【作者】 黄景玲；

【导师】 陈一戎；

【作者基本信息】 兰州大学 ， 生物化学及分子生物学， 2010， 博士

【摘要】 研究目的(1)探讨脐血间充质干细胞可否成功的在体外分离、培养、扩增并诱导成心肌样细胞；探讨脐血来源的间充质干细胞的分离鉴定,经体外扩增、纯化的方法以及定向分化成心肌样细胞的诱导剂5-氮胞苷(5-AZA)作用后的生物学特性；(2)观察脐血间充质干细胞与心肌细胞在体外共同培养,与脐血MSCs共同培养的心肌细胞是否具有抵抗缺氧引起的凋亡;(3)观察体外进行长期的培养和扩增脐血MSCs,以及对细胞的遗传物质有修饰作用的试剂5-AZA进行体外诱导后的脐血MSCs的生物安全性；(4)进行重组人粒细胞集落刺激因子(G-CSF)对进行骨髓动员,然后分离纯化其外周血单个核细胞,将单核细胞经心导管在梗死相关动脉内注射治疗的急性心肌梗死患者进行临床指标的观察。方法与结果第一部分：提取健康妊娠产妇分娩的胎儿脐带血,分离单个核细胞进行培养、传代,取第3代以后细胞,用流式细胞仪直接标记分析CD34、CD44、CD90细胞表面标志。用10μmmol/L的5-AZA诱导4周后,倒置显微镜下观察细胞呈类心肌细胞样改变、透视电镜观察：细胞核呈椭圆形,胞浆内可见成束的肌丝,肌丝内可见梭形密体、丰富的糖原颗粒、大量的线粒体、内质网发达。免疫荧光组化染色体可见：GATA4, cTnI表达阳性的细胞。并检测细胞β-肌球蛋白重链(β-MyHC)基因的表达。5-AZZ作用后细胞进行生长周期分析以及生长曲线描记。结果显示体外纯化扩增后的脐血间充质干细胞含有与骨髓来源的间充质干细胞相同的细胞表面标志,第三代以后细胞纯度较高,经5-AZA在体外诱导培养,可形成心肌样细胞,在诱导剂量作用后,基本的生物学特性未发生改变。第二部分：将人的心肌细胞置于95%N2-5%CO2缺氧环境下培养造成模拟心肌缺血损伤模型。实验结果显示细胞凋亡与缺氧呈时间依赖性,将人的心肌细胞和人脐血MSCs在缺氧环境下共同培养,脐血MSCs具有抵抗缺氧引起的心肌细胞凋亡的能力。第三部分：通过对体外长期培养、扩增的脐血MSCs细胞和经5-AZA诱导作用28天后的MSCs细胞的生长曲线和生长周期分析、细胞核型分析、端粒酶活性检测、细胞周期相关基因表达的检测以及细胞成瘤实验来评价试验用脐血MSCs是否发生恶性转化。结果证实：经离体培养、扩增脐血MSCs和经5-AZA诱导分化成心肌样细胞后不存在基因突变,不具有成瘤性。第四部分：选择6例急性心肌梗死的病人,在移植前1周,应用干细胞动员剂,重组人粒细胞集落刺激因子(G-CSF)对进行骨髓动员,然后用细胞分离仪对外周血单个核细胞分离纯化。分离纯化的单核细胞经心导管在梗死相关动脉内注射治疗。干细胞移植术后进行随访1、3、6月的心电图、心脏超声测定左室射血分数(LVEF)、心肌核素显像。同时与对照组进行比较。随访结果显示：经干细胞移植治疗后的患者心功能明显改善、心电图上心肌梗死部位有小的R波、心脏内存活细胞较多。结论：(1)脐血成为继骨髓之外的另一个MSCs来源。脐血MSCs为心肌细胞移植提供了良好的细胞供体,是心肌干细胞另一来源；(2)心肌细胞凋亡与缺氧呈时间相关性。人脐血MSCs和人的心肌细胞共同培养,能有效抵抗缺氧引起的心肌细胞凋亡；(3)通过我们的实验建立了从细胞水平评价心肌细胞凋亡的方法；(4)脐血MSCs在体外进行长期培养具有正常MSCs细胞的生长速度；(5)在体外进行长期培养扩增的脐血MSCs和经过5-AZA向心肌细胞诱导后的脐血MSCs没有发生恶性转化；(6)干细胞移植治疗后的患者心功能明显改善、心电图上心肌梗死部位有小的R波、心脏内存活细胞较多。更多还原

