雌激素对高血压脑出血患者内皮祖细胞的影响及其机制研究
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摘要
背景和目的:脑出血在我国占急性脑血管病的30%左右。急性期病死率是急性脑血管病中最高(30%～40%)。脑出血以高死亡率、高致残率一直是世界各国医学研究的重点。现在研究表明脑卒中后神经可以一定程度再生,但卒中后2-6周内约80%或更多的新生神经细胞逐渐死亡。这就提示我们如果没有适合新生神经元存活、生长的微环境,缺乏适宜的营养支持和连接,新生的神经元将被直接暴露在损伤周围有害的环境中,神经组织的修复和再生会受到极大的限制。血管再生和修复可能是有效的神经再生的基础。两者的关系可能是相互依赖、相互促进的。
Asahara等于1997年发现了内皮祖细胞(endothelial progenitorcells,EPCs)。EPCs是内皮细胞的前体细胞,可以表达多种内皮标志,并可以整合到缺血区域的新生血管中,在血管损伤后修复和血管再生中起着重要的作用。近年来的研究表明生理、病理、药理等多种条件均能影响机体EPC的动员和功能。脑出血患者大多数为老年,往往具有高血压等心脑血管疾病的危险因素。研究发现随着年龄的增加外周血中EPCs数目和功能下降。高血压也降低动物和人循环中EPCs数目和功能,且EPCs易于衰老。这就势必会影响脑卒中危险人群和患者的损伤血管修复和疾病后的血管再生,增加血管性疾病的发生/复发几率,使其预后不良。同时研究还表明急性心脏、肢体、脑缺血和血管损伤,手术急性期等可以诱导EPCs动员和功能增强,提示应激状态下机体EPCs数目和功能可能暂时性升高。这可能和VEGF的生成和增多有关,研究发现在诸如缺血等情况可以上调EPCs和其他分细胞表达和分泌VEGF,VEGF是动员、激活EPCs的关键因子,是内皮细胞增殖、迁移、成熟的重要因子,并在生理和病理情况下血管修复和再生起着非常关键的作用。高血压脑出血急性期会不会也引起EPCs功能增强,机体或EPCs VEGF表达和分泌能否上调,EPCs是否易于衰老,国内外尚未见报道。因此我们对高血压脑出血患者急性期外周血循环中的EPCs功能研究,并与高血压患者对比,以探讨高血压脑出血急性期EPCs的功能变化及其可能的机理。
研究表明成人外周血中EPCs数目含量少,整合到损伤和缺血区域率较低,这就有必要寻找合适的药物或措施来增加EPCs的增殖和功能。研究显示他汀类药、雌激素、促红细胞生成素等可以促进EPCs的数目增加、功能增强、延缓EPCs的衰老。Imanishi研究发现雌激素可以促进健康人的EPCs分泌VEGF,通过增强端粒酶的活性来延缓EPCs的衰老,从而增强EPCs的功能,其作用和PI3K/AKt信号途径有关。PI3K/AKt途径在细胞生存和增殖中起关键性的作用。但雌激素对老年高血压脑出血患者的EPCs有何影响以及其影响的可能机制,目前国内外尚未见报道。因此我们应用不同浓度雌激素干预急性期高血压脑出血患者的EPCs,观察其对高血压脑出血急性期患者EPCs功能和衰老的影响,并探讨其可能的影响机理。
材料和方法
1.病人资料老年高血压男性患者和急性期高血压脑出血男性患者,高血压病史5年以上,排除可能对EPCs有影响的药物应用史和其他近期疾病史;高血压脑出血患者基底节出血量为20ml-30ml,发病时间1-3天内,并排除其他非高血压所引起的脑出血疾病。
2.外周血内皮祖细胞的分离、培养、鉴定取空腹外周静脉血15ml,密度梯度离心法获得单个核细胞,用含20%胎牛血清和生长因子的M199培养基培养。第7天与Dil标记的乙酰化低密度脂蛋白(DilacLDL)和FITC标记的荆豆凝集素Ⅰ(FITC-UEA-I)孵育,多波长激光共聚焦显微镜鉴定,FITC-UEA-I和Dil-acLDL双染色阳性细胞为正在分化的EPCs。
3.实验分组高血压脑出血患者和高血压患者EPCs功能对比实验分组:分为高血压组和高血压脑出血组:培养基为含20%胎牛血清和生长因子的M199培养基;培养第7天进行进一步实验。雌激素干预高血压脑出血患者EPCs实验分组:在培养第6天,将高血压脑出血患者EPCs各自分为5组,换用成分同上另含相应浓度雌激素的培养基。①高血压脑出血组;②雌激素10nmol/L组;③雌激素100nmol/L组;④雌激素1μmol/L组;⑤雌激素1μmol/L+PI3K抑制剂(LY294002)组:先LY294002(1μmol/L)孵育1h,再用1μmol/L雌激素组培养基。24h后进行进一步实验(VEGF相关实验无第⑤组)。
4.内皮祖细胞集落和细胞计数、粘附、增殖、迁移能力检测第7天随机计数各组EPC集落和细胞数目,然后消化、收集部分培养的EPCs分别进行下列实验。将细胞密度调整到1×10~5个/ml,接种到纤维连接蛋白(1ng/μl)包被的培养板,培养30min,计数贴壁细胞。将细胞密度调整到2×10~3个/ml,用MTT细胞增殖及细胞毒性试剂盒测定EPCs的增殖能力。将细胞密度调整到2×10~4个/ml,用改良的Boyden小室法培养24小时,计数迁移到小室下层的细胞数。
5.内皮祖细胞VEGF mRNA和蛋白质表达检测,血清和内皮祖细胞培养上清液VEGF含量测定第7天Trizol法提取各组EPCs总RNA;细胞蛋白裂解液裂解提取EPCs蛋白质。以RT-PCR法半定量测定VEGF mRNA表达;Western blot法半定量测定VEGF蛋白质表达。取患者血清和第7天培养上清液(第4天换用不含生长因子的各组相应培养基培养72小时);应用血管内皮细胞生长因子VEGFELISA试剂盒检测VEGF含量。
6.内皮祖细胞衰老检测自第一次换液时改用各组相应培养基换液,培养2周,采用细胞衰老β-半乳糖苷酶染色试剂盒检测各组衰老EPCs并计数。
