高低淋巴道转移力小鼠肝癌细胞间抑制性消减杂交文库的建立  

Construction of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

作　　者：侯力[1] 唐建武[1] 崔晓楠[1] 王波[1] 宋波[1] 孙雷[1] 

机构地区：[1]大连医科大学病理学教研室,辽宁大连116027

出　　处：《中华肿瘤防治杂志》2006年第1期11-14,共4页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金资助项目(30371583)

摘　　要：目的:以抑制性消减杂交技术分离高低淋巴道转移力小鼠肝癌细胞HcaF和HcaP间差异表达的基因。方法:采用抑制性消减杂交技术,以高低转移力小鼠肝癌细胞HcaF为检测子,以低转移力HcaP细胞为驱赶子,分离高转移力癌细胞中高表达的基因的cDNA片段。将差异表达基因的cDNA片段插入T/A克隆载体,并通过热转化的方法转化大肠埃希菌DH5α,建立了高低淋巴道转移力小鼠肝癌细胞间抑制性消减杂交文库。以菌液PCR的方法检测随机挑选的白色菌落中插入片段的出现频率。提取14个重组质粒进行序列分析。结果:在消减文库中共获800个白色菌落,对随机挑选的160个白色菌落进行菌液PCR扩增,发现95%的白色菌落中含有100～700bp的插入片段。对重组质粒的序列分析和同源性比对发现,所获的14个序列中3个为已知基因,分别来自小鼠热休克蛋白、ribosomalproteinS13、ethanolinduced6基因;4个可能来源于新基因;3个与3个不同的EST同源。结论:成功构建了高低淋巴道转移力小鼠肝癌细胞间抑制性消减杂交文库,为今后高通量筛选癌淋巴道转移相关基因,奠定了坚实的基础。OBJECTIVE：To detect the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca- P with different lymphatic metastasis potential using the suppression subtractive hybridization （SSH） method in order to make the molecular mechanism of lymphatic metastasis clearly. METHODS： cDNA of Hca-F cells was used as tester and cDNA of Hca-P cells was used as driver. The cDNAs which were highly expressed in Hca-F cells were isolated by using SSH method. The isolated cDNA were cloned into T/A cloning vector. The ligation products were transformed into DH5α competent cells. Individual 160 clones were selected randomly and then used for clone PCR amplification. The vector DNA from positive clones were isolated for sequencing. RESULTS： There were 800 positive bacteria clones in the amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95 percent clones contained 100-700 bp inserts. Analysis of 14 sequenced cDNA clones randomly picked from the SSH library revealed 3 known genes （mouse heat shock protein 80 KD, ribosomal protein $13, ethanol induced 6）and 3 expressed sequence tags （ESTs）. Four cDNAs showed no homology and presumably represent unknown genes. CONCLUSIONS： A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for searching new lymphatic metastasis related genes.

关 键 词：肝肿瘤 试验性/病理学 肿瘤转移 癌基因 杂交 

分 类 号：R735.7[医药卫生—肿瘤] R730.2[医药卫生—临床医学]