to investigate: (1) the biodistribution and myocardial localization of intracoronarily delivered bone marrow-derived mononuclear cells (MNCs) after acute myocardial infarction (AMI) in vivo, and (2) the beneficial effects of intracoronary infusion of autologous MNCs on post-infarction hearts of swine.
Methods: Fourteen Chinese swine were divided into two groups, including group control (n=7) and group 2 (intracoronary delivery of MNCs, n=7), and AMI models were made by occlusion of left anterior descending coronary artery for 90 minutes. Bone marrow-derived MNCs (1×10~9 cells per animal) were radiolabeled with 185 MBq ~(18)F-fluoro-deoxy-glucose (~(18)F-FDG, specific activity, 18.5 MBq/ml=SmCi/ml) and infused into the infarct-related coronary artery (n=6) 1 week after AMI. MNC biodistribution after intracoronary infusion was determined by dual-nuclide single photon emission computed tomography (SPECT), including ~(99m)Tc-sestamibi imaging for localization and size of perfusion defect and ~(18)F-FDG imaging for anatomic localization and distribution of radiolabeled MNCs. The potentials of differentiation of implanted cells in vivo and capillary density in both infarcted and peri-infarct region were detected by immunofluorescent analysis. Cardiac function and area of perfusion defect were detected at baseline (1 week after myocardial infarction) and endpoint (6 weeks after transplantation) by magnetic resonance imaging (MRI) and ~(99m)Tc-sestamibi SPECT.
Results: More than 90% of the total radioactivity was cell bound. Cell viability of radiolabeled MNCs was more than 97%. One hour after cell infusion, all animals underwent dual-nuclide SPECT imaging. After intracoronary transfer, 6.8±1.8% of ~(18)F-FDG-labeled MNCs was detected in the infarcted myocardium; the remaining activity was found primarily in liver and spleen. Dual-nuclide SPECT showed that MNCs predominantly engrafted in the under-perfused region. The retention rate of infused cells in hearts has a significantly positive correlation with under-perfused area (P=0.001, Pearson correlation=0.973). At endpoint, there were severe fibrosis and inflammatory cell infiltration observed in infarcted zone with seldom surviving myocardium in group control, in contrast, there were less fibrosis and inflammatory cell infiltration in group 2 with more surviving myocardium. In group 2, the capillary density was significantly more than that in group control in both infarcted zone (P＜0.003) and peri-infarct zone (P＜0.003). MRI showed that all parameters at baseline were not significantly different between 2 groups (all P＞0.05). At endpoint, regional wall thickening, left ventricular ejection fraction were increased, while left ventricular mass index, dyskinetic segments, end-systolic volume and infarcted size were decreased in group 2 compared with control group (all P＜0.05). SPECT also showed that the area of perfusion defect significantly decreased in group 2 at endpoint compared with control group (P＜0.0001).
Conclusion: The present study demonstrate: (1) ~(18)F-FDG labeling and dual-nuclide SPECT imaging can be used to monitor biodistribution and myocardial homing of MNCs, and (2) intracoronary delivery of MNCs can decrease infarcted area, improve cardiac function and prevent ventricular remodeling after AMI.
Objectives: To study whether drug-facilitation in the perioperative period of mesenchymal stem cells (MSCs) transplantation can improve survival, differentiation and subsequent activities of implanted cells in swine hearts with acute myocardial infarction (AMI).
Methods: Fifty-six Chinese mini-pigs were divided into 8 groups (n=7 in every group), including group 1 (control), group 2 (MSCs transplantation alone), group 3 (Tongxinluo administration, TXL), group 4 (TXL + MSCs), group 5 (simvastatin),group 6 (simvastatin + MSCs), group 7 (atorvastatin), group 8 (atorvastatin + MSCs), and AMI models were made by occlusion of left anterior descending coronary artery for 90 minutes. Then autologous bone marrow MSCs (3×10~7 cells per animal) were injected into infarcted and peri-infarct myocardium immediately after AMI and reperfusion. The survival and differentiation of implanted cells in vivo were detected with immunofluorescent staining. The data of cardiac function were obtained at baseline (1 week after transplantation) and endpoint (6 weeks after transplantation) with single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). The cellular apoptosis in the peri-infarction region was detected with TUNEL assay and oxidative stress level was investigated in the post-infarct myocardium. Inflammatory cytokines within the post-infarcted myocardium, including interleukin-1β(IL-1β), IL-6 and tumor necrosis factor (TNF-α), were detected with Western blotting at the protein level.
Results: Baseline SPECT showed that the area of perfusion defect was not significantly different among all groups (51.8±16.5%, 46.5±9.2%, 47.9±12.2%, 52.7±15.5%, 49.7±16.8%, 53.7±14.9%3 48.9±15.9% vs 50.7±14.5%, all P＞0.05). At endpoint, perfusion defect in group MSCs and TXL was not different compared with group control (47.3±13.2%, 44.8±13.9% vs 47.8±11.1%, both P＞0.05). However, it was significantly decreased in group simvastatin, atorvastatin and three drug-facilitating MSCs groups compared with group control (39.3±12.4%, 38.7±13.1%, 27.6±7.4%, 28.3±7.7%, 29.3±9.0% vs 47.8±11.1%, all P＜0.05).
