【作者】 王存；

【导师】 谭玉珍； 王海杰；

【作者基本信息】 复旦大学 ， 人体解剖与组织胚胎学， 2009， 硕士

【摘要】 目的研究不同年龄大鼠MCSCs超微结构和衰老相关指标的差异,明确年龄因素对MCSCs增殖、存活和向心肌细胞分化的影响,探讨IGF-1对干细胞衰老的调节作用及其机制。方法从幼年、成年和老年雄性SD大鼠骨髓中分离MMSCs,通过单克隆培养技术、c-kit免疫荧光标记和RT-PCR检测心肌早期转录因子Nkx2.5的表达,从MMSCs中筛选出具有心肌特异分化潜能的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ROS染色检测细胞衰老变化,流式细胞术分析细胞周期。建立体外无血清缺氧模型,Annexin V/PI双标流式细胞术和Hochest 33342染色检测MCSCs存活和凋亡。用BMP-2诱导不同年龄组MCSCs向心肌细胞分化,RT-PCR检测诱导后细胞心肌早期转录因子和心肌特异基因的表达,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cTnT表达的变化。用IGF-1处理老年组MCSCs,SA-β-半乳糖苷酶染色和ROS染色检测细胞衰老指标差异,流式细胞仪分析细胞周期。Hochest 33342染色检测IGF-1处理前后,老年组MCSCs在无血清缺氧培养条件下凋亡细胞比例。用BMP-2诱导各实验组MCSCs向心肌细胞定向分化,RT-PCR检测诱导后细胞Nkx2.5、GATA-4和cTnT、Cx-43 mRNA的表达,免疫细胞化学染色检测细胞内cTnT表达的变化。通过RT-PCR检测IGF-1处理前后细胞中p53、p21和bax mRNA的表达变化以及细胞VEGF、HGF和FGF mRNA的表达差异。结果从MMSCs克隆中筛选出表达心肌早期转录因子Nkx2.5的c-kit~+MMSCs克隆。透射电镜显示老年组MCSCs核浆比减小,胞浆内髓样小体增多。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。在无血清缺氧条件下培养12h后,老年组凋亡细胞比例明显高于幼年组。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,BMP-2诱导后幼年组细胞cTnT表达明显,老年组细胞表达较弱。老年组MCSCs经IGF-1处理后,β-半乳糖苷酶阳性细胞数目和ROS阳性细胞数目减少,处于增殖期的细胞比例明显增高。在无血清缺氧条件下培养12h后,老年IGF-1处理组MCSCs凋亡细胞比例较未处理组下降,差异有显著性意义。用BMP-2诱导后4周,老年IGF-1处理组细胞的Nkx2.5、GATA-4和cTnT、Cx-43 mRNA表达较未处理组增强。经IGF-1处理后,老年组MCSCs的p53、p21和bax mRNA表达下调,VEGF、HGF和FGFmRNA表达上调。结论随着年龄增长,MCSCs发生衰老变化,其增殖、存活和向心肌细胞分化能力下降。IGF-1能在一定程度上抑制MCSCs衰老并提高其增殖、存活和向心肌细胞分化的能力。更多还原

【Abstract】 Objective To investigate the changes in ultrastructure and senescence of the marrow-derived cardiac stem cells(MCSCs) from rats at different ages,clarify the impacts of age on proliferation,survival and differentiation of the cells and explore the regulatory effects of IGF-1 on senescence of MCSCs.Methods MMSCs were isolated from bone marrow of young,adult and aged male SD rats.MCSCs were selected from MMSCs with single-cell cloning culture,c-kit immunoflourence staining and RT-PCR analysis.Ultrastructural changes of the cells were viewed with a transmission electron microscope.The senescence-associated changes were examined with SA-P-galactosidase staining and ROS staining.Distribution of cell cycle of the cells was evaluated with flow cytometric analysis.The cells were treated with serum deprivation and hypoxia for 12 hours.The survived and apoptotic cells were determined by Annexin V/PI flow cytometric analysis and Hochest 33342 staining.Differentiation of 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 was examined with immunocytochemistry.In another experiment,the MCSCs of the aged group were treated with IGF-1.The senescence-associated changes were examined with SA-P-galactosidase staining and ROS staining.Distribution of cell cycle was evaluated with flow cytometric analysis.The cells were treated with serum deprivation and hypoxia for 12 hours and the apoptotic cells were determined by Hochest 33342 staining.After induction with BMP-2,expression of cardiac transcription factors and cardiac specific genes in IGF-1 treated group and untreated group were determined with RT-PCR.cTnT expression of the cells was examined with immunocytochemistry.Expression of p53,p21 and bax mRNA and expression of VEGF,HGF and bFGF mRNA were examined with RT-PCR.Results The nucleus/plasma ratio of MCSCs from the aged rats decreased and there were some myelin bodies in the cells of the aged group.With increasing of age,the cells in S+G2/M phase reduced,andβ-galactosidase-positive cells and ROS-positive cells increased.After being treated with serum deprivation and hypoxia for 12 hours, survival rate of the cells from the aged rats was 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 in the young group increased significantly.In adult and aged group,the level of expression of the cardiac transcription factors and cardiac specific genes was lower than that of the cells in young group.In immunocytochemical staining,cTnT expression of the cells in the young group was obvious after induction with BMP-2.Being compared with young group,cTnT expression of cells in the aged group was weak after induction.After treatment with IGF-1,the cells in S+G2/M phase increased in the aged group,β-galactosidase-positive cells and ROS-positive cells reduced.After being treated with serum deprivation and hypoxia for 12 hours,rates of the apoptotic cells in IGF-1 treated group was lower than that of the cells in the untreated group.Being compared with the IGF-1 untreated group,expression of the cardiac transcription factors and cardiac specific genes in IGF-1 treated group was strong after induction with BMP-2.Expression of VEGF,HGF and bFGF mRNA in IGF-1 treated group was higher than that of the cells in untreated group and expression of p53,p21 and bax mRNA in IGF-1 treated group decreased significantly.Conclusion With increasing of age,MCSCs become senescent,the abilities of proliferation,survival and differentiation of the cells decrease.IGF-1 may attenuate senescence of the aged cells and improve their potential of proliferation,survival and differentiation.更多还原