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汉族人肾透明细胞癌细胞系的建立
翟羽佳1△,谭晓洁1△,侯建国2,肖建如3,徐丹枫4,余永伟5,常文军1,林丽萍1,曹广文1*
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(.第二军医大学卫生勤务学系流行病学教研室,上海 200433;.第二军医大学长海医院泌尿外科,上海 200433;3.第二军医大学长征医院骨科,上海 200003;4.第二军医大学长征医院泌尿外科,上海 200003;5.第二军医大学长海医院病理科,上海 200433)
摘要:
目的:取汉族人临床标本,建立肾透明细胞癌(ccRCC)细胞系,初步研究该细胞系的生物学特性。方法:2005~2007年间共采集43例ccRCC新鲜手术标本(包括肾原位肿瘤、骨转移、淋巴结转移肿瘤及癌栓),于手术后30~60 min内用植块法进行体外培养,记录传代超过50代的细胞株的生长曲线,测定集落形成率,对细胞进行染色体分析,病理学检查,流式细胞术检测及NOD-SCID鼠荷瘤实验。结果:绝大部分原代细胞传代次数在5代内,其中5例可连续传代5代以上,4例传代超过10代。仅1例来源于骨转移组织的原代细胞(命名为RCC05-TXJ)及1例来源于原位ccRCC的原代细胞(命名为RCC05-ZYJ)可稳定连续传代。RCC05-TXJ经历21个月传110代,群体倍增时间19.2 h,染色体众数为75,集落形成率为41%;RCC05-ZYJ经历18个月传160代,群体倍增时间为16.5 h,染色体众数为55,集落形成率为37%。免疫组化显示CA9及CD133阳性,流式细胞术分析发现随细胞传代增多CA9及CD133表达明显增高(P<0.05)。两株细胞在NOD-SCID鼠体内均有良好的致瘤及转移性,但是转移倾向明显不同。结论:应用汉族人肾透明细胞癌组织成功建立了2个转移潜能不同的细胞系,随着传代次数增加,肿瘤干细胞比例增加。
关键词:  肾透明细胞癌  细胞系  汉族
DOI:10.3724/SP.J.1008.2008.00479
投稿时间:2007-12-29修订日期:2008-03-20
基金项目:国家自然科学基金(30370788,30571609).
Establishment of clear cell renal cell carcinoma lines of Han nationality in China
ZHAI Yu-jia1△,TAN Xiao-jie1△,HOU Jian-guo2,XIAO Jian-ru3,XU Dan-feng4,YU Yong-wei5,CHANG Wen-jun1,LIN Li-ping1,CAO Guang-wen1*
(1.Department of Epidemiology,Faculty of Medical Services,Second Military Medical University,Shanghai 200433,China;2.Department of Urology,Changhai Hospital,Second Military Medical University,Shanghai 200433;3.Department of Orthopedics,Changzheng Hospital,Second Military Medical University,Shanghai 200003;4.Department of Urology,Changzheng Hospital,Second Military Medical University,Shanghai 200003;5.Department of Pathology,Changhai Hospital,Second Military Medical University,Shanghai 200433)
Abstract:
Objective:To establish clear cell renal cell carcinoma (ccRCC) cell lines from clinical ccRCC specimens of Han nationality in China and to characterize the biological features.Methods: From 2005 to 2007,fresh surgical samples of ccRCC were obtained from 43 patients; the samples included primary tumor in situ,osseous metastasis,lymph node metastasis,and cancerous embolus.The samples were cultured in vitro using explant-culture method within 30-60 min after surgery.Analysis on cell growth and colony-forming efficiency was recorded for the lines which were passaged for over 50 generations.Chromosome examination,pathological examination and tumorigenesis in NOD-SCID mice were used to determine their malignancy.Flow cytometry was used to determine expression of CA9 and CD133.Results: Most of the primary cells could only be passaged for less than 5 generations; 5 lines could be serially passaged for over 5 passages,3 lines for over 10 passages, and only 2 lines could be stably passaged.One line,named RCC05-TXJ, was from osseous metastatic ccRCC and had been serially passaged for 110 generations in 21 months; the average doubling time was 19.2 h,average chromosome number was 75,and colony forming efficiency was 41%.Another line,named RCC05-ZYJ,was from primary ccRCC specimen and had been serially passaged for 160 generations in 18 months; the average doubling time was 16.5 h,average chromosome number was 55,and the colony forming efficiency was 37%.Immunohistological analysis demonstrated that both lines expressed CA9 and CD133.Flow cytometry analysis found that expression levels of CA9 and CD133 increased with the passages.Both RCC05-ZYJ and RCC05-TXJ lines were able to form tumor and to metastasize in NOD-SCID mice; however,their metastatic ability was obviously different.Conclusion: We have established 2 ccRCC cell lines with different metastatic potentials from the clinical ccRCC specimens of Han nationality in China.The ratio of tumor stem cells increases with the passages.
Key words:  clear cell renal cell carcinoma  cell line  Han nationality in China