Baseline MRI indicated that there were not significant differences in cardiac function among all groups (all P＞0.05). At endpoint, every cardiac function parameter in group MSCs and TXL was not significantly different compared with group control (all P＞0.05), however, the number of dyskinetic segments (P=0.039) and infarcted size (P=0.0061) in group simvastatin, and the number of dyskinetic segments (P=0.04) in group atorvastatin, were remarkably decreased compared with group control. Except end-diastolic volume (EDV), other parameters of cardiac function in the three drug-facilitating MSCs transplantation groups were significantly improved compared with group control (all P＜0.05).
H&E and Masson's Trichrome staining showed that there were serious and extensive fibrosis and inflammatory cell infiltration with seldom surviving myocardium within infarcted regions in group control, MSCs and TXL. However, fibrosis and inflammation were slighter in group simvastatin and atorvastatin than that in group control. Furthermore, inflammation and fibrosis were significantly decreased in the three drug-facilitating MSCs transplantation groups. The implanted MSC survivals in the three drug-facilitating MSCs transplantation groups were significantly more that that in group MSCs (223.1±26.9, 310.6±83.8, 373.9±90.3 vs 73.2±21.3/HPF, all P＜0.0001), and were the same as the cardiomyogenic differentiation efficiencies (55.6±12.1%, 46.0±5.1%, 44.4±12.5% vs 8.7±2.4%, all P＜0.0001) and connexin 43 expression (22.4±6.6, 17.0±1.6, 14.2±2.6 vs 4.7±1.8, all P＜0.0001). In addition, the capillary densities within infarcted and peri-infarction regions in group MSCs, TXL, simvastatin and atorvastatin were not significantly different from that in group control (all P＞0.05). However, they were significantly more in the three drug-facilitating MSCs transplantation groups than that in group control (all P＜0.0001).
At endpoint, the apoptotic index in group control was significantly increased compared with that in group sham (10.1±1.8 vs 0.9±0.3, P＜0.0001). There was not significant difference in the apoptotic index between group control and MSCs (9.2±1.4, P＞0.05), however, it was remarkably decreased in group TXL (5.6±1.7), simvastatin (5.7±1.5) and atorvastatin (5.2±1.9) compared with group control (all P＜0.0001). Based on the drug application, it was less in the three drug-facilitating MSCs transplantation groups than that in group control (2.4±0.5, 2.3±0.3, 1.9±0.3, P＜0.0001). In addition, Western blotting indicated that Bax increased and Bcl-2 decreased significantly in group control compared with group sham (both P＜0.0001). However, Bax was decreased and Bcl-2 increased in group MSCs, three drug application and drug-facilitating MSCs transplantation groups (all P＜0.0001).
In addition, SOD activity in group control was significantly decreased compared with group sham (82.5±8.2 vs 122.5±12.2 U/mg protein, P＜0.0001) and MDA contents decreased (9.0±0.8 vs 5.5±0.9nmol/mg protein, P＜0.0001). They were not remarkably different between group control and MSCs (P＞0.05). However, the three drug application and three drug-facilitating MSCs transplantation significantly increased SOD activity (all P＜0.0001) and decreased MDA contents (all P＜0.0001).
Furthermore, the expressions of IL-1β, IL-6 and TNF-αin group control were significantly higher than that in group sham (all P＜0.0001), however, they were remarkably decreased by the three drug application and three drug-facilitating MSCs transplantation (all P＜0.0001).
Conclusion: Our study demonstrated (1) immediately intramyocardial injection of MSCs after AMI and reperfusion resulted in limited survival and differentiation potential of implanted cells in vivo, without significant benefits in cardiac function; (2) TXL-, simvastatin- and atorvastatin-facilitation resulted in significantly powerful survival and differentiation potential of implanted cells in vivo via inhibition of apoptosis, oxidative stress and inflammatory responses, accompanying by significant benefits in cardiac function.
引文
1. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction: experimental observations and clinical implications. Circulation. 1990; 81(4): 1161-1172.
2. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001; 344(23): 1750-1757.
3. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002; 418(6893): 41-49.
4. Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet. 2004; 363(9419): 1439-1441.
5. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284(5411): 143-147.
6. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature. 2001; 410(6829): 701-705.
7. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest. 2002; 109(3): 337-346.
8. Harris RG, Herzog EL, Bruscia EM, et al. Lack of a fusion requirement for development of bone marrow-derived epithelia. Science. 2004; 305(5680): 90-93.
9. Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation. 1999; 100(19 suppl): II 247-256.
10. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature. 2003; 425(6961): 968-973.
11. Liu Y, Song J, Liu W, et al. Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res. 2003; 58(2): 460-468.
12. Kadivar M, Khatami S, Mortazavi Y, et al. In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells. Biochem Biophys Res Commun. 2006; 340(2): 639-647.
13. Xu W, Zhang X, Qian H, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. Exp Biol Med. 2004; 229(7): 623-631.
14. Xu M, Wani M, Dai YS, et al. Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes. Circulation. 2004; 110(17): 2658-2665.
15. Planat-Benard V, Menard C, Andre M, et al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res. 2004; 94(2): 223-229.
16. Grinnemo KH, Mansson-Broberg A, Leblanc K, Human mesenchymal stem cells do not differentiate into cardiomyocytes in a cardiac ischemic xenomodel. Ann Med. 2006; 38(2):144-153.
17. Sartore S, Lenzi M, Angelini A, et al. Amniotic mesenchymal stem cells autotransplanted in a porcine model of cardiac ischemic do not differentiate to cardiogenic phenotype. Eur J Cardiothorac Surg. 2005; 28(5):677-684.
18. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997; 276(5309): 71-74.
19. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol. 1999; 181(1): 67-73.
20. Moscoso I, Centeno A, Lopez E, et al. Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation. Transplant Proceed. 2005; 37(1): 481-482.