结果:
1.内皮祖细胞形态学观察结果刚分离的细胞呈个体均一较小的圆形,于培养过程中体积逐渐变大、形态变为梭形;高血压脑出血组于第3天开始出现EPCs集落,并与培养过程中可见多层条索或类血管结构;高血压组EPCs出现较晚(第5天),细胞密度较少,未见条索或类血管结构。应用不同浓度的雌激素干预后,EPCs集落和细胞数目增多体积增大。
2.内皮祖细胞鉴定结果所培养细胞从开始的个体较小的圆形,逐渐体积变大形态变成梭形,典型EPCs集落形成;细胞呈DiI-acLDL、FITC-UEA-I双染色阳性。
3.高血压脑出血和高血压患者内皮祖细胞集落和细胞计数、粘附、增殖、迁移功能结果高血压脑出血组EPCs集落和细胞计数、粘附、增殖、迁移能力均较高血压组明显增加,具有显著性统计学差异(P＜0.05或P＜0.01)。
4.高血压脑出血和高血压患者血清和内皮祖细胞培养上清液VEGF含量、内皮祖细胞VEGF mRNA和蛋白质表达结果高血压脑出血组患者血清和EPCs培养上清液VEGF含量、内皮祖细胞VEGFmRNA和蛋白质表达、均较高血压组明显增加,具有显著性统计学差异(P＜0.05或P＜0.01)。
5.高血压脑出血和高血压患者衰老内皮祖细胞计数高血压脑出血和高血压患者相比,衰老EPCs数目无明显变化,不具有统计学差异(P＞0.05)。
6.雌激素干预后高血压脑出血患者内皮祖细胞集落计数、粘附、增殖、迁移功能结果不同浓度的雌激素均能增加高血压脑出血患者EPCs集落计数、粘附、增殖、迁移能力,具有显著性统计学差异(P＜0.01);且随着雌激素浓度的增加其作用效果递增,不同浓度雌激素组间相比均有显著性统计学差异(P＜0.01),在雌激素1μmol/L时达到最大;当用PI3K拮抗剂LY294002预处理后,再以1μmol/L雌激素干预,此种增强作用被明显抑制,与雌激素1μmol/L组相比,具有显著性统计学差异(P＜0.01)。
7.雌激素干预后高血压脑出血患者内皮祖细胞培养上清液VEGF含量、VEGF mRNA和蛋白质表达结果不同浓度的雌激素均能增加高血压脑出血患者EPCs培养上清液VEGF含量、VEGF mRNA和蛋白质表达,具有显著性统计学差异(P＜0.01);且随着雌激素浓度的增加其作用效果递增,不同浓度雌激素组间相比均有显著性统计学差异(P＜0.01),在雌激素1μmol/L时达到最大。
8.雌激素干预后高血压脑出血患者衰老内皮祖细胞计数结果不同浓度的雌激素均能抑制高血压脑出血患者EPCs衰老,具有显著性统计学差异(P＜0.01);且随着雌激素浓度的增加其抑制作用递增,不同浓度雌激素组间相比均有显著性统计学差异(P＜0.01),在雌激素1μmol/L时达到最大。当用PI3K拮抗剂LY294002预处理后,再以1μmol/L雌激素干预,其抑制作用被明显抑制,与雌激素1μmol/L组相比,具有显著性统计学差异(P＜0.01)。
结论:
1.脑出血急性期高血压患者内皮祖细胞的数目和功能增强。EPCsVEGF在mRNA和蛋白质水平表达上调,EPCs和机体分泌VEGF增加是其可能机理之一。但EPCs易衰老的转归没有明显改变。
2.雌激素在体外呈浓度依赖性增强急性期高血压脑出血患者EPCs的数目和功能。这可能和其增加EPCs VEGF mRNA、蛋白质表达和分泌有关,同时也可能和抑制EPCs衰老、改善其易衰老的转归有关。
3.雌激素在体外增强EPCs数目和功能,延缓EPCs衰老的作用可能与PI3K途径有关。
Background and Purpose:Stroke is one of the three major diseases threatening human health and the leading cause of death and disabilities in many countries and districts.Primary cerebral hemorrhage refers to non-traumatic hemorrhage of brain parenchyma,the proportion of cerebral hemorrhage(CH)is about 30%of total stroke. The mortality of CH during acute stage is about 30-40%,which is the highest among all kinds of cerebrovascular diseases.So many studies focused on CH and many discoveries about the pathogenesis,treatment, especially neurogenesis after stroke emerged.Studies showed evidences of neurogenesis related to neural stem cell after stroke but about 80%of new neurons died within 2 to 6 weeks after onset of stroke.The increasing evidences indicated that neurogensis after stroke is limited by inadequate angiogenesis/vasculogensis,which provide nutrition and proper microenviroment that facilitate new neurons' survival and growth.
