利用抑制性消减杂交技术构建太空诱变宫颈癌细胞cDNA消减文库  

Construct a subtractive library of Caski cell lines induced by exposing to the space environment by suppression subtractive hybridization

作　　者：张志杰[1] 郭锋杰[1] 童永清[1] 李跃辉[1] 谢平丽[1] 李官成[1] 

机构地区：[1]中南大学肿瘤研究所,湖南长沙410078

出　　处：《中国医师杂志》2009年第12期1592-1595,共4页Journal of Chinese Physician

基　　金：973研究前期专项2003（CA04200）,国家自然科学基金（No.30672352）

摘　　要：目的利用抑制性消减杂交技术构建太空诱变宫颈癌细胞的消减文库，为进一步从基因表达水平研究空间特殊环境影响肿瘤细胞生物学行为改变的分子机制奠定基础。方法采用SuperSMART和SSH技术，以太空诱变的宫颈癌48A9细胞株作为Tester，地面正常对照组宫颈癌细胞株作为Driver，分离太空诱变的宫颈癌48A9细胞中差异表达的基因片段；连接T载体，转化宿主菌，构建太空诱变的宫颈癌48A9细胞cDNA抑制性消减文库。结果经蓝白筛选后随机挑取300个阳性克隆，以接头1和2R内侧序列为引物进行菌液PCR扩增鉴定阳性克隆，结果显示其中288个克隆有插入片段，阳性率为96％，大小分布在0．2—1．0kb间。结论本实验利用抑制性消减杂交技术成功的构建了高质量的太空诱变宫颈癌细胞消减文库，为进一步从基因水平阐明太空特殊环境对肿瘤细胞生物学行为改变的分子机制奠定了基础。Objectives Construct a subtractive library of Caski cell line induced by exposing to the space environment by suppression subtractive hybridization and pave the way to explain the molecular mechanisms of the changes at the gene level. Methods Super SMART cDNA synthesis and suppression subtractive hybridization （SSH） were performed to isolate differentially expressed cDNA fragments from strains subclonal 48A9 cell line. cDNA from the 48A9 cell line were used as ＂tester＂, and the other from the control Caski cell line as ＂driver＂. Subtractive products were directly inserted into T/A cloning vector, and then transformed into host bacteria to set up a suhtractive cDNA library of specially or highly expressed genes in strains subclonal 48A9 cell line. Results mRNA were directly extracted and purified with good quality. Double strand cDNA were reverse transcripted integratedly, and then cut by Rsa I into even length short segments. Ligation was identified as high effective. After two hybridizations, a subtractive library of differentially expressed genes in strains subclonal 48A9 cell line was successfully constructed by SSH. Conclusion SSH is an effective approach to isolate differentially expressed genes.

关 键 词：杂交 遗传 宫颈肿瘤/遗传学 基因文库 

分 类 号：R737[医药卫生—肿瘤]