【摘要】 目的构建人肝细胞生长因子(hHGF)真核表达载体,在L02细胞中表达hHGF并筛选其中的差异表达基因。方法构建pcDNA3.1(-)-hHGF载体,测序鉴定;其转染L02细胞后进行蛋白免疫印迹检测;将pcDNA3.1(-)-hHGF和pcDNA3.1(-)载体分别转染L02细胞后,提取总mRNA并逆转录为cDNA,运用基因表达谱芯片技术分析差异表达基因。结果构建的hHGF真核表达载体pcDNA3.1(-)-hHGF经酶切和测序鉴定正确;转染L02细胞后经蛋白免疫印迹证实hHGF存在明确表达;pcDNA3.1(-)及pcDNA3.1(-)-hHGF分别转染L02细胞后提取总RNA,经基因表达谱芯片分析发现,表达水平显著上调和下调的基因分别有404个和145个,在表达显著上调的基因中包含7种锌指基因。结论筛选出了hHGF转染L02细胞后的差异表达基因,为其作用机制及与锌指蛋白相关性的研究提供了重要依据。更多还原

【Abstract】 Objective To detect the hepatocyte growth factor(hHGF)expression and screen the differentially expressed genes in L02 cells transfected by hHGF.Methods pcDNA3.1(-)-hHGF expressive vector were constructed and confirmed by restriction enzyme digestion and DNA sequencing.The expression of hHGF protein in L02 cell line was tested by Western blot.mRNA of L02 cells transfected by pcDNA3.1(-)-hHGF and pcDNA3.1(-)were isolated and reverse transcripted to cDNA.Differentially expressed genes were analyzed by cDNA microarray technique.Results The expressive vector pcDNA3.1(-)-hHGF were constructed and confirmed by restriction enzyme digestion and DNA sequencing.hHGF protein expression were confirmed by Western blot.High quality mRNA and cDNA were prepared from L02 cells tranfcected by pcDNA3.1(-)and pcDNA3.1(-)-hHGF,respectively.Successful microarray screening were conducted.From the scanning results,404 genes were up-regulated(including 9 zinc finger genes)and 145 were down-regulated genes in L02 cell line transfected by hHGF.Conclusions cDNA microarray technology was successfully used to screen the genes differentially expressed in L02 cell line transfected by hHGF,which brought some new clues for the regulation mechanism study on hHGF in liver cells.更多还原