应用抑制性消减杂交技术克隆肾癌转移相关基因  

Differential screening of human metastasis-associated genes of renal cell carcinoma by suppression subtractive hybridization

作　　者：陈磊[1] 于德新[1] 施浩强[1] 方卫华[1] 江山[1] 唐亮[1] 

机构地区：[1]安徽医科大学附属医院泌尿外科,合肥230022

出　　处：《临床泌尿外科杂志》2008年第4期303-306,共4页Journal of Clinical Urology

摘　　要：目的:应用抑制性消减杂交技术构建人高侵袭性肾癌细胞和低侵袭性肾癌细胞间差异表达的cD-NA消减文库,筛选并克隆肾细胞癌转移相关基因。方法:采用涂有Matrigel胶的Transwell小室分离回收高、低侵袭性肾癌细胞,分别从高侵袭性肾癌细胞与低侵袭性肾癌细胞中提取polyA+RNA,合成双链cDNA,经RsaI酶切后将高侵袭性肾癌细胞cDNA分为两组并加上接头1和接头2,再与过量低侵袭性肾癌细胞cDNA进行两次消减杂交及两次抑制性PCR,PCR产物与pMD18-T载体连接并转化JM109大肠杆菌构建成cDNA消减文库,文库扩增后随机挑取克隆进行测序及同源性分析。结果:文库共包含185个阳性克隆,随机挑取50个阳性克隆分析,其中45个克隆有插入片段。将其中15个有插入片段的克隆进行测序,表明源于7个已知基因。结论:利用抑制性消减杂交技术,从一对同源的高低转移表型差异的细胞株中获得了7条可能与肾癌转移相关的cD-NA序列,他们可能在促进肾癌转移中起到重要作用。Objective：To construct different invasion cells＇ cDNA library of human renal cell carcinoma（RCC） by means of suppression subtractive hybridization（SSH）, then to screen and clone metastasis-associated genes of RCC. Methods： In vitro invasion assay the Transwells coating Matrigel were performed for separation and recovery of high invasive and low invasive renal cell carcinoma cells. PolyA＋ RNA was isolated from high invasive cells and matched low invasive cell , respectively. Then double-strand cDNA were synthesized and restricted by RsaⅠ. High invasive renal cell carcinoma cell cDNA were divided into two groups and ligated with either adaptor1 or adaptor2. After High invasive RCC cDNA hybridized with low invasive cell cDNA twice and underwent nested PCR, the PCR products were cloned in pMD18-T Vector and transformed E. coli JM109. Some positive clones that randomly picked up were sequenced and analyzed. Results： The SSH library contained 185 clones with SSH cDNA fragments. Among 50 clones sequenced randomly,45 have insert-fragment. 15 of 45 clones were sequencd and the result show they derived from 7 known genes. Conclusions：Through using SSH , we have identified seven cDNA fragments which are much higher levels in the high metastasis cancer cell clone than low metastasis variety, therefore ,it is supported that they might play an important role in the metastasis of renal cell carcinoma and may very well potential application in the diagnosis and treatment of metastasis.

关 键 词：肾肿瘤 基因 抑制性消减杂交 肿瘤转移 

分 类 号：R737.11[医药卫生—肿瘤]