人成体心源间充质样细胞表型分析及向心脏谱系诱导分化潜能的研究

Characterization and cardiac lineage differentiation potential of cardiogenic mesenchymal-like cells from human adults

目的探讨成人心源性间充质样细胞 (CDMCs)的分子表型及向心脏谱系的分化潜能。方法实验分为:不同培养时间CDMCs (第3、5、7代),并以脐带间充质干细胞 (UCMSCs)为对照。分析各细胞分子表型并向心脏谱系诱导分化。显微镜观察细胞形态;计算生长倍增时间并绘制细胞生长曲线;流式细胞术分析表面标志抗原表达;实时定量PCR和Western blot分别测干细胞多能分子及组织特异性分子mRNA和蛋白表达。结果采用重复测量资料方差分析、单因素方差分析和配对t检验。结果 CDMCs具有UCMSCs形态特征与增殖能力,体外培养1 ~ 7 d,与UCMSCs比较,P3、5、7代CDMCs增殖能力差异无统计学意义 (P> 0.05)。与UCMSCs相比,不同培养时间CDMCs表面标志抗原 (CD90)表达 (冻存前:97.13%±2.00%比59.87%±34.14%、38.83%±11.04%、34.77±14.78%;冻存后:99.83%±0.17%比56.00%±19.47%、47.48±11.88%、41.15±8.68%)降低(P< 0.05)。与UCMSCs相比,不同培养时间CDMCs中Rex1 (0.00±0.00比0.68±0.50、0.29±0.17、0.38±0.50)、Oct3/4 (1.00±0.02比5.28±0.78、3.88±0.95、3.63±0.34)、Nanog(1.00±0.16比7.57±4.69、5.40±3.58、5.34±0.76)以及心脏特异转录因子Nkx2.5 (1.00±0.12比30.60±22.43、19.69±9.65、8.82±4.94)、Gata4 (1.00±0.85比60467±25266、44350±25800、35067±23113)表达均增高,差异有统计学意义 (P均< 0.05)。与诱导前比较,向心肌诱导分化15 d后,不同培养时间CDMCs中cTnT蛋白表达水平 (0.40±0.13比0.98±0.16、0.38±0.18 比0.69±0.15、0.17±0.11比0.70±0.17)增高 (P< 0.05)。结论 CDMCs不仅具备部分干细胞和间充质细胞表型,还具有心脏组织特异性。其具备心脏谱系分化潜能,心肌细胞分化能力可能优于UCMSCs。

Objective To investigate the molecular phenotype of cardiac derived mesenchymal-like cells(CDMCs)and explore the differentiation potential into cardiac lineage.Methods The experiment was divided into:different culture time of CDMCs(passage 3,5,7),and umbilical cord mesenchymal stem cells(UCMSCs)were used as control.Molecular characterization and cardiac lineage differentiation were carried out in each group.Cell morphological changes were observed by microscopy.Growth doubling time and population growth curves were generated.Surface marker antigen expressions were analyzed by flow cytometry.Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expressions of pluripotent and tissue-specific molecules.Statistical analysis included repeated measures ANOVA,one-way analysis of variance and paired t-test.Results CDMCs had morphological characteristics and proliferative ability of UCMSCs,and there was no significant difference in the proliferation rate among four groups(P>0.05).Compared with UCMSCs,surface marker antigen(CD90)expression decreased in passage 3,5,7 CDMCs(before cryopreservation:97.13%±2.00%vs59.87%±34.14%,38.83%±11.04%,34.77%±14.78%;after cryopreservation:99.83%±0.17%vs56.00%±19.47%,47.48%±11.88%,41.15%±8.68%,P<0.05).The expressions of Rex1(0.00±0.00vs0.68±0.50,0.29±0.17,0.38±0.50),Oct3/4(1.00±0.02vs5.28±0.78,3.88±0.95,3.63±0.34)and Nanog(1.00±0.16 vs 7.57±4.69,5.40±3.58,5.34±0.76)were significantly increased in CDMCs at different culture time compared with UCMSCs(P<0.05).Compared with UCMSCs,Nkx2.5(1.00±0.12 vs 30.60±22.43,19.69±9.65,8.82±4.94)and Gata4(1.00±0.85 vs 60467±25266,44350±25800,35067±23113)were highly expressed in each culture times of CDMCs(P<0.05).Compared with pre-differentiation,the expression of cTnT protein in each groups of CDMCs(0.40±0.13 vs 0.98±0.16,0.38±0.18 vs 0.69±0.15,0.17±0.11 vs 0.70±0.17)was significantly higher after 15 days myocardial differentiation(P<0.05).Conclusion CDMCs exhibited partial stem cell and mesenchymal features.In addition,it possessed cardiac tissue specificity.CDMCs could differentiate into cardiac lineage,and the differentiation potential of cardiac myocytes may be superior to UCMSCs.