人食管癌间质干样细胞分离培养及生物学特性鉴定

 摘要：目的 探讨人食管癌间质干样细胞（mesenchymal stem-like cells from human esophageal carcinoma,hEC-MSCs）体外分离培养方法,并对其生物学特性进行鉴定。方法 用胶原酶消化法分离培养人食管癌间质干样细胞,RT-PCR 检测基因表达,流式细胞仪检测表面标记和细胞周期,绘制生长曲线,检测染色体核型,成骨诱导和心肌诱导检测其多向分化潜能。结果 胶原酶消化法分离的hEC-MSCs呈长梭形样,细胞增殖迅速,表达间质干细胞（MSCs）相关基因Oct-4、Nanog、vimentin、N-cadherin,不表达上皮细胞相关基因E-cadherin;表达MSCs相关表面标记CD13、CD29、CD44、CD105,不表达CD33、CD45、CD133、CD14、CD34及HLA-DR。细胞周期分析显示多数细胞处于G0/G1期,少数在S和G2/M期。细胞内含有 46 条染色体, 形态正常;具有分化为成骨细胞及心肌细胞的潜能。结论 胶原酶消化法能有效分离hEC-MSCs,对进一步研究食管癌的微环境具有重要意义。

Isolation of mesenchymal stem-like cells from human esophageal carcinoma and identification of their biological characteristics

Objective To isolate mesenchymal stem-like cells from human esophageal carcinoma and identify their biological characteristics.Methods hEC-MSCs were isolated by collagenase digestion.Gene expression was detected by RT-PCR;surface antigen markers and cell cycle were determined by flow cytometry.Growth curve was portrayed and chromosme karyotype was assayed.Multilineage differentiation capacity of these cells was tested by inducing these cells differentiation into osteoblasts and cardiomyocytes.Results hEC-MSCs isolated by collagenase tended to be long and stringy in shape and multiplied rapidly.These cells expressed transcription factors Oct-4 and Nanog and stroma cell-related genes such as vimentin and N-cadherin,but not E-cadherin;FCM results showed that these cells expressed CD13,CD29,CD44 and CD105,but did not express CD33,CD45,CD133,CD14,CD34 and HLA-DR.Cell cycle analysis revealed that the majority of these cells stood in G0/G1 phase while a small population of cells was in S and G2/Mphase.These cells contain 46 chromosomes with normal karyotype and have differentiation potentials into osteoblasts and cardiomyocytes.Conclusions hEC-MSCs can be isolated efficiently by collagenase digestion.