过表达Glypican-3对高转移性肝癌细胞HCCLM3生物学行为的影响

发布时间：2019-06-28 17:59

【摘要】：旨在构建s GPC3/GPC3基因重组真核表达载体,研究过表达s GPC3/GPC3对HCCLM3人高转移性肝癌细胞生物学行为的影响。采用PCR法获得编码s GPC3/GPC3的基因片段并克隆到真核表达载体pc DNA3.0-GFP中,经脂质体介导转染HCCLM3肝癌细胞,RT-PCR和Western blot检测GPC3的表达情况;CCK-8法、克隆平板形成实验、细胞划痕实验、流式细胞术分别检测s GPC3/GPC3对HCCLM3生物学行为的影响。酶切分析、测序鉴定结果表明s GPC3/GPC3基因重组真核表达载体构建成功;RT-PCR和Western blot结果显示s GPC3/GPC3在HCCLM3中成功过表达。与正常组和转染空载体相比,过表达s GPC3/GPC3后,HCCLM3的增殖能力和克隆形成能力均受到明显抑制(P0.05),迁移率显著下降,细胞周期阻滞于G1期,细胞凋亡率增加。同时,过表达s GPC3对细胞的影响显著优于GPC3(P0.05)。s GPC3/GPC3表达上调抑制高转移性肝癌细胞HCCLM3生长和迁移,促进细胞凋亡以及周期再分布,在高转移性肝癌发生发展过程中起重要作用。
[Abstract]:The aim of this study was to construct a recombinant eukaryotic expression vector of s GPC3/GPC3 gene and to study the effect of overexpression of s GPC3/GPC3 on the biological behavior of HCCLM3 human highly metastatic hepatocellular carcinoma cells. The gene fragment encoding s GPC3/GPC3 was obtained by PCR and cloned into eukaryotic expression vector pc DNA3.0-GFP. The expression of GPC3 was detected by RT-PCR and Western blot, and the effect of s GPC3/GPC3 on the biological behavior of HCCLM3 was detected by CCK- 8, clone plate formation test, cell scratch test and flow cytometry, respectively. The results of restriction enzyme digestion and sequencing showed that the recombinant eukaryotic expression vector of s GPC3/GPC3 gene was successfully constructed, and the results of RT-PCR and Western blot showed that s GPC3/GPC3 was successfully overexpressed in HCCLM3. Compared with the normal group and the empty vector, the proliferation and clone formation ability of HCCLM3 were significantly inhibited after overexpression of s GPC3/GPC3 (P 0.05), the mobility decreased significantly, the cell cycle was blocked in G 1 phase, and the apoptosis rate increased. At the same time, the effect of overexpression of s GPC3 on cells was significantly better than that of GPC3 (P 0.05). S GPC3/GPC3 expression up-regulated inhibition of HCCLM3 growth and migration, promoting apoptosis and cycle redistribution, which played an important role in the occurrence and development of high metastasis HCC.
【作者单位】：广东药科大学基础学院广东省生物活性药物研究重点实验室;
【基金】：国家自然科学基金项目(81502520) 广东省自然科学基金项目(2016A030310299) 广东省医学科研基金项目(A2016091)
【分类号】：R735.7

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