CTP-NPRL2融合蛋白对肾癌原代细胞迁移侵袭能力的影响

发布时间：2018-03-16 10:16

本文选题：NPRL2　切入点：mTORC1　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的将原核表达的NPRL2(Nitrogen permease regulator 2-like)融合蛋白通过胞浆转导肽(Cytoplasmic transduction peptide,CTP)转入到肾癌原代细胞胞浆内,观察其对肾癌原代细胞迁移侵袭能力的影响,并观察其与肾癌的上皮间质转化(epithelial-mesenchymal transition,EMT)的关系。方法分别使用IV型胶原酶消化与在滤网上研磨的方法培养肾癌原代细胞;构建原核表达质粒p ET15b-CTP-NPRL2和p ET15b-NPRL2,表达融合蛋白CTP-NPRL2和NPRL2,并通过western blot鉴定融合蛋白的表达。通过免疫荧光实验检测融合蛋白的定位情况;通过迁徙侵袭实验检测导入融合蛋白后,对肾癌原代细胞的影响;Real-Time PCR检测Raptor、E-cadherin、Vimentin、Fibronectin m RNA表达水平;Western Blot检测Raptor、E-cadherin、Vimentin、Fibronectin蛋白表达水平。结果培养出肾癌原代细胞并通过流式细胞术与免疫组化鉴定;Western Blot结果提示重组质粒成功表达出目的融合蛋白;免疫荧光提示CTP-NPRL2融合蛋白可以穿过胞膜并定位于胞浆;迁移和侵袭实验显示CTP-NPRL2组肾癌细胞迁移侵袭能力明显降低;Real-Time PCR和Western Blot显示CTP-NPRL2组Raptor、Vimentin与Fibronectin的m RNA、蛋白表达水平与NPRL2组和BLANK组相比降低(P0.05),而E-cadherin的m RNA、蛋白表达水平与NPRL2组与BLANK组相比升高(P0.05)。结论 NPRL2可以影响肾癌原代细胞迁移能力,可能是通过对Raptor表达的影响,改变m TORC1活性,进而调节EMT相关蛋白的表达量,改变了EMT进程,进而影响肾癌原代细胞的迁移侵袭能力。
[Abstract]:Objective to transfer the NPRL2(Nitrogen permease regulator 2-like fusion protein into the primary cytoplasm of renal cell carcinoma (RCC) through cytoplasmic transduction peptide (Cytoplasmic transduction peptide CTP), and to observe its effect on the migration and invasion of primary RCC cells. The relationship between EMTs and epithelial-mesenchymal transition (EMT) of renal cell carcinoma was observed. Methods Primary cells of renal carcinoma were cultured by type IV collagenase digestion and grinding on the filter. Prokaryotic expression plasmids p ET15b-CTP-NPRL2 and pET15b-NPRL2 were constructed to express the fusion protein CTP-NPRL2 and NPRL2. The expression of the fusion protein was identified by western blot. The localization of the fusion protein was detected by immunofluorescence assay. The effect of Real-Time PCR on the expression of Raptorus E-cadherin fibronectin RNA and the expression of Raptorus E-cadherin PCR protein in primary RCC cells were detected by Real-Time PCR. Results the primary cell lines of RCC were cultured and identified by flow cytometry and immunohistochemistry. The results of Western Blot showed that the recombinant plasmids were constructed. The fusion protein was successfully expressed. Immunofluorescence showed that the CTP-NPRL2 fusion protein could pass through the cell membrane and be located in the cytoplasm. Migration and invasion assay showed that the migration and invasion ability of renal cancer cells in CTP-NPRL2 group was significantly lower than that in NPRL2 group and BLANK group, while the expression level of E-cadherin mRNA and protein in CTP-NPRL2 group was significantly lower than that in NPRL2 group and BLANK group. The expression level of E-cadherin mRNA and protein in CTP-NPRL2 group was significantly lower than that in NPRL2 group. The mRNA expression level of Fibronectin and Vimentin in CTP-NPRL2 group was significantly lower than that in NPRL2 group and BLANK group. Conclusion NPRL2 can affect the migration ability of primary RCC cells. It may be that the expression of Raptor changes the activity of m TORC1, and then regulates the expression of EMT related protein, which changes the process of EMT, and then affects the ability of migration and invasion of primary cell line of renal cell carcinoma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11

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