Since Asahara et al.reported that a subtype of hematopoietic progenitor cells from adults in 1997,namely "endothelial progenitor cells"(EPCs),showed endothelial cell feature,and play an important role in re-endothelialization during the neovascularization of ischemic organs.
The patients with stroke are always associated with ageing and hypertension.Hypertension and ageing may decrease the mobilization, migration and proliferative capacity of EPCs.EPCs of patients with hypertension and old age are easy to senescence.While increasing evidences show that the number,mobilization,migration and proliferative capacity of EPCs may be improved in acute period of ischemia diseases (such as ischemia diseases of heart,cerebral and limbs),heart surgery, and organ injury.This suggests that stressing of organ can augment the number and ability of EPCs.
Ischemia is believed to upregulate VEGF,which in turn are released to the circulation and induce mobilization of progenitor cells from the bone marrow.EPCs may act similar to monocytes/macrophages,which can increase arteriogenesis by providing cytokines and growth factors. Indeed,EPCs cultivated from different sources showed a marked expression of growth factors such as VEGF,HGF,and IGF-1.The release of growth factors in turn may influence the classical process of angiogenesis,namely the proliferation and migration as well as survival of mature endothelial cells.VEGF is a known chemokine critical for angiogenesis in adults.Intramuscular or intramyocardial VEGF gene transfer has been shown to increase EPC numbers in patients with limb ischemia or inoperable coronary disease.But it is unknown about EPCs functional activity change of old patients with CH associated with hypertension(CH-EH).And it's underlying mechanism needs further research.
EPCs remain extremely rare in adult peripheral blood(0.01%of mononuclear cells,under steady state conditions).And studies showed that the number of incorporated cells with an endothelial phenotype into injury or ischemic tissues is generally quite low.It is necessary to seek drugs or methods to augment the number and function of EPCs. Fortunately,studies showed that estrogen,erythropoietin,statins, angiotensin converting enzyme inhibitors can increase the number and functions of EPCs.Imanishi's study demonstrated that 17β-estradiol can improve the secretion of VEGF by EPCs,and delay the onset of senescence of EPCs in healthy human.PI3-K blockers could attenuate the effects of estrogen on EPCs differentiation and senescence and permitted the conclusion that PI3K/Akt signaling pathway is a possible regulator of EPCs proliferation.The PI3-kinase enzymes are widely expressed and play crucial roles in many biological responses including cell survival and proliferation.However,Imanishi's study also indicated that understan ding of the relationship of these factors to hypertension and its vascular sequelae must await further developments in clinical expertise. It is unknown that the effect of estrogen to EPCs of patients with CH-EH. So we aimed to investigate the effect of estrogen on EPCs of patients with CH-EH and explore the underlying molecular mechanism.
Methods:
1.Study Subjects:Each of patients in control group and CH-EH group had a hypertension history(more than 5 years),hadn't the history of diseases and drugs treatment that maybe affect EPCs.The CH diseases that not associated with hypertension must be excluded.The CH-EH patients were enrolled between 1-3 days after onset of stroke.The volumes of hemorrhage were 20-30ml.
2.Isolation and cultivation of EPCs:Peripheral blood(15 ml) was drawn by venipuncture,Mononuclear cells were isolated by density gradient method using Ficoll-Paque Plus.Cells were cultured with M199 medium,which contains 20%fetal-calf.The culture media were changed every 3 days beginning at day 4.
3.Characterization of EPCs:At day 7,EPCs were incubated with Dil- acLDL and FITC-UEA-I,Samples were examined with laser scanning con-focal microscope.Only cells exhibiting double positive were identified as EPCs.
4.Groups of experiment:Groups of the first experiment include hypertension group and CH-EH group.EPCs were cultured with the medium as described above.Groups of the second experiment include: 1).CH-EH EPCs group,2).CH-EH EPCs treated with 17β-estradiol10nmol/L group,3).CH-EH EPCs treated with 17β-estradiol100nmol/L group,4).CH-EH EPCs treated with 17β-estradiol 1μmol/L group,5).CH-EH EPCs treated with 17β-estradiol1μmol/L + LY294002 group.Every group of EPCs was cultured with the similar medium as above but also included different concentration of 17β-estradiol.EPCs treated with 17β-estradiol1μmol/L + LY294002 group were firstly cultured with LY294002 before culturing with 1μmol/L 17β-estradiol.
5.Numbers assay of EPCs colonies and EPCs:The numbers of EPCs and EPCs colonies were determined by counting 5 random high-power(×200)microscope fields per subject at day 7.After the numbers of EPC and EPC colony were counted,cells were assayed or harvested for further study.