21. Colter D, Class R, Di Girolamo CM, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A. 2000 97(7): 3213-3218.
22. Colter DC, Sekiya I, Prockop DJ, et al. Identification of a subpopulation of rapidly self renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A. 2001; 98(14): 7841-7845.
23. Zhang FB, Li L, Fang B, et al. Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun. 2005; 336(3): 784-792.
24. Zhang X, Soda Y, Takahashi K, et al. Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells. Biochem Biophys Res Commun. 2006; 351(4):853-859.
25. Pittenger, MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics.Circ Res. 2004; 95(1): 9-20.
26. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci. 2000;113(Pt 7): 1161-1166.
27. Dennis JE, Merriam A, Awadallah A, et al. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res. 1999; 14(5): 700-709.
28. Rao SG, Dravid G. Expansion of haematopoietic stem cells in vitro: A challenge to stem cell biologists. Indian J Exp Biol. 1999; 37(11): 1051-1052.
29. Shahdadfar A, Fronsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells. 2005; 23(9): 1357-1366.
30. Bruder SP, Jaiswal N, Haynesworth SE, et al. Growth kinetics, selfrenewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997; 64(2): 278-294.
31. Garcia-Verdugo JM, Penicaud L, Casteilla L et al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res. 2004; 94(2): 223-229.
32. Naruse K, Urabe K, Mukaida T, et al. Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation. Bone. 2004; 35(4): 850-858.
33. Yamada Y, Sakurada K, Takeda Y, et al. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells. Exp Cell Res. 2007; 313(4): 698-706.
34. Heng BC, Haider HKh, Sim EK, et al. Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. Cardiovasc Res. 2004; 62(1):34-42.
35. Fukuda K. Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes. C R Biol. 2002; 325(10): 1027-1038.
36. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest. 1999; 103(5): 697-705.
37. Molkentin JD. The zinc finger-containing transcription factors GATA-4,-5and -6, ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem. 2000; 275(50): 38949-38952.
38. Konieczny SF, Emerson CP. 5-azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell. 1984; 38(3): 791-800.
39. Taylor SM. 5-aza-2-deoxycytidine: cell differentiation and DNA methylation. Leukemia. 1993; 7(suppl 1): 3-8.
1. Fernandez-Aviles F, San Roman JA, Garcia-Frade J, et al. Experimental and clinical regenerative capability of human bone marrow cells after myocardial infarction. Circ Res. 2004; 95(7): 742-748.
2. Schuster MD, Kocher AA, Seki T, et al. Myocardial neovascularization by bone marrow angioblasts results in cardiomyocyte regeneration. Am J Physiol Heart Circ Physiol. 2004; 287(2): H 525-532.
3. Kocher A A, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 2001; 7(4): 430-436.
4. Tse HF, Kwong YL, Chan JK, et al. Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet. 2003; 361(9351): 47-49.
5. Perin EC, Dohmann HF, Borojevic R, et al. Improved exercise capacity and ischemia 6 and 12 months after transendocardial injection of autologous bone marrow mononuclear cells for ischemic cardiomyopathy. Circulation. 2004;110 (11 Suppl 1): 213-218.
6. Strauer BE, Brehm M, Zeus T, Kostering M, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 2002; 106(15): 1913-1918.
7. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation. 2003; 108(18): 2212-2218.
8. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomized controlled clinical trial. Lancet. 2004; 364(9429): 141-148.
9. Hofmann M, Wollert KC, Meyer GP, et al. Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation. 2005; 111(17): 2198-2202.
10. Hou D, Youssef EA, Brinton TJ, et al. Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery implications for current clinical trials. Circulation. 2005; 112 (suppl I): 1-150-156.
11. Kang WJ, Kang HJ, Kim HS, et al. Tissue distribution of 18F-FDG-labeled peripheral hematopoietic stem cells after intracoronary administration in patients with myocardial infarction. J Nucl Med. 2006; 47(8): 1295-1301.
12. Jonker JW, Freeman J, Bolscher E, Contribution of the ABC transporters Bcrp1 and Mdr1a/1b to the side population phenotype in mammary gland and bone marrow of mice. Stem Cells. 2005; 23(8): 1059-1065.
13. Macpherson H, Keir P, Webb S, et al. Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo. J Cell Sci. 2005; 118(Pt 11): 2441-2450.
14. Ojima K, Uezumi A, Miyoshi H, et al. Mac-1 (low) early myeloid cells in the bone marrow-derived SP fraction migrate into injured skeletal muscle and participate in muscle regeneration. Biochem Biophys Res Commun. 2004; 321(4): 1050-1061.
15. Asahara T, Murohara T, Sullivan A , et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275(5302): 964-967.
16. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995; 18(12): 1417-1426.
17. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast clonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970; 3(4): 393-403.
18. Quesenberry PJ, Dooner G, Dooner M, et al. The stem cell continuum: considerations on the heterogeneity and plasticity of marrow stem cells. Stem Cell Rev. 2005; 1(1): 29-36.
19. Allen TD, Dexter TM, Simmons PJ. Marrow biology and stem cells. Immunol Ser. 1990; 49(1): 1-38.
20. Wilson FD. Studies of the hematopoietic microenvironment. Hematopoietic stroma and microenvironment. Bibl Haematol. 1984; (48):210-292.
21. Allers C, Sierralta WD, Neubauer S, et al. Dynamic of distribution of human bone marrow- derived mesenchymal stem cells after transplantation into adult unconditioned mice. Transplantation. 2004; 78(4): 503-508.
