化学诱导剂5-氮杂胞苷及模拟生物微环境对骨髓间充质干细胞分化成心肌样细胞的影响

Effect of 5-azacytidine and simulated biological microenvironment on differentiation from bone marrow mesenchymal stem cells into myocardial-like cells

背景:研究显示干细胞有随环境发生分化即"环境依赖性分化"的特性,但骨髓间充质干细胞在心肌组织是否存在此种特性目前尚未形成公识。目的:观察化学诱导剂和模拟的生物微环境诱导对骨髓间充质干细胞向心肌样细胞分化的影响。设计、时间及地点:随机分组设计,细胞学体外对比观察,于2008-01/2009-02在苏州大学心血管内科干细胞移植实验室完成。材料:6～8周龄SD大鼠,用于骨髓间充质干细胞的培养;新生1～3d同种系SD大鼠,用于心肌细胞的培养。方法:取SD大鼠股骨骨髓,培养至第2代,经流式细胞仪检测骨髓间充质干细胞纯度97%以上,制成4×108L-1的细胞悬液,实验分为4组:单纯DMEM培养组:用含体积分数为10%胎牛血清的DMEM培养基继续培养;5-氮杂胞苷组:24h后培养液内加入10μmol/L的5-氮杂胞苷;心肌细胞裂解液组:将4倍于骨髓间充质干细胞数量的心肌细胞裂解液加入骨髓间充质干细胞培养瓶中;间接接触组:在培养体系中放入millicell插入式细胞培养皿,28d收获细胞。主要观察指标:免疫组织化学法检测诱导后骨髓间充质干细胞表达心肌特异性肌钙蛋白T、连接蛋白43、CD31的情况;RT-PCR法检测各组早期心肌细胞特异性转录因子GATA4、NKX2.5、β-肌球蛋白重链及α-肌球蛋白重链基因的表达情况。结果:免疫组织化学检测显示实验组诱导4周后特异性抗原呈阳性表达,实验组与单纯DMEM培养组相比,差异有显著性意义(P<0.05);间接接触组较心肌细胞裂解液组阳性细胞数目稍增多,但组间差异无显著性意义(P>0.05);CD31是血管内皮细胞的表面抗原标志,单纯DMEM培养组和5-氮杂胞苷组无表达,其他两组弱阳性表达,组间差异无显著性意义(P>0.05)。实验组诱导4周后,均表达心肌前体细胞特异性转录因子NKX-2.5和GATA4,同时表达胚胎期占优势的心肌细胞特异性β-肌球蛋白重链,而没有表达成年期占优势的心肌细胞特异性标志α-肌球蛋白重链。结论:5-氮杂胞苷可以诱导骨髓间充质干细胞向心肌样细胞分化,心肌细胞裂解液和细胞间接接触模拟的心肌微环境也可以诱导骨髓间充质干细胞分化成心肌样细胞,分化的细胞介于成熟的心肌细胞和心肌祖细胞之间的心肌细胞前体。

BACKGROUND：Research has been indicated that stem cells characterize by environment-depended differentiation;however,whether bone marrow mesenchymal stem cells have the same performance during differentiation in myocardial tissues remains poorly understood. OBJECTIVE：To investigate the effect of 5-azacytidine and simulated biological microenvironment on differentiation from bone marrow mesenchymal stem cells into myocardial-like cells. DESIGN,TIME AND SETTING：A randomized design and cytological contrast observation was performed at the Laboratory of Stem Cell Transplantation,Department of Cardiology,Soochow University from January 2008 to February 2009. MATERIALS：SD rats aged 6-8 months were collected for culture of bone marrow mesenchymal stem cells;additionally,SD rats aged 1-3 days were selected for culture of myocardial cells. METHODS：Femoral bone marrows were cultured to the second generation. The purity of the bone marrow mesenchymal stem cells which were examined by flow cytometry was 97% at least,and the cells were then made into cell suspension at density of 4×108/L. Thereafter,the cells were assigned into four groups：DMEM culture group（cells were cultured in DMEM culture media containing 10% fetal bovine serum） ,5-azacytidine group（cells were incubated in 10 μmol/L 5-azacytidine after 24 hours） ,myocardial cell lysate group（myocardial cells were cultured in culture bottle containing 4 times volume of bone marrow mesenchymal stem cells） ,and indirect contact group（cells were incubated in millicell Petri dish and collected after 28 days） . MAIN OUTCOME MEASURES：Expressions of troponin T,connexin 43,and CD31 were detected after induction of bone marrow mesenchymal stem cells using immunohistochemical method;expressions of transcription factor GATA4,NKX2.5,β-mysion heavy chain,and α-mysion heavy chain were determined using RT-PCR. RESULTS：Immunohistochemistry demonstrated that,4 weeks after culture,specific antigen expression was positive,and there was significant difference between 5-azacytidine group and DMEM culture group（P 0.05） . Positive cells were increased in indirect contact group compared with myocardial cell lysate group,but there was no significant difference between the two groups（P 0.05） . CD31 was an antigen marker on the surface of vascular endothelial cells. CD31 was not expressed in both DMEM culture group and 5-azacytidine group,but weakly expressed in indirect contact group and myocardial cell lysate group,and there was no significant difference between the two groups（P 0.05） . Four weeks after culture,both transcription factor NKX-2.5 and GATA4 were expressed in the 5-azacytidine group. Additionally,β-mysion heavy chain but not α-mysion heavy chain expression was observed. CONCLUSION：5-azacytidine induced the differentiation from bone marrow mesenchymal stem cells into myocardial-like cells;in addition,simulated biological microenvironment in both indirect contact group and myocardial cell lysate group also induced the same differentiation. The differentiated cells were cardiac possesses which were between mature cells and cardiac progenitor cells.