人胆囊癌SGC996细胞系侧群细胞的分离与体外培养.pdfVIP

population cells from human carcinoma of gallbladder SGC996 cell line, and cultured the two subpopulations in vitro, and preliminarily verified its characteristics of stem cells. Methods Flow cytometry （FACS ）was used to isolate side population cells and non-side population cells from human carcinoma of gallbladder SGC996 cell line, and cultured the two subpopulations in serum- free culture solution with EGF, bFGF and LIF and in serum culture solution respectively. Multiplication capacity in vitro of the two subpopulations was observed in serum culture solution by MTT. Results The proportion of side population cells in human carcinoma of gallbladder SGC996 cell line was 1.0% by flow cytometry （FACS ）, by addition of verapamil , it was 0.1%,which was greatly decreased. Side population cells exhibited higher multiplication capacity and were able to form tumor spheres in serum- free culture solution；while main population cells showed lower multiplication capacity and even died in the same condition. In serum culture solution, side population cells exhibited higher multiplication capacity on the 5th day, 6th day and 7th day by MTT. Conclusions Human carcinoma of gallbladder SGC996 cell line contained a small subpopulation of cells that could form tumor spheres and exhibited higher multiplication capacity in vitro. 【Key words 】 carcinoma of gallbladder；tumor stem cell；side population cell； culture in vitro 在肿瘤的发生、发展及浸润转移过程中，肿瘤干细胞（CSC）扮演着十分重 要的作用[1]，因此，研究肿瘤干细胞有助于进一步了解肿瘤发生的机制。目前， 分离肿瘤干细胞主要基于两种手段，一是通过筛选肿瘤干细胞特异性标志物；二 是利用肿瘤干细胞膜表面ABC家族蛋白的外排特性，从而在一整群细胞中分离出 外排染料Hoechst33342而显示低染特性一亚群细胞[2]，称之为“侧群细胞”。 所以，在缺乏特异性肿瘤干细胞标志物的情况下，分离侧群细胞成了研究肿瘤干 细胞重要的方法。本研究拟利用流式细胞荧光激活分选法（FACS）分离人胆囊癌 SGC996细胞系侧群细胞，并选用特定培养基行体外培养，初步探讨其相关肿瘤 学特性。 1材料与方法 1.1细胞和试剂 人胆囊癌细胞株SGC996购自上海中科院细胞库。细胞培养基RPMI1640、0.25 ％胰酶购自GIBCO公司，胎牛血清购自 Hyclone公司；EGF、bFGF、LIF购自