一种改进的肾透明细胞癌原代细胞培养方法  

Modified primary culturing of renal clear cell carcinoma

作　　者：宋雷[1] 叶文凌[2] 刘勇[1] 王伟[3] 黄睿[1] 

机构地区：[1]西北民族大学医学院医学机能学教研室,兰州730030 [2]河南大学医学院细胞生物学与遗传学教研室,河南开封475000 [3]兰州大学第二医院泌尿外科,兰州730000

出　　处：《第三军医大学学报》2016年第7期714-717,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金青年科学基金(81000961)~~

摘　　要：目的对肾透明细胞癌原代培养方法进行改进,以获得纯度及活力更高、遗传表型与来源更相近的肾透明细胞癌细胞系。方法将经过胶原酶消化法或机械法取得的肿瘤样本单细胞悬液,置入超低黏附培养板,用添加有丝分裂原等细胞因子的无血清培养基悬浮培养,离心滤除非肿瘤细胞和无成瘤能力肿瘤细胞,获得纯度高、异质性的肿瘤细胞囊球,囊球可以连续传代悬浮培养,也可转至常规培养条件培养,都可得到能连续传代的肿瘤细胞株。用流式细胞仪鉴定瘤球传代细胞,并与肾癌临床样本细胞进行表型比对。结果与单纯机械法或胶原蛋白酶消化法分离细胞及含血清培养基培养的方法相比,本法成功率更高,获得的肾透明细胞癌细胞保持与临床样本相似的异质性分化类型和表面抗原表达,并且无杂细胞污染。培养的细胞可经历稳定的连续传代并用于肿瘤相关研究。结论该方法简便易行,成功率高,培养的肾透明细胞癌细胞株可作为肾癌研究的良好实验模型。Objective To improve the method of primary culture of kidney cancer cells to get cells with high purity and similar phenotype. Methods Clinical specimens of renal clear cell carcinoma were digested and mechanically dissociated to prepare mono-cell suspension. The cells were subsequently cultured in ultra-low-adhesion plates,and tumor spheroids were formed after changing the medium to serum-free RPMI1640 medium（ SFM） supplemented with mitogens. Tumor spheroids could be digested and passaged to get successive generation. Monolayer adherent cells（ MACs） could be obtained from the digested tumor spheres and maintained as monolayer cultures in RPMI 1640 routine medium. Aliquots of the cell suspension were subjected to fluorescence-activated cell sorting to quantify the expression of cell surface antigen in the specimen cells and passaged cells. Results The improved method could dramatically increase the cells at high yield and high purity compared with traditional methods,and the cells obtained by serum-free culture had similar phenotype with primary cells and had no fibroblast cell incorporation. Conclusion The improved method is feasible and convenient and has high success rate. The cultured cells act as experimental model for research of renal cell carcinoma.

关 键 词：肾透明细胞癌 原代培养 无血清培养基 免疫表型 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学] R737.11[医药卫生—基础医学]