【Abstract】 Objectives1. Umbilical cord blood (UCB) as a source of hematopoietic stem cells has been successfully used in clinic for over 10 years. Could MSCs from UCB be isolated, cultured, amplified, and then induced into cardiomyocyte-like cells in vitro successfully? Using amplification and purification methods in vitro, we isolated and identified MSCs from UCB, and we investigated their biological properties after exposure to 5-azacytidine (5-AZA), a revulsant of promoting cardiomyocyte-like cell formation.2. We investigated whether cardiomyocytes resist to apoptosis caused by hypoxia after co-cultured with MSCs from UCB in vitro.3. MSCs from UCB were long-term cultured and amplified in vitro, their biological safety was observed after 5-AZA treatment.4. Granulocyte colony-stimulating factor (G-CSF) was used to mobilize bone marrow, after then, the mononuclear cells (MNCs) was isolated from peripheral blood to inject into the infarct site through cardiac catheterization. After treatment, the related indexes in those patients with acute MI were observed. Methods and ResultsMethods and resultsThe first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and The first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and passaged. Experiments were performed with cells from passage 3. Flow cytometry (FCM) was used to determine surface markers of cells (CD34, CD44, and CD90). After 10μmmol/L of 5-AZA treatment for 4 weeks, cardiomyocyte-like change was observed under inverted microscope. Transmission electron microscope (TEM) showed oval-shaped nucleus and bundles of filaments in the cytoplasm. Spindle dense bodies, abundant glycogen granules, large number of mitochondria and endoplasmic reticulum were found inside filaments. Cells with positive expression of GATA4 and cTnI were detected by immunofluorescence staining. Moreover, gene expression ofβ-myosin heavy chain (P-MyHC) was detected. After 5-AZZ treatment, cell growth cycle analysis and growth curve trace were performed.Results:The surface markers of MSCs from UCB after purification and amplification in vitro were in consistent with those of bone marrow counterpart. Cells had high purity from the 3rd generation. UCB-derived MSCs could differentiate into cardiomyocytes after 5-AZZ treatment in vitro, retaining their original characteristic with the inductive dose.The second part of the experiment:Human cardiac myocyte (HCM) ischemic injury model was established by exposure HCMs to hypoxia (95% N2-5% CO2). The results showed that hypoxia induced apoptosis of HCM in a time-dependent manner. Co-cultured with HCM in hypoxic conditions, UCB-derived MSCs could inhibit hypoxia-induced apoptosis.The third part of the experiment:Whether malignant transformation of UCB-derived MSCs occurred or not was determined by long-term cultivation and amplification in vitro, growth curve and growth cycle analysis after 5-AZA induction for 28 d, telomerase activity, G-banding patterns of chromosomal karyotpes, tumor formation, cell cycle-related gene and tumorigenicity. No abnormal chromosomal karyotpes were observed in UCB-derived MSCs after 5-AZA induction compared with that of un-induced MSCs. The fourth part of the experiment:Six patients with acute MI were selected in this study. One week before transplantation, G-CSF, a kind of stem cell mobilization reagent, was applied to mobilize bone marrow stem cells. MNCs were isolated and purified by cell isolation system, then were injected into the infarct site through cardiac catheterization. After stem cell transplantation, these patients were followed up for 1,3 or 6 months by ECG, left ventricular ejection fraction (LVEF) via cardiac ultrasound, and myocardial perfusion imaging (MPI). Compared with the control group, cardiac function in patients after stem cell transplantation improved significantly, and small R-waves appeared in infarct site determined by ECG, with more survival cells.Conclusions1. Umbilical cord blood is another source of MSCs other than bone marrow. UCB-derived MSCs provide a good cell donor for cardiomyocyte transplantation, which is another source of cardiac stem cells.2. Apoptosis of cardiomyocytes and hypoxia was time-related. Co-culture of human UCB-derived MSCs and HCM can resist against hypoxia-induced myocardial apoptosis effectively.3. The method of evaluating cardiomyocyte apoptosis at cellular level is established.4. UCB-derived MSCs in vitro have the similar growth rate with normal MSCs.5. No malignant transformation occurs in UCB-derived MSCs before and after 5-AZA treatment.6. The stem cell transplantation improves patients’ cardiac function significantly, and small R-waves are in infarct site on ECG, with more survival cells.更多还原