成人自体心肌干细胞体外高效扩增及特异性鉴定

Autologous adult cardiac stem cells amplification efficiency and specific identification in vitro

关键词:  自体组织  心肌干细胞  基因表达  流式细胞术  核型分析

Key words:  autologous tissue  cardiac stem cell  gene expression  flow cytometry  karyotyping

摘要:

[目的] 建立一种稳定、高效的成人自体心肌干细胞体外培养扩增及特异性鉴定方法。 [方法] 手术中取下的心脏组织标本，通过胶原酶消化的方法获得细胞，使用自体血清进行贴壁培养和传代的方法纯化细胞，细胞增殖至3代后进行RT-PCR基因表达检测，流式细胞表面标记检测及染色体核型分析的鉴定。 [结果] 通过本实验的方法可以在成人心肌组织中分离和纯化心肌干细胞，并在体外可大量增殖，至3代细胞可增殖至5×107。RT-PCR鉴定其具有全能干细胞因子Nanog、Oct-4、Sox-2、Rex-1的mRNA表达，及心肌转录因子Nkx2.5和GATA4的mRNA和蛋白表达；表达间质干细胞表面标记CD29、CD90、CD105；不表达造血干细胞表面标记CD45和人免疫抗原HLA-DR；且培养后46条染色体无移位和缺失等染色体突变。 [结论] 通过本研究确立了一种稳定、高效的成人自体心肌干细胞体外培养，扩增和特异性鉴定的方法，为自体心肌干细胞移植技术的临床应用奠定重要的基础。

Abstract:

[Objective] To establish a stable and efficient adult cardiac stem cells in vitro amplification and identification methods. [Methods] Heart tissue removed in surgery, the cells obtained by collagenase digestion method, using the adherent culture purified cells and passage. Cell proliferation to third-generation, gene expression detected by RT-PCR；cell surface markers detected by flow cytometry；and the karyotype identification analysised by karyotyping. [Results] Through this experiment, the cardiac stem cells in adult cardiac tissue were isolated and purified in vitro. Cells after culture maintained pluripotent stem cell factor Nanog, Oct-4，Sox-2 and Rex-1 mRNA expression，and cardiac transcription factor Nkx2.5 and GATA4 mRNA and protein expression; expressed CD29，CD90，CD105 stem cell surface marker, but not expressed the hematopoietic stem cell surface markers CD45 and human antigens HLA-DR; and chromosome mutation did not occur. [Conclusion] Through this study, establish a stable, efficient autologous adult cardiac stem cells amplification and specific identification method in vitro. And establish an important foundation of autologous cardiac stem cell transplantation for clinical application.