6.Adhesion assay of EPCs:1×10~5cells/ml EPCs of every group were replanted onto fibronectin-coated culture dishes and incubated at 37℃for 30 minutes.Adherent cells were counted 5 random high-power (×200)microscope fields per subject.
7.Migration assay of EPCs:2×10~4cells/ml EPCs of every group resuspended in 50ul M199 medium include different concentration of 17β-estradiol were placed in the upper chamber of a modified Boyden chamber.25ul M199 and human recombinant VEGF(50ng/ml)were placed in the lower compartment of the chamber.After 24h incubation at 37℃,cells migrating into the lower chamber were counted.
8.Proliferation assay of EPCs:2×10~3 cell/ml EPCs of every group were replanted 96-well culture dishes,MTT Cell Proliferation and Cytotoxicity Assay Kit were applied to assay proliferation of EPCs.The OD value was measured at 570nm.
9.Measurement of VEGF protein in serum and the culture medium:The VEGF protein content of serum of patients and culture medium(at day 7)were assayed by using VEGF ELISA Kit.The OD value was measured at 450nm.
10.VEGF mRNA and protein expression assay of EPCs:Total RNA of EPCs in every group was extracted by using Trizol RNA Extraction Kit,and proteins of EPCs in these groups were also extracted. Semi-quantitative reverse transcription- polymerase chain reaction (RT-PCR)analysis was performed to assay VEGF mRNA.VEGF protein was assessed by Western blotting.
11.Senescence-associatedβ-galactosidase activity assay: After the first medium changing,the EPCs of different groups were cultured with different medium which included different concentration of 17β-estradiol.EPCs at day 14 were harvested and senescenceassociatedβ-galactosidase activity was measured by using Senescenceβ-Galactosidase Staining Kit.Senescence cells among per 100 cells were counted 5 random high-power(×200)microscope fields per subject.
Results:
1.Morphological features of EPCs:Culture of total peripheral blood mononuclear cell resulted in the emergence of characteristic spindle-shaped cells and colonies consisting of peripheral spindle-shaped cells emanating from round central cells within 72 hours of culture in CH-EH group,bands or vessels like structure formed by layers of EPCs can be found in some fields.In hypertension group,the colonies of EPCs appeared at day 5,relative later to that of CH-EH.After intervention of different concentration of estrogen,the number and the size of EPCs colonies increased significantly.
2.Characterization of EPCs:These cells could be shown to uptake Dil- acLDL and FITC-UEA-I,and exhibiting double-positive. According the results of morphological features and double-positive cells, the cells that we cultured were EPCs.
3.Number assay of EPCs colonies and EPCs,adhesion, migration and proliferation of EPCs:Compared with hypertension group,the number of EPCs and EPCs colonies,adhesive migrating and proliferative faculty of EPCs in CH-EH group significantly increased (P＜0.01 or P＜0.05).After treated with different concentration 17β-estradiol,Compared with CH-EH group,except CH-EH EPCs treated with 17β-estradiol10nmol/L group's colonies counts,the number of EPCs and EPCs colonies,adhesive,migrating and proliferative faculty of EPCs in different concentration groups significantly increased (P＜0.01),and dose-dependently improved(P＜0.01)with a peak at 1umol/l concentration(P＜0.01).When EPCs were firstly incubated with LY294002 before cultured with medium of 17β-estradiol 1umol/l group,this effect of 17β-estradiol on EPCs of CH-EH was significantly inhibited compared with 17β-estradiol 1umol/l group(P＜0.01).
4.Measurement of VEGF protein in serum and the culture medium,VEGF mRNA and VEGF protein expression assay of EPCs: Compared with hypertension group,the content of VEGF protein in serum and the culture medium,VEGF mRNA and protein expression of EPCs,were all significantly increased in CH group(P＜0.01).After treated with different concentration 17β-estradiol,Compared with CH-EH group,the content of VEGF protein in culture medium,VEGF mRNA and protein expression of EPCs in different concentration groups significantly increased(P＜0.01),and dose-dependently improved(P＜0.01) with a peak at 1umol/l concentration(P＜0.01).
5.Senescence-associatedβ-galactosidase activity assay: Compared with hypertension group,the number of senescence EPCs in CH-EH group had no significant difference(P＞0.05).After treated with different concentration 17β-estradiol,compared with CH-EH group,the number of senescence EPCs in different concentration groups significantly decreased(P＜0.01),and dose-dependently improved (P＜0.01)with a peak at 1umol/l concentration(P＜0.01).When EPCs were firstly incubated with LY294002 before cultured with medium of 17β-estradiol 1umol/l group,this effect of 17β-estradiol on senescence EPCs of CH-EH was significantly inhibited compared with 17β-estradiol 1umol/l group(P＜0.01).
Conclusions:
1.The number of EPCs and EPCs colonies,adhesive,migrating and proliferative faculty of EPCs in acute period of CH-EH significantly increased.The content of VEGF protein in serum and the culture medium,VEGF mRNA and protein expression of EPCs,were all significantly increased.This may be the mechanism of acute cerebral hemorrhage augment EPCs numbers and function.The destiny of EPCs easy to senescence was not changed compared with EPCs of patients with hypertension.