22. Suzuki K, Murtuza B, Fukushima S, et al. Targeted cell delivery into infarcted rat hearts by retrograde intracoronary infusion: distribution, dynamics, and influence on cardiac function. Circulation. 2004; 110(11 Suppl 1): II 225-230.
23. Becker W, Meller J. The role of nuclear medicine in infection and inflammation. Lancet Infect Dis. 2001; 1(5): 326 -333.
24. Penicka M, Widimsky P, Kobylka P, et al. Early tissue distribution of bone marrow mononuclear cells after transcoronary transplantation in a patient with acute myocardial infarction. Circulation. 2005; 112(4): e63-e65.
25. Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation. 2001; 104(9): 1046-1052.
26. Orlic D, Kajustra J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001; 98(18): 10344-10349.
27. Tang YL, Zhao Q, Zhang YC, et al. Autologous mesenchymal stem cell transplantation induces VEGF and neovascularization in ischemic myocardium. Regul Pept. 2004; 117(1): 3-10.
28. Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003; 114(6): 763-776.
29. Leri A, Kajstura J, Anversa P. Myocyte proliferation and ventricular remodeling. J Card Fail. 2002; 8 (6 Suppl): S518 -525.
30. Urbanek K, Quaini F, Tasca G, et al. Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy. Proc Natl Acad Sci U S A. 2003; 100(18): 10440-10445.
31. Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A. 2003; 100(21): 12313- 12318.
32. Berry MF, Engler AJ, Woo YJ, et al. Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. Am J Physiol Heart Circ Physiol. 2006; 290(6): H2196-H2203.
33. Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature. 2004; 428(6983): 664-668.
34. Balsam LB, Wagers AJ, Christensen JL, et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004; 428(6983): 668-673.
35. Nadal-Ginard B, Kajstura J, Leri A, et al. Myocyte death, growth, and regeneration in cardiac hypertrophy and failure. Circ Res. 2003; 92(2): 139 -150.
36. Torella D, Rota M, Nurzynska D, et al. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004; 94(4): 514-524.
37. Kajstura J, Rota M, Whang B, et al. Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion. Circ Res. 2005; 96(1): 127-137.
1. Menasche P, Hagege AA, Vilquin JT, et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol. 2003; 41(7): 1078-1083.
2. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest. 1999; 103(5): 697-705.
3. Orlic D, Kajstura J,Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature.2001; 410(6829):701-705.
4. Strauer BE, Brehm M, Zeus T, et al. lntracoronary human autologous stem cell transplantation for myocardial regeneration followimg myocardial infarction. Dtsch Med Wochenschr. 2001; 126(34-35): 932-938.
5. Assmus B, Schachinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration: enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation. 2002; 106(24): 3009-3017.
6. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation. 2003; 108(18): 2212-2218.
7. Schachinger V, Assmus B, Britten MB, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol. 2004; 44(8): 1690-1699.
8. Schachinger V, Assmus B, Honold J, et al. Normalization of coronary blood flow in the infarct-related artery after intracoronary progenitor cell therapy: intracoronary Doppler substudy of the TOPCARE-AMI trial. Clin Res Cardiol. 2006; 95(1): 13-22.
9. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004; 364(9429): 141-148.
10．赵明中，高承海，张宇洋，等。通心络胶囊对缺血再灌注心肌细胞凋亡及相关基因蛋白表达的影像。中华心血管病杂志．2000；28(3)：206。
11．张子新 曾定尹 王绽菲。通心络对血管成形术后家兔丝裂素活化蛋白激酶表达的影响。中华心血管病杂志．2003；31(9)：690-693。
12．王华军，谢良地，姚恩辉，等。通心络治疗对自发性高血压大鼠心肌纤维化的影像。中西医结合心脑血管病杂志．2003；1(4)：189-190．。
13．王文健，傅晓东，陈伟华，等。通心络促血管生成作用的实验研究。疑难病杂志．2003；2(1)：2-5。
14．张艳慧，吉梅，张艳玲，等。通心络胶囊对血脂异常患者内皮功能影响的临床研究。疑难病杂志．2005；4(2)：92-93。
15．杨跃进，赵京林，荆志成，等。中药通心络对猪急性心肌梗死再灌注后无再流的影响。中华医学杂志．2005；85(13)：883—888。
16. Kwak B, Mulhaupt F, Myit S, et al. Statins as a newly recognized type of immunomodulator. Nature Med. 2000; 6(12): 1399-1402.
17. Sparrow CP, Burton CA, Hernandez M, et al. Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol. 2001; 21(1): 115-121.
18. Crisby M, Nordin-Fredriksson G, Shah PK, et al. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation. 2001; 103(7): 926-933.
19. Bates K, Ruggeroli CE, Goldman S, et al. Simvastatin restores endothelial NO-mediated vasorelaxation in large arteries after myocardial infarction. Am J Physiol Heart Circ Physiol. 2002; 283(2): H768-775.
20. Abe Y, Izumi T, Urabe A, et al. Pravastatin prevents myocardium from ischemia-induced fibrosis by protecting vascular endothelial cells exposed to oxidative stress. Cardiovasc Drugs Ther. 2006; 20(4): 273-280.
21. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995; 18(12): 1417-1426.
22. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast clonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970; 3(4): 393-403.
23. Quesenberry PJ, Dooner G, Dooner M, et al. The stem cell continuum: considerations on the heterogeneity and plasticity of marrow stem cells. Stem Cell Rev. 2005; 1(1): 29-36.
