核心蛋白聚糖（decorin）真核表达载体的构建及其对肝癌细胞（HepG2）作用机制的实验研究

发布时间：2018-01-19 08:52

本文关键词： 核心蛋白聚糖肝癌 P21~(WAF1/CIP1) TGF-β_1 caspase-3 caspase-8　出处：《吉林大学》2008年博士论文　论文类型：学位论文

【摘要】： 核心蛋白聚糖(decorin,DCN)属于分泌型、小分子、富含亮氨酸多糖基因扩展家族成员之一,主要存在于结缔组织中并且是一种与胶原纤维相关的蛋白多糖,研究发现它能抑制细胞增殖和抗纤维化,具有抗肿瘤的作用。目前国内外还没有decorin基因转染到肝癌细胞HepG2中以及研究decorin抗肝癌机制的报道,本实验采用体外基因重组技术构建了真核表达载体pcDNA3.1-decorin,并且转染到肝癌细胞HepG2中,探讨其对肝癌细胞的作用机理。本研究成功构建了真核表达载体pcDNA3.1-decorin,并转染到肝癌细胞HepG2中;结果发现decorin能够抑制肝癌细胞HepG2生长,FCM方法检测细胞周期发现pcDNA3.1-dec-HepG2组G0/G1期细胞比pcDNA3.1-HepG2组G0/G1期细胞明显升高、S期细胞显著降低,细胞生长停滞在G0/G1期; Caspase-3、Caspase-8活性检测发现pcDNA3.1-dec-HepG2组细胞比pcDNA3.1-HepG2组细胞显著升高,RT-PCR和western blot方法显示pcDNA3.1-dec-HepG2组细胞周期依赖激酶抑制剂P21 mRNA和蛋白质比pcDNA3.1-HepG2组细胞显著升高, RT-PCR方法显示pcDNA3.1-dec-HepG2组细胞转化生长因子TGF-β1比pcDNA3.1-HepG2组细胞显著降低。上述结果为进一步研究decorin抗肿瘤的作用机制奠定了基础,同时也为decorin的临床应用提供了理论依据。
[Abstract]:Core protein decorin (DCNs) belongs to secretory type, small molecule, rich in leucine polysaccharide gene expansion family members. It is mainly found in connective tissue and is a proteoglycan associated with collagen fiber. It has been found that it can inhibit cell proliferation and resist fibrosis. At present, there are no reports of decorin gene transfection into HCC HepG2 and study of the mechanism of decorin anti-HCC. In this study, eukaryotic expression vector pcDNA3.1-decorin was constructed by in vitro gene recombination technique and transfected into hepatoma cell HepG2. In this study, eukaryotic expression vector pcDNA3.1-decorin was constructed and transfected into hepatoma cell HepG2. The results showed that decorin could inhibit the growth of HepG2 cells. FCM assay showed that G0 / G1 phase cells in pcDNA3.1-dec-HepG2 group were significantly higher than those in pcDNA3.1-HepG2 group. S phase cells decreased significantly, cell growth stopped at G 0 / G 1 phase. The activity of Caspase-3 and Caspase-8 in pcDNA3.1-dec-HepG2 group was significantly higher than that in pcDNA3.1-HepG2 group. Expression of cell cycle dependent kinase inhibitor P21 in pcDNA3.1-dec-HepG2 group by RT-PCR and western blot. MRNA and protein were significantly higher than those in pcDNA3.1-HepG2 group. RT-PCR method showed that TGF- 尾 1 in pcDNA3.1-dec-HepG2 group was significantly lower than that in pcDNA3.1-HepG2 group. It lays a foundation for further study on the mechanism of anti-tumor effect of decorin. It also provides a theoretical basis for the clinical application of decorin.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

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