心肌梗死大鼠血清对大鼠骨髓间充质干细胞分化为心肌细胞的影响

背景:骨髓间充质干细胞的分化与微环境密切相关,心肌梗死后,心肌细胞发生坏死,补体活化,自由基产生,分泌各种细胞因子,患者的血清成分也发生改变,这些改变究竟对骨髓间质干细胞向心肌细胞分化存存怎样的影响?目的:观察急性心肌梗死大鼠血清在体外对大鼠骨髓间充质干细胞分化为心肌细胞的作用.设计、时间及地点:对比观察体外细胞学实验,于2005-09/2006-06在中山大学干细胞与组织工程中心实验室完成.材料:体质量50～80g三四周龄SD大鼠用于骨髓间质干细胞分离培养;体质量200～300 g 6～8周龄SD大鼠用于制作急性心肌梗死大鼠模型.方法:采用贴壁法分离培养大鼠骨髓间充质干细胞.结扎左冠状动脉前降支制作急性心肌梗死大鼠模型,并抽取正常大鼠与模型大鼠血清备用.取第3代骨髓间充质干细胞分6组培养:未诱导组、5-氮胞苷诱导组、5-氮胞苷+心肌梗死模型大鼠血清诱导组、5-氮胞苷+正常大鼠血清诱导组、心肌梗死模型大鼠血清诱导组、正常大鼠血清诱导组.主要观察指标:大鼠骨髓间充质干细胞经诱导后的形态学变化.大鼠骨髓间充质干细胞经诱导后心肌肌钙蛋白的表达情况.大鼠骨髓间充质干细胞经诱导后GATA-4和结蛋白mRNA表达水平.结果:大鼠骨髓间充质干细胞经5-氮胞苷联合血清诱导后.部分细胞渐伸长、变细,胞体中央形成球形,二三周后可见细胞呈球形、棒状,邻近细胞之间形成连接,镜下可见部分分化的细胞旱节律性跳动,晕心肌样细胞改变,其心肌肌钙蛋白、GATA-4、结蛋白表达阳性.对照组和单纯血清诱导组骨髓间充质干细胞未见形态改变.其心肌肌钙蛋白为阴性,GATA-4、结蛋白呈弱阳性.结论:单纯使用心肌梗死大鼠血清不能诱导骨髓间充质干细胞向心肌细胞分化,但心肌梗死血清能促进5-氮胞苷诱导的骨髓间充质干细胞向心肌细胞分化,并促进分化的心肌细胞成熟.

Effects of serum from myocardial infarction rats on rat bone marrow mesenchymal stem cell differentiation into cardiomyocytes

BACKGROUND: Differentiation of bone marrow mesenchymal stem calls(BMSCs) is associated with their microenvironment. After acute myocardial infarction (AMI), the necrosis of cardiomyocytes caused activation of complement, generation of free radical, and secretion of various cell factors. As a result, the ingredients of patients' serum also changed, so does the changes will influence the differentiation of BMSCs into cardiomyocytes? And what influence it will be? OBJECTIVE: To investigative effects of AMI rat serum on the differentiation of BMSCs into cardiomyocytes. DESIGN, TIME AND SETTING: The in vitro controlled cytology experiment was performed at the Laboratory of Center for Stem cells and Tissue Engineering, Sun Yat-sen University from September in 2005 to June in 2006. MATERIALS: SPF Sprague Dawley rats, weighing 50-80 g, aged 3-4 weeks, were used for culture of BMSCs, and SpragueDawley rats, weighing 200-300 g, aged 6-8 weeks, were used for making AMI models. METHODS: BMSCs were isolated and cultured with the adherent method. After making AMI rat models by deligating anterior descending of the left coronary artery, the serum were collected and centrifuged from AMI and normal rats. Then the passage 3 of BMSCs were divided into six groups, non-induction group, 5-azacytidine (5-aza) group, 5-aza plus serum from AMI rat group, 5-aza plus serum from normal rat group, serum from AMI rat group, and serum from normal rat group. MAIN OUTCOME MEASURES: Morphology changes in rat BMSCs after induction; Expression of cardiac troponin T of BMSCs after induction; Expression of GATA-4 and desmin mRNA of BMSCs after induction. RESULTS: After being induced by 5-aza and two kinds of serum, some cells became elongated and thinner. Two to three weeks after induction, some cells had a ball-like or rod-like appearance, and connection were formed between adjacent cells. It showed that some BMSC have differentiated into cardiac like cells. The expression of cardiac troponin T, GATA-4 and desmin were positive in cells differentiated from BMSCs. The troponin T expression in control group and simple serum induction group were negative, but GATA-4 and desmin expressed weakly. CONCLUSION: Serum from AMI rat cannot induce BMSCs to differentiate into cardiomyocytes alone, but it promotes BMSCs induced by 5-aza differentiating into cardiomyocytes and facilitates the differentiated calls into mature.