24. Allen TD, Dexter TM, Simmons PJ. Marrow biology and stem cells. Immunol Ser. 1990; 49(1): 1-38.
25. Wilson FD. Studies of the hematopoietic microenvironment: hematopoietic stroma and microenvironment. Bibl Haematol. 1984; (48):210-292.
26. Balsam LB, Wagers AJ, Christensen JL, et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004; 428(6983): 668-673.
27. Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature. 2004; 428(6983): 664-668.
28. Devine SM, Hoffman R. Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr Opin Hematol. 2000; 7(6):358-363.
29. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275(5302): 964-967.
30. Gehling UM, Ergun S, Schumacher U, et al. In vitro differentiation of endothelial cell from AC133 + progenitor cell. Blood. 2000; 95(10): 3106-3112.
31. Zhang S, Ge J, Sun A, et al. Comparison of various kinds of bone marrow stem cells for the repair of infarcted myocardium: single clonally purified non-hematopoietic mesenchymal stem cells serve as a superior source. J Cell Biochem. 2006; 99(4): 1132-1147.
32. Miyahara Y, Nagaya N, Kataoka M, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 2006; 12(4): 459-465.
33. Lim SY, Kim YS, Ahn Y, et al. The effects of mesenchymal stem cells transduced with Akt in a porcine myocardial infarction model. Cardiovasc Res. 2006; 70(3): 530-542.
34. Amado LC, Saliaris AP, Schuleri KH, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005; 102(32): 11474-11479.
35. Chen SL, Fang WW, Ye F, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol. 2004; 94(1): 92-95.
36. Katritsis DG, Sotiropoulou PA, Karvouni E, et al. Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium. Catheter Cardiovasc Interv. 2005; 65(3): 321-329.
37. Zhang M, Methot D, Poppa V, et al. Cardiomyocyte grafting for cardiac repair: graft cell death and antideath strategies. J Mol Cell Cardiol. 2001, 33(5): 907-921.
38. Muller-Ehmsen J, Krausgrill B, Burst V, et al. Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction. J Mol Cell Cardiol. 2006; 41(5): 876-884.
39. Tran N, Li Y, Maskali F, et al. Short-term heart retention and distribution of intramyocardial delivered mesenchymal cells within necrotic or intact myocardium. Cell Transplant. 2006; 15(4): 351-358.
40. Li RK, Mickle DA, Weisel RD, et al. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg. 2001; 72(6): 1957-1963.
41.Toma C, Pittenger MF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002; 105(1): 93-98.
42. Muller-Ehmsen J, Whittaker P, Kloner RA, et al. Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. J Mol Cell Cardiol. 2002; 34(2): 107-116.
43. Suzuki K, Smolenski RT, Jayakumar J, et al. Heat shock treatment enhances graft cell survival in skeletal myoblast transplantation to the heart. Circulation. 2000; 102(19 Suppl 3): III216-221.
44. Mangi AA, Noiseux N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med. 2003; 9(9): 1195-1201.
45. Jiang S, Haider HKh, Idris NM, et al. Supportive interaction between cell survival signaling and angiocompetent factors enhances donor cell survival and promotes angiomyogenesis for cardiac repair. Circ Res. 2006; 99(7):776-784.
46. Gnecchi M, He H, Noiseux N, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 2006; 20(6): 661-669.
47. Tang YL, Tang Y, Zhang C, et al. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated Heme Oxygenase-1 vector. J Am Coll Cardiol. 2005; 46(7): 1339-1350.
48. Jo JI, Nagaya N, Miyahara Y, et al. Transplantation of genetically engineered mesenchymal stem cells improves cardiac function in rats with myocardial infarction: benefit of a novel nonviral vector, Cationized Dextran. Tissue Eng. 2006; [Epub ahead of print].
49. Li W, Ma N, Ong LL, et al. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells. 2007; [Epub ahead of print].
50. Bartunek J, Croissant JD, Wijns W, et al. Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium. Am J Physiol Heart Circ Physiol. 2007; 292(2): H1095-1104.
51. Song H, Kwon K, Lim S, et al. Transfection of mesenchymal stem cells with the FGF-2 gene improves their survival under hypoxic conditions. Mol Cells. 2005; 19(3): 402-407
2. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001; 344(23): 1750-1757.
3. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002; 418(6893): 41-49.
4. Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet. 2004; 363(9419): 1439-1441.
5. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284(5411): 143-147.
6. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature. 2001; 410(6829): 701-705.
7. Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest. 2002; 109(3): 337-346.
8. Harris RG, Herzog EL, Bruscia EM, et al. Lack of a fusion requirement for development of bone marrow-derived epithelia. Science. 2004; 305(5680): 90-93.
9. Tomita S, Li RK, Weisel RD, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation. 1999; 100(19 suppl): II 247-256.
10. Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, et al. Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature. 2003; 425(6961): 968-973.
11. Liu Y, Song J, Liu W, et al. Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res. 2003; 58(2): 460-468.
12. Kadivar M, Khatami S, Mortazavi Y, et al. In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells. Biochem Biophys Res Commun. 2006; 340(2): 639-647.
13. Xu W, Zhang X, Qian H, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. Exp Biol Med. 2004; 229(7): 623-631.
14. Xu M, Wani M, Dai YS, et al. Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes. Circulation. 2004; 110(17): 2658-2665.
15. Planat-Benard V, Menard C, Andre M, et al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res. 2004; 94(2): 223-229.
