PEP-1介导的HNF4α重组蛋白转导对肝癌细胞的作用

发布时间：2018-04-22 13:32

本文选题：肝细胞癌 + HNF4α　；参考：《华东理工大学》2015年硕士论文

【摘要】：肝细胞核因子4a (HNF4a)是核受体超家族成员之一,在肝细胞的分化和功能维持中起着重要的作用。细胞穿膜肽(Cell Penetrating Peptides, CPPs)是一类由少于30个氨基酸组成的多肽,可以有效地将目标分子包括蛋白、RNA、DNA等导入细胞。本课题利用细胞穿膜肽PEP-1介导重组肝细胞核因子4a (HNF4a)蛋白进入肝癌细胞,并明确外源融合蛋白P-HNF4a对肝癌细胞的作用。我们构建了能够表达P-HNF4a的表达质粒pET28a-P-HNF4a,并优化了P-HNF4a的表达与纯化条件,经浓缩、透析后获得浓度为2.1μM、纯度为81%的融合蛋白P-HNF4a;成功将P-HNF4a转导进入肝癌细胞,利用Western blot检测其穿膜效率,发现穿膜效率高达69.5%,显示PEP-1可高效地携带HNF4a进入细胞；核浆分离和细胞免疫荧光检测表明P-HNF4a进入细胞后定位在细胞核,荧光素酶报告基因实验发现P-HNF4a可促进下游基因的表达,提示P-HNF4a具有转录因子的功能；P-HNF4a可促进Huh7细胞肝功能基因的表达,并抑制干细胞相关基因的表达；利用CCK-8法检测肝癌细胞的增殖,细胞划痕实验、小室迁移实验以及小室侵袭实验检测肝癌细胞转移能力,结果显示P-HNF4a显著抑制肝癌细胞的增殖、迁移和侵袭能力。以上结果提示P-HNF4a可诱导肝癌细胞向成熟肝细胞分化,抑制肝癌细胞的恶性生物学行为,表明利用P-HNF4a转导肝癌细胞是诱导分化治疗肝癌的潜在手段。
[Abstract]:HNF4a) is a member of the nuclear receptor superfamily and plays an important role in the differentiation and functional maintenance of hepatocytes. Cell Penetrating Peptides (CPPs) is a class of peptides consisting of less than 30 amino acids, which can effectively transfer target molecules, including protein RNA-DNA, into cells. In this study, recombinant hepatocyte nuclear factor 4a HNF4a (HNF4a) protein was transfected into hepatoma cells by transmembrane peptide PEP-1, and the effect of exogenous fusion protein P-HNF4a on hepatoma cells was clarified. We constructed the expression plasmid pET28a-P-HNF4aand optimized the expression and purification conditions of P-HNF4a. After dialysis, we obtained a fusion protein P-HNF4a with a concentration of 2.1 渭 M and a purity of 81%. P-HNF4a was successfully transduced into hepatoma cells. Using Western blot to detect the transmembrane efficiency, it was found that the transmembrane efficiency was as high as 69.5%, which showed that PEP-1 could efficiently carry HNF4a into the cells, and the nuclear and cytoplasmic separation and cellular immunofluorescence assay showed that P-HNF4a was located in the nucleus after entering the cell. Luciferase reporter gene experiment found that P-HNF4a could promote the expression of downstream genes, suggesting that P-HNF4a has the function of transcription factor, P-HNF4a can promote the expression of liver function genes in Huh7 cells, and inhibit the expression of stem cell related genes. CCK-8 assay was used to detect the proliferation of hepatoma cells, cell scratch assay, cell migration assay and compartment invasion assay. The results showed that P-HNF4a significantly inhibited the proliferation, migration and invasion of hepatoma cells. These results suggest that P-HNF4a can induce the differentiation of HCC cells into mature hepatocytes and inhibit the malignant biological behavior of HCC cells. It is suggested that P-HNF4a transduction of HCC cells is a potential means of inducing differentiation and treatment of HCC.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

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