不同年龄大鼠骨髓源性心肌干细胞生物学特性的比较  

Comparative study on biological characteristics of marrow-derived cardiac stem cells from rats at different Ages

作　　者：王存[1] 谭玉珍[1] 王海杰[1] 国海东[1] 张大勇[1] 崔晓军[1] 

机构地区：[1]复旦大学上海医学院人体解剖学与组织胚胎学系

出　　处：《解剖学报》2009年第4期573-579,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(30470883);高等学校博士点专项科研基金(200802460044)

摘　　要：目的研究不同年龄大鼠骨髓源性心肌干细胞（MCSCs）形态和衰老相关指标的差异,探讨年龄因素对MCSCs增殖、存活和向心肌细胞分化的影响。方法利用单细胞克隆培养技术从幼年、成年和老年雄性SD大鼠骨髓中筛选MCSCs,透射电镜下观察细胞超微结构改变。SA-β-半乳糖苷酶染色和活性氧（ROS）染色检测细胞衰老变化,流式细胞术分析细胞周期分布。Annexin V/PI双标流式细胞术和Hochest33342染色检测细胞存活和凋亡。用骨形态发生蛋白-2（BMP-2）诱导不同年龄组MCSCs向心肌细胞分化,通过RT-PCR检测诱导后心肌早期转录因子和心肌特异基因的表达,并通过免疫细胞化学染色检测各年龄组细胞经（BMP-2）诱导后细胞内心肌特异性肌钙蛋T（cTnT）表达的变化。结果老年组MCSCs核质比减小,胞质内可见髓样小体。随着年龄增长,处于增殖期的MCSCs比例下降,β-半乳糖苷酶和ROS阳性细胞数目增多。老年组存活细胞比例较幼年组降低。用BMP-2诱导后4周,幼年组细胞的Nkx2.5、GATA-4和cTnT、Cx-43 mRNA表达明显,成年组和老年组细胞表达低于幼年组,差异有显著性意义。免疫细胞化学染色显示,BM...Objective To investigate the changes in morphology and senescence-associated markers of the marrow-derived cardiac stem cells （MCSCs） from rats at different ages and to explore the impacts of age on proliferation, survival and differentiation of MCSCs. Methods With single-cell cloning culture, MCSCs were selected from the bone marrow of young, adult and aged male SD rats respectively. Ultrastructural changes of the cells were viewed under a transmission electron microscope. The senescence-associated changes were examined with SA-β-galactosidase staining and reactive oxygen species （ROS） staining. Distribution of cell cycle of MCSCs from different age groups was evaluated with flow cytometric analysis. Rates of the survived and apoptotic cells were determined by Annexin V/PI double-labeled flow cytometric analysis and Hochest33342 staining. Differentiation of the MCSCs toward cardiomyocytes was induced with BMP-2. Expression of cardiac transcription factors and cardiac specific genes of the cells after induction were examined with RT-PCR. CTnT expression of the cells also be examined with immunocytochemistry. Results The nucleus/plasma ratio of the cells from aged rats decrease and there are some myelin bodies in the cells of aged group. With increasing of age, the MCSCs in S ＋ G2/M phase reduce, while β-galactosidase-positive cells and ROS-positive cells increase. Survival rate of the cells from aged rats is lower than that of the cells from young rats. At four week after induction with BMP-2, expression of Nkx2.5, GATA-4, cTnT mRNA and Cx-43 mRNA of the cells of young group increase significantly. In adult and aged group, expression of the cardiac transcription factors and cardiac specific genes is lower than that of the cells in young group. In immunocytochemical staining, cTnT expression of the cells in young group is stronger after induction with BMP-2. As compared with that of the cells in young group, cTnT expression of the cells in aged group is weak after induction. Conclusion With increasing

关 键 词：骨髓源性心肌干细胞 衰老 骨形态发生蛋白-2 细胞分化 Β-半乳糖苷酶 活性氧 流式细胞术 

分 类 号：Q255[生物学—细胞生物学] R318[医药卫生—生物医学工程]