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背景与目的已有的研究表明,nm23-H1基因是一个重要的肺癌转移抑制基因,为了筛选与该基因表达相关的差异表达基因,本研究拟构建nm23-H1基因转染前后人大细胞肺癌细胞株(L9981和L9981-nm23-H1)差异表达基因的抑制消减cDNA文库,为进一步筛选、克隆与nm23-H1转移相关的基因奠定基础。方法利用抑制消减杂交(suppressive subtractive hybridization,SSH)技术构建nm23-H1基因转染前后人大细胞肺癌细胞株(L9981和L9981-nm23-H1)间差异表达基因的正向和反向消减cDNA文库,经蓝白菌落筛选克隆,并PCR反应鉴定。结果成功构建了该两株细胞株差异表达基因的正向和反向消减cDNA文库。经蓝白菌落筛选,正向消减文库总共获得约300个白斑克隆,反向消减文库总共获得约400个白斑克隆,从正反向消减文库中各挑选96个克隆进行PCR扩增检测是否有插入片段,结果显示在挑选的正向文库中有84个克隆有插入片段,反向文库中有83个克隆有插入片段,其片段大小范围为(300-750)bp。结论抑制消减杂交是克隆差异表达基因的有效方法,我们应用SSH法和T/A克隆技术成功建立了nm23-H1基因转染前后人大细胞肺癌细胞株(L9981和L9981-nm23-H1)差异表达基因的抑制消减cDNA文库。nm23-H1基因在肺癌细胞中的表达可能影响某些转移相关基因的差异表达。
BACKGROUND & OBJECTIVE Studies have shown that nm23-H1 gene is an important lung cancer metastasis suppressor gene. In order to screen differentially expressed genes related to the gene expression, we constructed a human lung adenocarcinoma cell line before and after transfection of nm23-H1 gene (L9981 and L9981-nm23-H1) differentially expressed genes in the subtractive cDNA library for further screening, cloning and nm23-H1 metastasis-related genes laid the foundation. Methods Forward and reverse subtracted cDNA libraries of differentially expressed genes in human large cell lung cancer cell lines (L9981 and L9981-nm23-H1) before and after transfection with nm23-H1 gene were constructed by suppression subtractive hybridization (SSH) Blue and white colonies were screened for clones and identified by PCR reaction. Results The forward and reverse subtracted cDNA libraries of differentially expressed genes of the two cell lines were successfully constructed. A total of about 300 leukoplakia were obtained from the blue-and-white colonies, and about 400 leukoplakia were obtained from the forward subtracted library. A total of about 400 leukoplakia were obtained from the reverse-subtracted library and 96 clones from the positive and negative subtracted libraries, respectively. The results showed that there were 84 clones with insert in the selected forward library and 83 clones in the reverse library with insert size ranging from (300-750) bp. Conclusion Suppression subtractive hybridization is an effective method for cloning differentially expressed genes. We successfully established the differentially expressed genes of human large cell lung cancer cell line (L9981 and L9981-nm23-H1) before and after transfection with nm23-H1 gene using SSH and T / A cloning techniques Inhibition subtractive cDNA library. The expression of nm23-H1 gene in lung cancer cells may affect the differential expression of some metastasis-related genes.