2.Estrogen can increase the numbers and function of EPCs of patients with CH-EH in vitro,and this effect is dose-dependence. Estrogen can improve VEGF mRNA and protein expression and VEGF secretion of EPCs,and reduce EPCs senescence.The effect is dose-dependent.These may be mechanisms of estrogen augment numbers and function of EPCs of patients with CH-EH.
3.The role of Estrogen augments number and function of EPCs, reduces EPCs senescence of patients with CH-EH in vitro is related to PI3K/Akt signaling pathway.
Asahara等于1997年发现了内皮祖细胞(endothelial progenitorcells,EPCs)。EPCs是内皮细胞的前体细胞,可以表达多种内皮标志,并可以整合到缺血区域的新生血管中,在血管损伤后修复和血管再生中起着重要的作用。近年来的研究表明生理、病理、药理等多种条件均能影响机体EPC的动员和功能。脑出血患者大多数为老年,往往具有高血压等心脑血管疾病的危险因素。研究发现随着年龄的增加外周血中EPCs数目和功能下降。高血压也降低动物和人循环中EPCs数目和功能,且EPCs易于衰老。这就势必会影响脑卒中危险人群和患者的损伤血管修复和疾病后的血管再生,增加血管性疾病的发生/复发几率,使其预后不良。同时研究还表明急性心脏、肢体、脑缺血和血管损伤,手术急性期等可以诱导EPCs动员和功能增强,提示应激状态下机体EPCs数目和功能可能暂时性升高。这可能和VEGF的生成和增多有关,研究发现在诸如缺血等情况可以上调EPCs和其他分细胞表达和分泌VEGF,VEGF是动员、激活EPCs的关键因子,是内皮细胞增殖、迁移、成熟的重要因子,并在生理和病理情况下血管修复和再生起着非常关键的作用。高血压脑出血急性期会不会也引起EPCs功能增强,机体或EPCs VEGF表达和分泌能否上调,EPCs是否易于衰老,国内外尚未见报道。因此我们对高血压脑出血患者急性期外周血循环中的EPCs功能研究,并与高血压患者对比,以探讨高血压脑出血急性期EPCs的功能变化及其可能的机理。
研究表明成人外周血中EPCs数目含量少,整合到损伤和缺血区域率较低,这就有必要寻找合适的药物或措施来增加EPCs的增殖和功能。研究显示他汀类药、雌激素、促红细胞生成素等可以促进EPCs的数目增加、功能增强、延缓EPCs的衰老。Imanishi研究发现雌激素可以促进健康人的EPCs分泌VEGF,通过增强端粒酶的活性来延缓EPCs的衰老,从而增强EPCs的功能,其作用和PI3K/AKt信号途径有关。PI3K/AKt途径在细胞生存和增殖中起关键性的作用。但雌激素对老年高血压脑出血患者的EPCs有何影响以及其影响的可能机制,目前国内外尚未见报道。因此我们应用不同浓度雌激素干预急性期高血压脑出血患者的EPCs,观察其对高血压脑出血急性期患者EPCs功能和衰老的影响,并探讨其可能的影响机理。
材料和方法
1.病人资料老年高血压男性患者和急性期高血压脑出血男性患者,高血压病史5年以上,排除可能对EPCs有影响的药物应用史和其他近期疾病史;高血压脑出血患者基底节出血量为20ml-30ml,发病时间1-3天内,并排除其他非高血压所引起的脑出血疾病。
2.外周血内皮祖细胞的分离、培养、鉴定取空腹外周静脉血15ml,密度梯度离心法获得单个核细胞,用含20%胎牛血清和生长因子的M199培养基培养。第7天与Dil标记的乙酰化低密度脂蛋白(DilacLDL)和FITC标记的荆豆凝集素Ⅰ(FITC-UEA-I)孵育,多波长激光共聚焦显微镜鉴定,FITC-UEA-I和Dil-acLDL双染色阳性细胞为正在分化的EPCs。
3.实验分组高血压脑出血患者和高血压患者EPCs功能对比实验分组:分为高血压组和高血压脑出血组:培养基为含20%胎牛血清和生长因子的M199培养基;培养第7天进行进一步实验。雌激素干预高血压脑出血患者EPCs实验分组:在培养第6天,将高血压脑出血患者EPCs各自分为5组,换用成分同上另含相应浓度雌激素的培养基。①高血压脑出血组;②雌激素10nmol/L组;③雌激素100nmol/L组;④雌激素1μmol/L组;⑤雌激素1μmol/L+PI3K抑制剂(LY294002)组:先LY294002(1μmol/L)孵育1h,再用1μmol/L雌激素组培养基。24h后进行进一步实验(VEGF相关实验无第⑤组)。
4.内皮祖细胞集落和细胞计数、粘附、增殖、迁移能力检测第7天随机计数各组EPC集落和细胞数目,然后消化、收集部分培养的EPCs分别进行下列实验。将细胞密度调整到1×10~5个/ml,接种到纤维连接蛋白(1ng/μl)包被的培养板,培养30min,计数贴壁细胞。将细胞密度调整到2×10~3个/ml,用MTT细胞增殖及细胞毒性试剂盒测定EPCs的增殖能力。将细胞密度调整到2×10~4个/ml,用改良的Boyden小室法培养24小时,计数迁移到小室下层的细胞数。
5.内皮祖细胞VEGF mRNA和蛋白质表达检测,血清和内皮祖细胞培养上清液VEGF含量测定第7天Trizol法提取各组EPCs总RNA;细胞蛋白裂解液裂解提取EPCs蛋白质。以RT-PCR法半定量测定VEGF mRNA表达;Western blot法半定量测定VEGF蛋白质表达。取患者血清和第7天培养上清液(第4天换用不含生长因子的各组相应培养基培养72小时);应用血管内皮细胞生长因子VEGFELISA试剂盒检测VEGF含量。
