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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在怎样的影响?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200～300g6～8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呈节律性跳动,呈心肌样细胞改变,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。
BACKGROUND: Differentiation of bone marrow mesenchymal stem cells is closely related to the microenvironment. After myocardial infarction, myocardial cells undergo necrosis, activation of complement, generation of free radicals, secretion of various cytokines, and changes of serum components of patients. Whether these changes affect bone marrow Mesenchymal stem cells differentiate into cardiomyocytes? Aim: To observe the effect of serum of rat with acute myocardial infarction on differentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes in vitro. DESIGN, TIME AND SETTING: The comparative in vitro cytology experiment was performed at the Laboratory of Stem Cell and Tissue Engineering Center, Sun Yat-sen University from September 2005 to June 2006. MATERIALS: Three-week-old SD rats weighing 50 ~ 80g were used for isolation and culture of bone marrow mesenchymal stem cells. SD rats with body weight of 200 ~ 300g and 6 ~ 8 weeks old were used to make acute myocardial infarction rat model. Methods: Rat bone marrow mesenchymal stem cells were isolated and cultured by adherent method. Ligation of the left anterior descending coronary artery rat model of acute myocardial infarction, and normal rats and rat serum samples were taken for later. The third generation of BMSCs was divided into 6 groups: untreated group, 5-azacytidine-induced group, 5-azacytidine + myocardial infarction model rat serum induction group, 5-azacytidine + normal rat Serum induction group, myocardial infarction model rats induced by serum, normal rat serum induction group. MAIN OUTCOME MEASURES: Morphological changes of rat bone marrow-derived mesenchymal stem cells after induction. Expression of cardiac troponin after induced by rat bone marrow mesenchymal stem cells. Rat bone marrow mesenchymal stem cells after induction of GATA-4 and desmin mRNA expression levels. Results: After induced by 5-azacytidine combined with serum, some MSCs gradually grew and became thinner, and the central part of the cell body formed a sphere. After two or three weeks, the cells were spherical, rod-shaped and formed adjacent cells Connected, microscopically visible part of the differentiated cells rhythmic beating, showing cardiac myocyte changes in cardiac troponin, GATA-4, knot protein expression was positive. The bone marrow mesenchymal stem cells of the control group and the pure serum-induced group showed no morphological changes, and the cardiac troponins were negative, while the GATA-4 and the desmin were weakly positive. Conclusion: Serum from myocardial infarction rats can not induce the differentiation of BMSCs into cardiomyocytes, but serum from myocardial infarction can promote the differentiation of BMSCs into cardiomyocytes and promote the differentiation of cardiomyocytes mature.