【作者】 张晓芳；

【导师】 季晓平；

【作者基本信息】 山东大学 ， 内科学， 2005， 硕士

【摘要】 目的 建立大鼠骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)体外分离、培养、传代及诱导分化的实验方法;观察MSCs能否经体外5-氮胞苷(5-azacytidine,5-aza)诱导分化为心肌样细胞:探讨MSCs同种异体移植治疗急性心肌梗死(Acute Myocardial Infarction,AMI)的可行性。 方法 2月龄健康雌性Wistar大鼠55只,5只用于提供MSCs;余50只用于建立AMI模型,随机分为两组,MSCs移植组和对照组各25只。 取大鼠双侧股骨、胫骨、肱骨,用DMEM培养液反复冲洗骨髓腔,将冲洗液移入培养瓶培养。以贴壁筛选法分离、纯化MSCs。当MSCs达80%融合时消化传代。MSCs传至第3代覆盖瓶底达50%时开始加入终浓度为10μmol/L的5-aza诱导,作用24h。以后每代细胞覆盖瓶底达50%时均给予5-aza诱导1次。用荧光相差显微镜观察各代细胞的形态改变和生长分化情况。以传至第15代达80%融合、5-aza诱导13次、培养达4个月之久的MSCs作为移植细胞备用。少量移植细胞用于透射电镜观察和免疫组化鉴定心肌特异性蛋白,余者均在5-溴脱氧尿嘧啶(5-Bromodeoxyuridine,BrdU)标记后用于移植。 应用液氮冷冻法制作大鼠AMI模型,选择梗死灶及其周围共四个位点为注射点,移植组每点注入MSCs悬液70μl(4～5×10~6个细胞),对照组注入等量DMEM液。分别于术前1天、术后1周、术后4周行组织多普勒超声心动图(Tissue Doppler Echocardiography,TDE)检查,以收缩期峰值速度(Systolic Peak Velocity,Vs)、左室舒张末容积(Left Ventricle End Diastolic Volume,LVEDV)和左室射血分数(Left Ventricle Ejection Fraction,LVEF)为指标评价心功能。移植组于更多还原

【Abstract】 Objective To establish the experimental methodology of isolating, culturing, proliferating and inducing rat bone marrow mesenchymal stem cells (MSCs) in vitro;observe whether or not MSCs induced by 5-azacytidine (5-aza) in vitro can differentiate into cardiomyocyte-like cells;and investigate the feasibility of acute myocardial infarction (AMI) treatment by allogeneic transplantation of MSCs.Methods Fifty-five healthy female Wistar rats aged two months were selected. Five were donors for MSCs. The others would be used as AMI animal models randomly separated into two groups including the transplanted group (n-25) and the control group (n=25).Bone marrow taken out from bilateral femora, tibiae and humeri was cultured with DMEM culture solution in culture bottles. MSCs were isolated and purified by the wall-adhering method. MSCs were digested and transferred to the next generation once they achieved 80% fusion. The third generation of MSCs began to be induced by 5-aza (10 μmol/L), and the following generations were all induced in the same way. The inducement started every time the new generation occupied 50% of the bottle bottom, and every inducement persisted for 24 hours. The morphology and growth of every generation were observed under fluorescence phase-contrast microscope. The fifteenth generation with 80% fusion was prepared for transplantation. These transplant cells had been induced 13 times by 5-aza and cultured for four months. Few transplant cells were observed under electron microscope and identified by immunohistochemistry. The other cells were labeled by 5-bromodeoxyuridine (BrdU) and transplanted into the AMI models.Myocardial infarctions were made by liquid nitrogen freezing. The prepared cellslabeled by BrdU were injected into the infarcted parts of the transplanted group. The same volume of DMEM solution was injected into the infarcted parts of the control group. Tissue Doppler echocardiography (TDE) was applied one day preoperation, one week postoperation and four weeks postoperation. Cardiac function was evaluated by systolic peak velocity (Vs), left ventricle end diastolic volume (LVEDV) and left ventricle ejection fraction (LVEF). The transplanted rats were sacrificed four weeks postoperation and the transplanted parts were harvested for BrdU immunofluorescent stain.Results The transplant cells (i.e. the fifteenth generation of MSCs) were same as cardiomyocytes in morphology, and expressed the cardiac-specific proteins, a-actin and troponin T, showing that MSCs induced by 5-azacytidine (5-aza) in vitro could differentiate into cardiomyocyte-like cells. Ten rats of the transplanted group died with 40% mortality. Twelve rats of the control group died with 48% mortality. The difference of the mortality between both groups had no statistical significance (P>0.05). In the control group (?=13), Vs, LVEDV, LVEF was respectively 13.97 + 2.06 cm/s, 0.31 ±0.06 ml, (77.50+4.81)% one day preoperation, 6.79+1.41 cm/s, 1.13 + 0.14 ml, (40.33 + 10.51)% one week postoperation, and 8.81 + 1.83 cm/s, 1.10 + 0.15 ml, (45.77 + 7.56)% four weeks postoperation. In the control group, the difference of the cardiac function among preoperation, one week postoperation and four weeks postoperation had statistical significance (.PO.05). The cardiac function four weeks postoperation was better than one week postoperation, but worse than preoperation. In the transplanted group (?=15), Vs, LVEDV, LVEF was respectively 14.01 + 1.03 cm/s, 0.30±0.05 ml, (76.83+4.52)% one day preoperation, 7.02 ±2.01 cm/s, 1.16 ±0.13 ml, (42.56 ±9.58)% one week postoperation, and 11.15 ± 1.78 cm/s, 0.67 + 0.11 ml, (65.38±9.57)% four weeks postoperation. In the transplanted group, the difference of the cardiac function among preoperation, one week postoperation and four weeks postoperation had statistical significance (PO.05). The cardiac function four weeks postoperation was better than one week postoperation, but worse than preoperation. The differences of the cardiac function preoperation and one week postoperation between both groups had no statistical significance (P>0.05), while the difference of the cardiac function four weeks postoperation between both groups hadstatistical significance (P<0.05). The transplanted group had better cardiac function than the control four weeks postoperation. The BrdU-labeled cells were found in the histological sections of the transplanted parts under fluorescent microscope, which suggested the transplant cells survived in the host cardiac muscles.Conclusion Rat MSCs cultured generation by generation and repeatedly induced by 5-aza in vitro can differentiate into cardiomyocyte-like cells, which if transplanted into the rat infarcted cardiac muscles will survive and help improve the host’s cardiac function. Our study provides experimental support for clinical treatment of AMI by MSCs transplantation.更多还原