16. Grinnemo KH, Mansson-Broberg A, Leblanc K, Human mesenchymal stem cells do not differentiate into cardiomyocytes in a cardiac ischemic xenomodel. Ann Med. 2006; 38(2):144-153.
17. Sartore S, Lenzi M, Angelini A, et al. Amniotic mesenchymal stem cells autotransplanted in a porcine model of cardiac ischemic do not differentiate to cardiogenic phenotype. Eur J Cardiothorac Surg. 2005; 28(5):677-684.
18. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997; 276(5309): 71-74.
19. Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol. 1999; 181(1): 67-73.
20. Moscoso I, Centeno A, Lopez E, et al. Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation. Transplant Proceed. 2005; 37(1): 481-482.
21. Colter D, Class R, Di Girolamo CM, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci U S A. 2000 97(7): 3213-3218.
22. Colter DC, Sekiya I, Prockop DJ, et al. Identification of a subpopulation of rapidly self renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A. 2001; 98(14): 7841-7845.
23. Zhang FB, Li L, Fang B, et al. Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun. 2005; 336(3): 784-792.
24. Zhang X, Soda Y, Takahashi K, et al. Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells. Biochem Biophys Res Commun. 2006; 351(4):853-859.
25. Pittenger, MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics.Circ Res. 2004; 95(1): 9-20.
26. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci. 2000;113(Pt 7): 1161-1166.
27. Dennis JE, Merriam A, Awadallah A, et al. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res. 1999; 14(5): 700-709.
28. Rao SG, Dravid G. Expansion of haematopoietic stem cells in vitro: A challenge to stem cell biologists. Indian J Exp Biol. 1999; 37(11): 1051-1052.
29. Shahdadfar A, Fronsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells. 2005; 23(9): 1357-1366.
30. Bruder SP, Jaiswal N, Haynesworth SE, et al. Growth kinetics, selfrenewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997; 64(2): 278-294.
31. Garcia-Verdugo JM, Penicaud L, Casteilla L et al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res. 2004; 94(2): 223-229.
32. Naruse K, Urabe K, Mukaida T, et al. Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation. Bone. 2004; 35(4): 850-858.
33. Yamada Y, Sakurada K, Takeda Y, et al. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells. Exp Cell Res. 2007; 313(4): 698-706.
34. Heng BC, Haider HKh, Sim EK, et al. Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. Cardiovasc Res. 2004; 62(1):34-42.
35. Fukuda K. Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes. C R Biol. 2002; 325(10): 1027-1038.
36. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest. 1999; 103(5): 697-705.
37. Molkentin JD. The zinc finger-containing transcription factors GATA-4,-5and -6, ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem. 2000; 275(50): 38949-38952.
38. Konieczny SF, Emerson CP. 5-azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell. 1984; 38(3): 791-800.
39. Taylor SM. 5-aza-2-deoxycytidine: cell differentiation and DNA methylation. Leukemia. 1993; 7(suppl 1): 3-8.
1. Fernandez-Aviles F, San Roman JA, Garcia-Frade J, et al. Experimental and clinical regenerative capability of human bone marrow cells after myocardial infarction. Circ Res. 2004; 95(7): 742-748.
2. Schuster MD, Kocher AA, Seki T, et al. Myocardial neovascularization by bone marrow angioblasts results in cardiomyocyte regeneration. Am J Physiol Heart Circ Physiol. 2004; 287(2): H 525-532.
3. Kocher A A, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 2001; 7(4): 430-436.
4. Tse HF, Kwong YL, Chan JK, et al. Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet. 2003; 361(9351): 47-49.
5. Perin EC, Dohmann HF, Borojevic R, et al. Improved exercise capacity and ischemia 6 and 12 months after transendocardial injection of autologous bone marrow mononuclear cells for ischemic cardiomyopathy. Circulation. 2004;110 (11 Suppl 1): 213-218.
6. Strauer BE, Brehm M, Zeus T, Kostering M, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 2002; 106(15): 1913-1918.
7. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation. 2003; 108(18): 2212-2218.
8. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomized controlled clinical trial. Lancet. 2004; 364(9429): 141-148.
9. Hofmann M, Wollert KC, Meyer GP, et al. Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation. 2005; 111(17): 2198-2202.
10. Hou D, Youssef EA, Brinton TJ, et al. Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery implications for current clinical trials. Circulation. 2005; 112 (suppl I): 1-150-156.
11. Kang WJ, Kang HJ, Kim HS, et al. Tissue distribution of 18F-FDG-labeled peripheral hematopoietic stem cells after intracoronary administration in patients with myocardial infarction. J Nucl Med. 2006; 47(8): 1295-1301.
12. Jonker JW, Freeman J, Bolscher E, Contribution of the ABC transporters Bcrp1 and Mdr1a/1b to the side population phenotype in mammary gland and bone marrow of mice. Stem Cells. 2005; 23(8): 1059-1065.
13. Macpherson H, Keir P, Webb S, et al. Bone marrow-derived SP cells can contribute to the respiratory tract of mice in vivo. J Cell Sci. 2005; 118(Pt 11): 2441-2450.
14. Ojima K, Uezumi A, Miyoshi H, et al. Mac-1 (low) early myeloid cells in the bone marrow-derived SP fraction migrate into injured skeletal muscle and participate in muscle regeneration. Biochem Biophys Res Commun. 2004; 321(4): 1050-1061.
15. Asahara T, Murohara T, Sullivan A , et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275(5302): 964-967.
16. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995; 18(12): 1417-1426.
17. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast clonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970; 3(4): 393-403.
