摘要
背景与目的筛选肺癌转移相关基因有助于阐明肺癌侵袭转移的分子机理。为了筛选不同转移潜能肺癌的转移相关差异表达基因,本研究构建不同转移潜能的人大细胞肺癌细胞株NL9980和L9981间差异表达基因的消减cDNA文库,并对消减文库进行初步筛选。方法应用抑制消减杂交技术构建人大细胞肺癌高低转移株NL9980与L9981间差异表达基因的正向消减cDNA文库和反向消减cDNA文库,利用α互补原理,经蓝白菌落初步筛选获得阳性克隆,应用斑点杂交的方法对正向和反向消减cDNA文库进行杂交筛选。结果成功构建了人大细胞肺癌高低转移株差异表达基因的正向消减cDNA文库和反向消减cDNA文库。经蓝白菌落筛选和斑点杂交,正向消减文库获得了307个阳性克隆,反向消减文库获得了78个阳性克隆。结论抑制消减杂交是克隆差异表达基因的有效方法,利用此方法可以成功构建人大细胞肺癌高低转移株差异表达基因消减cDNA文库。不同转移潜能人大细胞肺癌细胞株中存在可能与转移相关的差异表达基因。
Background and objective Screening metastatic-related genes of lung cancer is helpful to understand the molecular mechanisms of lung cancer invasion and metastasis. In order to screen the differential expression genes related to metastasis of lung cancer, we constructed and preliminarily screened the subtracted cDNA libraries of human large cell lung cancer cell lines with different metastatic potentials in this study. Methods Subtracted cDNA library was constructed in the different metastastic potential cell lines NL9980 and L9981 by suppression subtractive hybridization (SSH) method. The positive clones were preliminarily screened by blue-white colony based on the α-complementary principal, and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot to obtain the clones corresponding to differential expression segments. Results The subtracted cDNA libraries were successfully constructed in the different metastastic potential cell lines NL9980 and L9981. Three hundred and seven positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained by the dot blot method. Conclusion SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse subtracted cDNA libraries of different metastastic potential cell lines are constructed by this method. The differential expression genes related to tumor metastasis might exist in the human large cell lung cancer cell lines with different metastasis potential.
出处
《中国肺癌杂志》
CAS
2007年第3期163-167,共5页
Chinese Journal of Lung Cancer
基金
国家自然科学基金重点项目(No.30430300)
天津市科技支撑计划重点项目(No.06YFSZSF05300)资助~~
关键词
肺肿瘤
转移相关基因
抑制消减杂交
斑点杂交
Lung neoplasms Metastatic-related genes Suppression subtractive hybridization Dot blot