6.内皮祖细胞衰老检测自第一次换液时改用各组相应培养基换液,培养2周,采用细胞衰老β-半乳糖苷酶染色试剂盒检测各组衰老EPCs并计数。
结果:
1.内皮祖细胞形态学观察结果刚分离的细胞呈个体均一较小的圆形,于培养过程中体积逐渐变大、形态变为梭形;高血压脑出血组于第3天开始出现EPCs集落,并与培养过程中可见多层条索或类血管结构;高血压组EPCs出现较晚(第5天),细胞密度较少,未见条索或类血管结构。应用不同浓度的雌激素干预后,EPCs集落和细胞数目增多体积增大。
2.内皮祖细胞鉴定结果所培养细胞从开始的个体较小的圆形,逐渐体积变大形态变成梭形,典型EPCs集落形成;细胞呈DiI-acLDL、FITC-UEA-I双染色阳性。
3.高血压脑出血和高血压患者内皮祖细胞集落和细胞计数、粘附、增殖、迁移功能结果高血压脑出血组EPCs集落和细胞计数、粘附、增殖、迁移能力均较高血压组明显增加,具有显著性统计学差异(P＜0.05或P＜0.01)。
4.高血压脑出血和高血压患者血清和内皮祖细胞培养上清液VEGF含量、内皮祖细胞VEGF mRNA和蛋白质表达结果高血压脑出血组患者血清和EPCs培养上清液VEGF含量、内皮祖细胞VEGFmRNA和蛋白质表达、均较高血压组明显增加,具有显著性统计学差异(P＜0.05或P＜0.01)。
5.高血压脑出血和高血压患者衰老内皮祖细胞计数高血压脑出血和高血压患者相比,衰老EPCs数目无明显变化,不具有统计学差异(P＞0.05)。
6.雌激素干预后高血压脑出血患者内皮祖细胞集落计数、粘附、增殖、迁移功能结果不同浓度的雌激素均能增加高血压脑出血患者EPCs集落计数、粘附、增殖、迁移能力,具有显著性统计学差异(P＜0.01);且随着雌激素浓度的增加其作用效果递增,不同浓度雌激素组间相比均有显著性统计学差异(P＜0.01),在雌激素1μmol/L时达到最大;当用PI3K拮抗剂LY294002预处理后,再以1μmol/L雌激素干预,此种增强作用被明显抑制,与雌激素1μmol/L组相比,具有显著性统计学差异(P＜0.01)。
7.雌激素干预后高血压脑出血患者内皮祖细胞培养上清液VEGF含量、VEGF mRNA和蛋白质表达结果不同浓度的雌激素均能增加高血压脑出血患者EPCs培养上清液VEGF含量、VEGF mRNA和蛋白质表达,具有显著性统计学差异(P＜0.01);且随着雌激素浓度的增加其作用效果递增,不同浓度雌激素组间相比均有显著性统计学差异(P＜0.01),在雌激素1μmol/L时达到最大。
8.雌激素干预后高血压脑出血患者衰老内皮祖细胞计数结果不同浓度的雌激素均能抑制高血压脑出血患者EPCs衰老,具有显著性统计学差异(P＜0.01);且随着雌激素浓度的增加其抑制作用递增,不同浓度雌激素组间相比均有显著性统计学差异(P＜0.01),在雌激素1μmol/L时达到最大。当用PI3K拮抗剂LY294002预处理后,再以1μmol/L雌激素干预,其抑制作用被明显抑制,与雌激素1μmol/L组相比,具有显著性统计学差异(P＜0.01)。
结论:
1.脑出血急性期高血压患者内皮祖细胞的数目和功能增强。EPCsVEGF在mRNA和蛋白质水平表达上调,EPCs和机体分泌VEGF增加是其可能机理之一。但EPCs易衰老的转归没有明显改变。
2.雌激素在体外呈浓度依赖性增强急性期高血压脑出血患者EPCs的数目和功能。这可能和其增加EPCs VEGF mRNA、蛋白质表达和分泌有关,同时也可能和抑制EPCs衰老、改善其易衰老的转归有关。
3.雌激素在体外增强EPCs数目和功能,延缓EPCs衰老的作用可能与PI3K途径有关。
Background and Purpose:Stroke is one of the three major diseases threatening human health and the leading cause of death and disabilities in many countries and districts.Primary cerebral hemorrhage refers to non-traumatic hemorrhage of brain parenchyma,the proportion of cerebral hemorrhage(CH)is about 30%of total stroke. The mortality of CH during acute stage is about 30-40%,which is the highest among all kinds of cerebrovascular diseases.So many studies focused on CH and many discoveries about the pathogenesis,treatment, especially neurogenesis after stroke emerged.Studies showed evidences of neurogenesis related to neural stem cell after stroke but about 80%of new neurons died within 2 to 6 weeks after onset of stroke.The increasing evidences indicated that neurogensis after stroke is limited by inadequate angiogenesis/vasculogensis,which provide nutrition and proper microenviroment that facilitate new neurons' survival and growth.
