人肺鳞癌干细胞的体外分离培养和鉴定  

In vitro isolation,culture and identification of lung cancer stem cells in patients with lung squamous carcinoma

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28期4526-4530,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。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,并计数第4代细胞CD133+、CD44+及CD133/CD44双阳性细胞的阳性率。结果与结论:接种后0.5 h细胞贴壁生长,培养第4天形成典型的细胞集落,第7天呈铺路石样外观,细胞数量级可达108。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。说明实验初步从肺鳞癌患者新鲜肺组织标本中分离培养并快速扩增SP细胞,并从中获取更多更纯的干细胞样肺鳞癌细胞,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 cel s can be isolated from lung cancer cel lines. But there are few reports about in vitro isolation, culture and identification of lung cancer stem cel s in patients with lung squamous carcinoma. OBJECTIVE：To explore the feasible methods of harvesting lung cancer stem cel s from fresh lung cancer tissue in patients with lung squamous carcinoma. METHODS：Side population cel s were isolated by col agenase digestion, Ficol density gradient centrifugation and Hoechst 33342 solution. The isolated cel s were suspended in conditioned medium for isolated culture. Flow cytometry method was used to detect lung cancer stem cel s based on the cel surface markers CD133 and CD44, and the positive rates of CD133＋, CD44＋and CD133＋/CD44＋cel s were recorded. RESULTS AND CONCLUSION：Cel s adhered at 0.5 hour after incubation;typical cel colony was formed at 4 days of culture;cel s showed paving stone-shape at 7 days in a total number of 10 8. The positive rates of CD133＋, CD44＋and CD133＋/CD44＋cel s at passage 4 were increased significantly. These findings indicate that stemcel-like lung cancer cel s were obtained from fresh lung cancer tissue in patients with lung squamous carcinoma, which were stably and rapidly amplified in vitro, laying the foundation for the further study on the heterogeneity and resistance of lung cancer stem cel s in the future.

关 键 词：干细胞 肿瘤干细胞 肺鳞癌 肺癌干细胞 原代培养 细胞鉴定 表型 诱导分化 流式细胞术 湖南省自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]