人肺鳞癌来源肺癌干细胞原代培养:数量及功能变化  

Changes of lung cancer stem cells in patients with lung squamous carcinoma during primary culture

作　　者：刘哲亮[1] 邬娇[2] 王林纤[3] 陈跃军[1] 吴冠宇[1] 肖高明[1] 

机构地区：[1]中南大学湘雅医学院附属肿瘤医院胸外一科,湖南省长沙市410013 [2]中南大学湘雅医学院附属肿瘤医院麻醉科,湖南省长沙市410013 [3]中南大学湘雅医学院附属肿瘤医院检验科,湖南省长沙市410013

出　　处：《中国组织工程研究》2015年第32期5172-5176,共5页Chinese Journal of Tissue Engineering Research

基　　金：湖南省自然科学基金资助项目(14JJ7092);湖南省卫生厅科研计划项目(C2013-027)~~

摘　　要：背景:研究显示可从肺癌细胞系中分离出肺癌干细胞,但对于从新鲜肺鳞癌标本中原代培养肺癌干细胞的报道较少。目的:探索从新鲜肺鳞癌组织标本中分离肺鳞癌干细胞的可行方法,及其在原代培养过程中的数量和功能变化。方法:借助胶原酶消化法、密度梯度离心法和Hoechst33342染料外排法初筛SP细胞,将初筛细胞置于条件培养基分离培养,后续应用流式细胞分析及分选技术,进一步分离纯化肺鳞癌干细胞。实验结合CD133和CD44鉴定目标细胞,计数第1代和第4代肺癌干细胞的CD133+、CD44+和CD133/CD44双阳性细胞数;检测第1代和第4代肺癌干细胞的单细胞克隆形成能力、平板集落形成能力和细胞球形成能力。结果与结论:传代培养第4代肺癌干细胞的CD133+、CD44+和CD133/CD44双阳性细胞数明显增多,流式细胞检测结果显示,第4代和第1代的CD133+和CD133/CD44双阳性细胞表达率差异有显著性意义,第4代和第1代肺癌干细胞的CD44+细胞表达率差异无显著性意义。第4代细胞的单细胞克隆形成能力,平板集落形成能力和细胞球形成能力比均明显强于第1代肺癌干细胞。实验初步从肺鳞癌患者新鲜肺组织标本中分离培养并获取干细胞样肺鳞癌细胞,且在体外稳定快速扩增,为进一步研究肺鳞癌干细胞的异质性和耐药性奠定基础。BACKGROUND：Studies have shown that lung cancer stem cels can be isolated from the lung cancer cel lines, But there are few reports on in vitro isolation, culture and identification of lung cancer stem cels in patients with lung squamous carcinoma. OBJECTIVE：To establish the feasible methods of harvesting lung cancer stem cels from fresh lung cancer tissues in patients with lung squamous carcinoma, and to investigate the alterations in cel number and function during primary culture. METHODS： Side population cels were isolated by colagenase digestion, Ficol density gradient centrifugation and Hoechst 33342 efflux properties. The isolated cels were isolated and cultured in conditioned medium. Flow cytometry method was used to detect lung cancer stem cels based on the cel surface markers CD133 and CD44, and the positive rates of CD133＋, CD44＋ and CD133＋/CD44＋ were recorded. The single cel clones assay, flat colony formation assay and the cel sphere formation assay were used to identify the stem-like characteristics of lung cancer stem cels between the first and fourth generations. RESULTS AND CONCLUSION：The positive rates of CD133＋, CD44＋ and CD133＋/CD44＋ cels at the fourth generation were increased significantly, and the positive rates of CD133＋ and CD133＋/CD44＋ cels at passage 4 were significantly higher than those at the first generation. The abilities of single cel clone formation, the flat colony formation and the cel sphere formation in the fourth-generation cels were greatly enhanced compared with the first-generation cels. Experimental findings showed that stem cel-like lung cancer cels were obtained from fresh lung cancer tissue in patients with lung squamous carcinoma, which stably and rapidly amplified in vitro, laying the foundation for the further study of the heterogeneity and drug resistance of lung cancer stem cels.

关 键 词：干细胞 肿瘤干细胞 肺癌干细胞 原代培养 细胞鉴定 湖南省自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]