18. Quesenberry PJ, Dooner G, Dooner M, et al. The stem cell continuum: considerations on the heterogeneity and plasticity of marrow stem cells. Stem Cell Rev. 2005; 1(1): 29-36.
19. Allen TD, Dexter TM, Simmons PJ. Marrow biology and stem cells. Immunol Ser. 1990; 49(1): 1-38.
20. Wilson FD. Studies of the hematopoietic microenvironment. Hematopoietic stroma and microenvironment. Bibl Haematol. 1984; (48):210-292.
21. Allers C, Sierralta WD, Neubauer S, et al. Dynamic of distribution of human bone marrow- derived mesenchymal stem cells after transplantation into adult unconditioned mice. Transplantation. 2004; 78(4): 503-508.
22. Suzuki K, Murtuza B, Fukushima S, et al. Targeted cell delivery into infarcted rat hearts by retrograde intracoronary infusion: distribution, dynamics, and influence on cardiac function. Circulation. 2004; 110(11 Suppl 1): II 225-230.
23. Becker W, Meller J. The role of nuclear medicine in infection and inflammation. Lancet Infect Dis. 2001; 1(5): 326 -333.
24. Penicka M, Widimsky P, Kobylka P, et al. Early tissue distribution of bone marrow mononuclear cells after transcoronary transplantation in a patient with acute myocardial infarction. Circulation. 2005; 112(4): e63-e65.
25. Kamihata H, Matsubara H, Nishiue T, et al. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation. 2001; 104(9): 1046-1052.
26. Orlic D, Kajustra J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001; 98(18): 10344-10349.
27. Tang YL, Zhao Q, Zhang YC, et al. Autologous mesenchymal stem cell transplantation induces VEGF and neovascularization in ischemic myocardium. Regul Pept. 2004; 117(1): 3-10.
28. Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003; 114(6): 763-776.
29. Leri A, Kajstura J, Anversa P. Myocyte proliferation and ventricular remodeling. J Card Fail. 2002; 8 (6 Suppl): S518 -525.
30. Urbanek K, Quaini F, Tasca G, et al. Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy. Proc Natl Acad Sci U S A. 2003; 100(18): 10440-10445.
31. Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci U S A. 2003; 100(21): 12313- 12318.
32. Berry MF, Engler AJ, Woo YJ, et al. Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. Am J Physiol Heart Circ Physiol. 2006; 290(6): H2196-H2203.
33. Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature. 2004; 428(6983): 664-668.
34. Balsam LB, Wagers AJ, Christensen JL, et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004; 428(6983): 668-673.
35. Nadal-Ginard B, Kajstura J, Leri A, et al. Myocyte death, growth, and regeneration in cardiac hypertrophy and failure. Circ Res. 2003; 92(2): 139 -150.
36. Torella D, Rota M, Nurzynska D, et al. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004; 94(4): 514-524.
37. Kajstura J, Rota M, Whang B, et al. Bone marrow cells differentiate in cardiac cell lineages after infarction independently of cell fusion. Circ Res. 2005; 96(1): 127-137.
1. Menasche P, Hagege AA, Vilquin JT, et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol. 2003; 41(7): 1078-1083.
2. Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest. 1999; 103(5): 697-705.
3. Orlic D, Kajstura J,Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature.2001; 410(6829):701-705.
4. Strauer BE, Brehm M, Zeus T, et al. lntracoronary human autologous stem cell transplantation for myocardial regeneration followimg myocardial infarction. Dtsch Med Wochenschr. 2001; 126(34-35): 932-938.
5. Assmus B, Schachinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration: enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation. 2002; 106(24): 3009-3017.
6. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation. 2003; 108(18): 2212-2218.
7. Schachinger V, Assmus B, Britten MB, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol. 2004; 44(8): 1690-1699.
8. Schachinger V, Assmus B, Honold J, et al. Normalization of coronary blood flow in the infarct-related artery after intracoronary progenitor cell therapy: intracoronary Doppler substudy of the TOPCARE-AMI trial. Clin Res Cardiol. 2006; 95(1): 13-22.
9. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004; 364(9429): 141-148.
10．赵明中，高承海，张宇洋，等。通心络胶囊对缺血再灌注心肌细胞凋亡及相关基因蛋白表达的影像。中华心血管病杂志．2000；28(3)：206。
11．张子新 曾定尹 王绽菲。通心络对血管成形术后家兔丝裂素活化蛋白激酶表达的影响。中华心血管病杂志．2003；31(9)：690-693。
12．王华军，谢良地，姚恩辉，等。通心络治疗对自发性高血压大鼠心肌纤维化的影像。中西医结合心脑血管病杂志．2003；1(4)：189-190．。
13．王文健，傅晓东，陈伟华，等。通心络促血管生成作用的实验研究。疑难病杂志．2003；2(1)：2-5。
14．张艳慧，吉梅，张艳玲，等。通心络胶囊对血脂异常患者内皮功能影响的临床研究。疑难病杂志．2005；4(2)：92-93。
15．杨跃进，赵京林，荆志成，等。中药通心络对猪急性心肌梗死再灌注后无再流的影响。中华医学杂志．2005；85(13)：883—888。
16. Kwak B, Mulhaupt F, Myit S, et al. Statins as a newly recognized type of immunomodulator. Nature Med. 2000; 6(12): 1399-1402.
17. Sparrow CP, Burton CA, Hernandez M, et al. Simvastatin has anti-inflammatory and antiatherosclerotic activities independent of plasma cholesterol lowering. Arterioscler Thromb Vasc Biol. 2001; 21(1): 115-121.
