人骨髓间充质干细胞体外定向诱导分化为心肌样细胞
发布时间：2018-07-27 19:38
【摘要】： 目的:探讨体外诱导人骨髓间充质干细胞(human bone marrow-derived mesenchymal stem cells, hBMSCs)定向诱导分化为心肌样细胞的实验条件。 方法:取成人弃骨骨髓,通过全骨髓贴壁法分离培养hBMSCs。在传至第三代时,筛选具有心肌特异性分化潜能的c-kit~+的hBMSCs细胞克隆,用含20%胎牛血清的低糖DMEM培养液继续克隆培养。当细胞达到70%融合时,分别用10μmol/l5-氮胞苷(5-azacytidine crystalline,5-aza),100mg/l参麦注射液,10μmol/l5-氮胞苷和100mg/l参麦注射液3组诱导剂诱导hBMSCs。24小时后,更换为含10%胎牛血清的低糖DMEM培养液继续培养。在诱导3周后,通过显微镜下观察其形态变化。用免疫细胞化学方法检测心肌特异性标记抗原Desmin和肌钙蛋白T的表达;用RT-PCR方法检测心肌特异基因心房利尿肽(atrial natriuretic peptide,ANP)和脑钠肽(brain natriuretic peptide,BNP)表达。 结果:骨髓中含有多种hBMSCs细胞克隆,其中成纤维细胞样克隆表达c-kit~+。分别用3组诱导剂诱导c-kit~+细胞15天后,部分细胞胞体明显增粗,可见有些细胞间有融合样现象发生。诱导3周后,3组都有类似肌管样细胞出现,细胞短粗,呈不规则的圆柱形,有分支,互相连成网。苏木精-伊红(hematoxylin-eosin HE)染色后,3组细胞都排列规则,类似于心肌细胞的形态,可见部分细胞有排列规则的横纹样结构。免疫细胞化学显示诱导3周后的hBMSCs,Desmin阳性细胞数目分别是:5-aza组(65.3±4.7%),参麦注射液组(62.3±3.9%),5-aza和参麦注射液组(68.5±2.1%),3组Desmin阳性细胞表达率无显著差异; cTnT阳性细胞数目分别是:5-aza组(62.2±4.1%),参麦注射液组(63.8±3.4%), 5-aza和参麦注射液组(66.0±6.2%),3组Desmin阳性细胞表达率无显著差异。阴性对照组和未分化的hBMSCs均无Desmin和cTnT的表达。RT-PCR显示3种不同的诱导剂诱导hBMSCs3周后都有ANP,BNP的表达,未分化的hBMSCs无ANP,BNP的表达。 结论:5-aza和中药可以诱导c-kit~+的hBMSCs细胞克隆分化为心肌样细胞。体外诱导成的心肌样细胞表现为未成熟心肌细胞的结构和功能特征。证明骨髓中含有多种干细胞克隆,c-kit可作为筛选hBMSCs向心肌样细胞分化的一项检测指标。筛选具有心肌特异性分化潜能的hMSCs可提高诱导分化率。
[Abstract]:Objective: To investigate the experimental conditions of induced differentiation of human bone marrow mesenchymal stem cells (human bone marrow-derived mesenchymal stem cells, hBMSCs) into cardiomyocytes in vitro.
Methods: the bone marrow of adult discarded bone was taken from the whole bone marrow adherent method to isolate and culture hBMSCs. in the third generation, and the c-kit~+ hBMSCs cell clones with the specific differentiation potential of the myocardium were screened. The low sugar DMEM culture medium containing 20% fetal bovine serum was continued to be cloned and cultured. When the cells reached 70% fusion, 10 micron mol/l5- azacytidine (5-azacytidine) was used respectively. Crystalline, 5-aza), 100mg/l Shenmai injection, 10 mol/l5- azacytidine and 100mg/l Shenmai injection 3 inducers were induced for hBMSCs.24 hours, and changed into low sugar DMEM culture solution containing 10% fetal bovine serum. After 3 weeks of induction, the morphological changes were observed under the microscope. The specific markers of cardiac muscle were detected by immunocytochemical method. The expression of antigen Desmin and troponin T; the expression of cardiac specific gene atrial natriuretic peptide (ANP) and brain natriuretic peptide (brain natriuretic peptide, BNP) by RT-PCR method.
Results: there were a variety of hBMSCs cell clones in the bone marrow, in which the fibroblast like clone and expression c-kit~+. induced c-kit~+ cells with 3 groups of inducers for 15 days, and some of the cell bodies were obviously thickened, and some of the cells had the phenomenon of fusion. After 3 weeks of induction, the 3 groups had the appearance of myotube like cells, and the cells were rough and irregular. A cylindrical, branched, interconnected network. After hematoxylin eosin (hematoxylin-eosin HE) staining, the 3 groups of cells were arranged regularly, similar to the morphology of the cardiac myocytes, and some cells were arranged in a regular pattern. The immunocytochemistry showed that the number of hBMSCs in the 3 weeks after induction was: the number of Desmin positive cells was (65.3 + 4.7%) in the 5-aza group, respectively. In the group of Mai Injection (62.3 + 3.9%), 5-aza and Shenmai injection group (68.5 + 2.1%), there was no significant difference in the expression rate of Desmin positive cells in the 3 groups, and the number of cTnT positive cells were in group 5-aza (62.2 + 4.1%), Shenmai injection group (63.8 + 3.4%), 5-aza and Shenmai injection group (66 + 6.2%), and there was no significant difference in the expression rate of Desmin positive cells in the 3 group. No Desmin and cTnT expression.RT-PCR in both group and undifferentiated hBMSCs showed that 3 different inducers were induced to have ANP, BNP expression, and undifferentiated hBMSCs without ANP and BNP expression after hBMSCs3 weeks.
