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目的:构建hHGF真核表达载体,并在HepG2细胞中表达,筛选其对HepG2细胞的差异表达基因.方法:构建pcDNA3.1(-)-hHGF载体,测序鉴定;转染HepG2细胞后蛋白免疫印迹检测;运用基因表达谱芯片技术,筛选pcDNA3.1(-)-hHGF和pcDNA3.1(-)载体分别转染HepG2细胞后,提取mRNA并逆转录为cDNA,经表达谱芯片分析差异表达基因.结果:构建的表达载体经酶切和测序确定;转染HepG2细胞后hHGF表达经蛋白免疫印迹证实;经20000个基因的表达谱芯片分析发现,其中有430个基因表达水平显著上调,88个基因表达水平显著下调.结论:筛选出HGF转染HepG2细胞后的差异表达基因,对其在肝细胞中多种作用机制研究提供了重要依据.
OBJECTIVE: To construct eukaryotic expression vector of hHGF and express it in HepG2 cells, and to screen differentially expressed genes of HepG2 cells.Methods: The pcDNA3.1 (-) - hHGF vector was constructed and identified by sequencing; Western blotting was performed after transfection with HepG2 cells After transfecting HepG2 cells with pcDNA3.1 (-) - hHGF and pcDNA3.1 (-) vectors respectively, the mRNA was extracted and reverse transcribed into cDNA. The differentially expressed genes were analyzed by expression profile microarray. Results: The constructed expression vector was confirmed by restriction enzyme digestion and sequencing. The expression of hHGF in HepG2 cells was confirmed by Western blotting. The gene expression profile of 20000 genes was significantly up-regulated by the analysis of 20000 genes, and 88 genes The expression level was significantly down-regulated.Conclusion: The differentially expressed genes after HGF transfection into HepG2 cells were screened out, which provided an important basis for the study of multiple mechanisms of action in HepG2 cells.