18. Crisby M, Nordin-Fredriksson G, Shah PK, et al. Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization. Circulation. 2001; 103(7): 926-933.
19. Bates K, Ruggeroli CE, Goldman S, et al. Simvastatin restores endothelial NO-mediated vasorelaxation in large arteries after myocardial infarction. Am J Physiol Heart Circ Physiol. 2002; 283(2): H768-775.
20. Abe Y, Izumi T, Urabe A, et al. Pravastatin prevents myocardium from ischemia-induced fibrosis by protecting vascular endothelial cells exposed to oxidative stress. Cardiovasc Drugs Ther. 2006; 20(4): 273-280.
21. Wakitani S, Saito T, Caplan AI. Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve. 1995; 18(12): 1417-1426.
22. Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast clonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 1970; 3(4): 393-403.
23. Quesenberry PJ, Dooner G, Dooner M, et al. The stem cell continuum: considerations on the heterogeneity and plasticity of marrow stem cells. Stem Cell Rev. 2005; 1(1): 29-36.
24. Allen TD, Dexter TM, Simmons PJ. Marrow biology and stem cells. Immunol Ser. 1990; 49(1): 1-38.
25. Wilson FD. Studies of the hematopoietic microenvironment: hematopoietic stroma and microenvironment. Bibl Haematol. 1984; (48):210-292.
26. Balsam LB, Wagers AJ, Christensen JL, et al. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 2004; 428(6983): 668-673.
27. Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature. 2004; 428(6983): 664-668.
28. Devine SM, Hoffman R. Role of mesenchymal stem cells in hematopoietic stem cell transplantation. Curr Opin Hematol. 2000; 7(6):358-363.
29. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275(5302): 964-967.
30. Gehling UM, Ergun S, Schumacher U, et al. In vitro differentiation of endothelial cell from AC133 + progenitor cell. Blood. 2000; 95(10): 3106-3112.
31. Zhang S, Ge J, Sun A, et al. Comparison of various kinds of bone marrow stem cells for the repair of infarcted myocardium: single clonally purified non-hematopoietic mesenchymal stem cells serve as a superior source. J Cell Biochem. 2006; 99(4): 1132-1147.
32. Miyahara Y, Nagaya N, Kataoka M, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 2006; 12(4): 459-465.
33. Lim SY, Kim YS, Ahn Y, et al. The effects of mesenchymal stem cells transduced with Akt in a porcine myocardial infarction model. Cardiovasc Res. 2006; 70(3): 530-542.
34. Amado LC, Saliaris AP, Schuleri KH, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005; 102(32): 11474-11479.
35. Chen SL, Fang WW, Ye F, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol. 2004; 94(1): 92-95.
36. Katritsis DG, Sotiropoulou PA, Karvouni E, et al. Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium. Catheter Cardiovasc Interv. 2005; 65(3): 321-329.
37. Zhang M, Methot D, Poppa V, et al. Cardiomyocyte grafting for cardiac repair: graft cell death and antideath strategies. J Mol Cell Cardiol. 2001, 33(5): 907-921.
38. Muller-Ehmsen J, Krausgrill B, Burst V, et al. Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction. J Mol Cell Cardiol. 2006; 41(5): 876-884.
39. Tran N, Li Y, Maskali F, et al. Short-term heart retention and distribution of intramyocardial delivered mesenchymal cells within necrotic or intact myocardium. Cell Transplant. 2006; 15(4): 351-358.
40. Li RK, Mickle DA, Weisel RD, et al. Optimal time for cardiomyocyte transplantation to maximize myocardial function after left ventricular injury. Ann Thorac Surg. 2001; 72(6): 1957-1963.
41.Toma C, Pittenger MF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation. 2002; 105(1): 93-98.
42. Muller-Ehmsen J, Whittaker P, Kloner RA, et al. Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. J Mol Cell Cardiol. 2002; 34(2): 107-116.
43. Suzuki K, Smolenski RT, Jayakumar J, et al. Heat shock treatment enhances graft cell survival in skeletal myoblast transplantation to the heart. Circulation. 2000; 102(19 Suppl 3): III216-221.
44. Mangi AA, Noiseux N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med. 2003; 9(9): 1195-1201.
45. Jiang S, Haider HKh, Idris NM, et al. Supportive interaction between cell survival signaling and angiocompetent factors enhances donor cell survival and promotes angiomyogenesis for cardiac repair. Circ Res. 2006; 99(7):776-784.
46. Gnecchi M, He H, Noiseux N, et al. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 2006; 20(6): 661-669.
47. Tang YL, Tang Y, Zhang C, et al. Improved graft mesenchymal stem cell survival in ischemic heart with a hypoxia-regulated Heme Oxygenase-1 vector. J Am Coll Cardiol. 2005; 46(7): 1339-1350.
48. Jo JI, Nagaya N, Miyahara Y, et al. Transplantation of genetically engineered mesenchymal stem cells improves cardiac function in rats with myocardial infarction: benefit of a novel nonviral vector, Cationized Dextran. Tissue Eng. 2006; [Epub ahead of print].
49. Li W, Ma N, Ong LL, et al. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells. 2007; [Epub ahead of print].
50. Bartunek J, Croissant JD, Wijns W, et al. Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium. Am J Physiol Heart Circ Physiol. 2007; 292(2): H1095-1104.
51. Song H, Kwon K, Lim S, et al. Transfection of mesenchymal stem cells with the FGF-2 gene improves their survival under hypoxic conditions. Mol Cells. 2005; 19(3): 402-407