Conclusion: 5-aza and traditional Chinese medicine can induce the hBMSCs cell clone of c-kit~+ to differentiate into myocardial like cells. The induced cardiomyocytes in vitro are characterized by the structure and function of immature cardiomyocytes. It is proved that there are a variety of stem cell clones in the bone marrow, and c-kit can be used as a detection index for screening the differentiation of hBMSCs centripetal muscle like cells. HMSCs with cardiac specific differentiation potential can increase the differentiation rate.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
本文编号：2148925
[Abstract]:Objective: To investigate the experimental conditions of induced differentiation of human bone marrow mesenchymal stem cells (human bone marrow-derived mesenchymal stem cells, hBMSCs) into cardiomyocytes in vitro.
Methods: the bone marrow of adult discarded bone was taken from the whole bone marrow adherent method to isolate and culture hBMSCs. in the third generation, and the c-kit~+ hBMSCs cell clones with the specific differentiation potential of the myocardium were screened. The low sugar DMEM culture medium containing 20% fetal bovine serum was continued to be cloned and cultured. When the cells reached 70% fusion, 10 micron mol/l5- azacytidine (5-azacytidine) was used respectively. Crystalline, 5-aza), 100mg/l Shenmai injection, 10 mol/l5- azacytidine and 100mg/l Shenmai injection 3 inducers were induced for hBMSCs.24 hours, and changed into low sugar DMEM culture solution containing 10% fetal bovine serum. After 3 weeks of induction, the morphological changes were observed under the microscope. The specific markers of cardiac muscle were detected by immunocytochemical method. The expression of antigen Desmin and troponin T; the expression of cardiac specific gene atrial natriuretic peptide (ANP) and brain natriuretic peptide (brain natriuretic peptide, BNP) by RT-PCR method.
Results: there were a variety of hBMSCs cell clones in the bone marrow, in which the fibroblast like clone and expression c-kit~+. induced c-kit~+ cells with 3 groups of inducers for 15 days, and some of the cell bodies were obviously thickened, and some of the cells had the phenomenon of fusion. After 3 weeks of induction, the 3 groups had the appearance of myotube like cells, and the cells were rough and irregular. A cylindrical, branched, interconnected network. After hematoxylin eosin (hematoxylin-eosin HE) staining, the 3 groups of cells were arranged regularly, similar to the morphology of the cardiac myocytes, and some cells were arranged in a regular pattern. The immunocytochemistry showed that the number of hBMSCs in the 3 weeks after induction was: the number of Desmin positive cells was (65.3 + 4.7%) in the 5-aza group, respectively. In the group of Mai Injection (62.3 + 3.9%), 5-aza and Shenmai injection group (68.5 + 2.1%), there was no significant difference in the expression rate of Desmin positive cells in the 3 groups, and the number of cTnT positive cells were in group 5-aza (62.2 + 4.1%), Shenmai injection group (63.8 + 3.4%), 5-aza and Shenmai injection group (66 + 6.2%), and there was no significant difference in the expression rate of Desmin positive cells in the 3 group. No Desmin and cTnT expression.RT-PCR in both group and undifferentiated hBMSCs showed that 3 different inducers were induced to have ANP, BNP expression, and undifferentiated hBMSCs without ANP and BNP expression after hBMSCs3 weeks.
Conclusion: 5-aza and traditional Chinese medicine can induce the hBMSCs cell clone of c-kit~+ to differentiate into myocardial like cells. The induced cardiomyocytes in vitro are characterized by the structure and function of immature cardiomyocytes. It is proved that there are a variety of stem cell clones in the bone marrow, and c-kit can be used as a detection index for screening the differentiation of hBMSCs centripetal muscle like cells. HMSCs with cardiac specific differentiation potential can increase the differentiation rate.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329
【参考文献】
相关期刊论文 前3条
1 谭玉珍,王海杰;心肌干细胞的提交分化及其调控机制[J];国外医学.生物医学工程分册;2004年01期
2 孙丽莉,谭玉珍,王海杰,贺其志;骨髓间充质干细胞的克隆培养及其向心肌细胞的诱导分化[J];复旦学报(医学版);2003年06期
3 汪蕾,林国生,郭军,蒋学俊,杨波;直接与间接接触对诱导骨髓间充质干细胞成心肌样分化的影响[J];现代医学;2004年05期
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