一种基于RNA-seq验证的人肾癌细胞和正常细胞原代培养方法  

A primary culture method of human renal carcinoma cells and normal cells based on RNA-seq validation

作　　者：周晓光[1] 王浩舟 李彦生[1] 宋黎明[1] ZHOU Xiaoguang;WANG Haozhou;LI Yansheng;SONG Liming(Department of Urology,Beijing Chaoyang Hospital,Capital Medical University,Beijing100020,China)

机构地区：[1]首都医科大学附属北京朝阳医院泌尿外科,北京100020

出　　处：《中国医药导报》2020年第26期4-7,25,F0003,共6页China Medical Herald

基　　金：国家自然科学基金青年科学基金项目(81502196)。

摘　　要：目的建立更加快速便捷的肾脏肿瘤及正常肾小管上皮细胞原代培养方法。方法选取2019年1—3月首都医科大学附属北京朝阳医院行肾癌根治性切除术和肾部分切除术后患者新鲜的肾透明细胞癌组织和肾脏皮质组织。采用机械分解,胶原酶消化和细胞连续筛分提纯的方法提纯肿瘤和正常原代细胞。分别通过转录组测序(RNA-seq)技术鉴定两种原代细胞的遗传背景,免疫细胞化学鉴定细胞标志物的表达,透射电镜观察原代细胞的超微结构。最后采用裸鼠荷瘤模型鉴定肿瘤细胞的成瘤特性。结果共计分离纯化3株人近端肾小管上皮原代细胞,8株肾透明细胞癌原代细胞。RNA-seq显示测序结果符合两类细胞的遗传学背景,免疫细胞化学可鉴定出近端肾小管上皮细胞标志蛋白的表达。透射电镜可观察到两类细胞特异性超微结构。裸鼠荷瘤模型证实了肾癌原代细胞的成瘤特性。结论成功建立并确定一种快速便捷的肾癌细胞和肾小管上皮细胞原代培养的流程和方法。Objective To establish a more rapid and convenient method for primary culture of renal tumor and normal renal tubular epithelial cells.Methods Fresh clear renal cell carcinoma tissue and renal cortical tissue from patients undergoing radical nephrectomy and partial nephrectomy in Beijing Chaoyang Hospital,Capital Medical University from January to March 2019 were selected.The tumor and normal primary cells were purified by mechanical segmentation,collagenase digestion and continuous cell screening and purification.The genetic background of the two primary cells was identified by transcriptomic sequencing(RNA-seq)technique,the expression of cell markers was identified by immunocytochemistry,and the ultrastructure of the primary cells was observed by transmission electron microscopy.Finally,the tumorigenesis characteristics of tumor cells were identified by the model of nude mice bearing tumors.Results A total of three human proximal tubular epithelial primary cells and eight renal clear cell carcinoma primary cells were isolated and purified.RNA-seq showed that the sequencing results were consistent with the genetic background of the two types of cells,and immunocytochemistry could identify the expression of marker proteins in the proximal renal tubular epithelial cells.Two kinds of cell specific ultrastructure could be observed by transmission electron microscope.The tumor bearing model of nude mice confirmed the tumorigenic properties of primary renal cell carcinoma cells.Conclusion A rapid and convenient processes and methods for primary culture of renal carcinoma cells and renal tubular epithelial cells is successfully established.

关 键 词：原代细胞培养 肾细胞癌 近端肾小管上皮细胞 转录组测序 

分 类 号：R737.11[医药卫生—肿瘤]