Since Asahara et al.reported that a subtype of hematopoietic progenitor cells from adults in 1997,namely "endothelial progenitor cells"(EPCs),showed endothelial cell feature,and play an important role in re-endothelialization during the neovascularization of ischemic organs.
The patients with stroke are always associated with ageing and hypertension.Hypertension and ageing may decrease the mobilization, migration and proliferative capacity of EPCs.EPCs of patients with hypertension and old age are easy to senescence.While increasing evidences show that the number,mobilization,migration and proliferative capacity of EPCs may be improved in acute period of ischemia diseases (such as ischemia diseases of heart,cerebral and limbs),heart surgery, and organ injury.This suggests that stressing of organ can augment the number and ability of EPCs.
Ischemia is believed to upregulate VEGF,which in turn are released to the circulation and induce mobilization of progenitor cells from the bone marrow.EPCs may act similar to monocytes/macrophages,which can increase arteriogenesis by providing cytokines and growth factors. Indeed,EPCs cultivated from different sources showed a marked expression of growth factors such as VEGF,HGF,and IGF-1.The release of growth factors in turn may influence the classical process of angiogenesis,namely the proliferation and migration as well as survival of mature endothelial cells.VEGF is a known chemokine critical for angiogenesis in adults.Intramuscular or intramyocardial VEGF gene transfer has been shown to increase EPC numbers in patients with limb ischemia or inoperable coronary disease.But it is unknown about EPCs functional activity change of old patients with CH associated with hypertension(CH-EH).And it's underlying mechanism needs further research.
EPCs remain extremely rare in adult peripheral blood(0.01%of mononuclear cells,under steady state conditions).And studies showed that the number of incorporated cells with an endothelial phenotype into injury or ischemic tissues is generally quite low.It is necessary to seek drugs or methods to augment the number and function of EPCs. Fortunately,studies showed that estrogen,erythropoietin,statins, angiotensin converting enzyme inhibitors can increase the number and functions of EPCs.Imanishi's study demonstrated that 17β-estradiol can improve the secretion of VEGF by EPCs,and delay the onset of senescence of EPCs in healthy human.PI3-K blockers could attenuate the effects of estrogen on EPCs differentiation and senescence and permitted the conclusion that PI3K/Akt signaling pathway is a possible regulator of EPCs proliferation.The PI3-kinase enzymes are widely expressed and play crucial roles in many biological responses including cell survival and proliferation.However,Imanishi's study also indicated that understan ding of the relationship of these factors to hypertension and its vascular sequelae must await further developments in clinical expertise. It is unknown that the effect of estrogen to EPCs of patients with CH-EH. So we aimed to investigate the effect of estrogen on EPCs of patients with CH-EH and explore the underlying molecular mechanism.
Methods:
1.Study Subjects:Each of patients in control group and CH-EH group had a hypertension history(more than 5 years),hadn't the history of diseases and drugs treatment that maybe affect EPCs.The CH diseases that not associated with hypertension must be excluded.The CH-EH patients were enrolled between 1-3 days after onset of stroke.The volumes of hemorrhage were 20-30ml.
2.Isolation and cultivation of EPCs:Peripheral blood(15 ml) was drawn by venipuncture,Mononuclear cells were isolated by density gradient method using Ficoll-Paque Plus.Cells were cultured with M199 medium,which contains 20%fetal-calf.The culture media were changed every 3 days beginning at day 4.
3.Characterization of EPCs:At day 7,EPCs were incubated with Dil- acLDL and FITC-UEA-I,Samples were examined with laser scanning con-focal microscope.Only cells exhibiting double positive were identified as EPCs.
4.Groups of experiment:Groups of the first experiment include hypertension group and CH-EH group.EPCs were cultured with the medium as described above.Groups of the second experiment include: 1).CH-EH EPCs group,2).CH-EH EPCs treated with 17β-estradiol10nmol/L group,3).CH-EH EPCs treated with 17β-estradiol100nmol/L group,4).CH-EH EPCs treated with 17β-estradiol 1μmol/L group,5).CH-EH EPCs treated with 17β-estradiol1μmol/L + LY294002 group.Every group of EPCs was cultured with the similar medium as above but also included different concentration of 17β-estradiol.EPCs treated with 17β-estradiol1μmol/L + LY294002 group were firstly cultured with LY294002 before culturing with 1μmol/L 17β-estradiol.
5.Numbers assay of EPCs colonies and EPCs:The numbers of EPCs and EPCs colonies were determined by counting 5 random high-power(×200)microscope fields per subject at day 7.After the numbers of EPC and EPC colony were counted,cells were assayed or harvested for further study.
6.Adhesion assay of EPCs:1×10~5cells/ml EPCs of every group were replanted onto fibronectin-coated culture dishes and incubated at 37℃for 30 minutes.Adherent cells were counted 5 random high-power (×200)microscope fields per subject.
7.Migration assay of EPCs:2×10~4cells/ml EPCs of every group resuspended in 50ul M199 medium include different concentration of 17β-estradiol were placed in the upper chamber of a modified Boyden chamber.25ul M199 and human recombinant VEGF(50ng/ml)were placed in the lower compartment of the chamber.After 24h incubation at 37℃,cells migrating into the lower chamber were counted.
8.Proliferation assay of EPCs:2×10~3 cell/ml EPCs of every group were replanted 96-well culture dishes,MTT Cell Proliferation and Cytotoxicity Assay Kit were applied to assay proliferation of EPCs.The OD value was measured at 570nm.
9.Measurement of VEGF protein in serum and the culture medium:The VEGF protein content of serum of patients and culture medium(at day 7)were assayed by using VEGF ELISA Kit.The OD value was measured at 450nm.
10.VEGF mRNA and protein expression assay of EPCs:Total RNA of EPCs in every group was extracted by using Trizol RNA Extraction Kit,and proteins of EPCs in these groups were also extracted. Semi-quantitative reverse transcription- polymerase chain reaction (RT-PCR)analysis was performed to assay VEGF mRNA.VEGF protein was assessed by Western blotting.
11.Senescence-associatedβ-galactosidase activity assay: After the first medium changing,the EPCs of different groups were cultured with different medium which included different concentration of 17β-estradiol.EPCs at day 14 were harvested and senescenceassociatedβ-galactosidase activity was measured by using Senescenceβ-Galactosidase Staining Kit.Senescence cells among per 100 cells were counted 5 random high-power(×200)microscope fields per subject.
Results:
1.Morphological features of EPCs:Culture of total peripheral blood mononuclear cell resulted in the emergence of characteristic spindle-shaped cells and colonies consisting of peripheral spindle-shaped cells emanating from round central cells within 72 hours of culture in CH-EH group,bands or vessels like structure formed by layers of EPCs can be found in some fields.In hypertension group,the colonies of EPCs appeared at day 5,relative later to that of CH-EH.After intervention of different concentration of estrogen,the number and the size of EPCs colonies increased significantly.
2.Characterization of EPCs:These cells could be shown to uptake Dil- acLDL and FITC-UEA-I,and exhibiting double-positive. According the results of morphological features and double-positive cells, the cells that we cultured were EPCs.
3.Number assay of EPCs colonies and EPCs,adhesion, migration and proliferation of EPCs:Compared with hypertension group,the number of EPCs and EPCs colonies,adhesive migrating and proliferative faculty of EPCs in CH-EH group significantly increased (P＜0.01 or P＜0.05).After treated with different concentration 17β-estradiol,Compared with CH-EH group,except CH-EH EPCs treated with 17β-estradiol10nmol/L group's colonies counts,the number of EPCs and EPCs colonies,adhesive,migrating and proliferative faculty of EPCs in different concentration groups significantly increased (P＜0.01),and dose-dependently improved(P＜0.01)with a peak at 1umol/l concentration(P＜0.01).When EPCs were firstly incubated with LY294002 before cultured with medium of 17β-estradiol 1umol/l group,this effect of 17β-estradiol on EPCs of CH-EH was significantly inhibited compared with 17β-estradiol 1umol/l group(P＜0.01).
4.Measurement of VEGF protein in serum and the culture medium,VEGF mRNA and VEGF protein expression assay of EPCs: Compared with hypertension group,the content of VEGF protein in serum and the culture medium,VEGF mRNA and protein expression of EPCs,were all significantly increased in CH group(P＜0.01).After treated with different concentration 17β-estradiol,Compared with CH-EH group,the content of VEGF protein in culture medium,VEGF mRNA and protein expression of EPCs in different concentration groups significantly increased(P＜0.01),and dose-dependently improved(P＜0.01) with a peak at 1umol/l concentration(P＜0.01).
5.Senescence-associatedβ-galactosidase activity assay: Compared with hypertension group,the number of senescence EPCs in CH-EH group had no significant difference(P＞0.05).After treated with different concentration 17β-estradiol,compared with CH-EH group,the number of senescence EPCs in different concentration groups significantly decreased(P＜0.01),and dose-dependently improved (P＜0.01)with a peak at 1umol/l concentration(P＜0.01).When EPCs were firstly incubated with LY294002 before cultured with medium of 17β-estradiol 1umol/l group,this effect of 17β-estradiol on senescence EPCs of CH-EH was significantly inhibited compared with 17β-estradiol 1umol/l group(P＜0.01).
Conclusions:
1.The number of EPCs and EPCs colonies,adhesive,migrating and proliferative faculty of EPCs in acute period of CH-EH significantly increased.The content of VEGF protein in serum and the culture medium,VEGF mRNA and protein expression of EPCs,were all significantly increased.This may be the mechanism of acute cerebral hemorrhage augment EPCs numbers and function.The destiny of EPCs easy to senescence was not changed compared with EPCs of patients with hypertension.
2.Estrogen can increase the numbers and function of EPCs of patients with CH-EH in vitro,and this effect is dose-dependence. Estrogen can improve VEGF mRNA and protein expression and VEGF secretion of EPCs,and reduce EPCs senescence.The effect is dose-dependent.These may be mechanisms of estrogen augment numbers and function of EPCs of patients with CH-EH.
3.The role of Estrogen augments number and function of EPCs, reduces EPCs senescence of patients with CH-EH in vitro is related to PI3K/Akt signaling pathway.
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