本文選題：NPRL2　切入點(diǎn)：mTORC1　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的 將原核表達(dá)的NPRL2(Nitrogen permease regulator 2-like)融合蛋白通過(guò)胞漿轉(zhuǎn)導(dǎo)肽(Cytoplasmic transduction peptide,CTP)轉(zhuǎn)入到腎癌原代細(xì)胞胞漿內(nèi),觀察其對(duì)腎癌原代細(xì)胞遷移侵襲能力的影響,并觀察其與腎癌的上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)的關(guān)系。方法 分別使用IV型膠原酶消化與在濾網(wǎng)上研磨的方法培養(yǎng)腎癌原代細(xì)胞;構(gòu)建原核表達(dá)質(zhì)粒p ET15b-CTP-NPRL2和p ET15b-NPRL2,表達(dá)融合蛋白CTP-NPRL2和NPRL2,并通過(guò)western blot鑒定融合蛋白的表達(dá)。通過(guò)免疫熒光實(shí)驗(yàn)檢測(cè)融合蛋白的定位情況;通過(guò)遷徙侵襲實(shí)驗(yàn)檢測(cè)導(dǎo)入融合蛋白后,對(duì)腎癌原代細(xì)胞的影響;Real-Time PCR檢測(cè)Raptor、E-cadherin、Vimentin、Fibronectin m RNA表達(dá)水平;Western Blot檢測(cè)Raptor、E-cadherin、Vimentin、Fibronectin蛋白表達(dá)水平。結(jié)果 培養(yǎng)出腎癌原代細(xì)胞并通過(guò)流式細(xì)胞術(shù)與免疫組化鑒定;Western Blot結(jié)果提示重組質(zhì)粒成功表達(dá)出目的融合蛋白;免疫熒光提示CTP-NPRL2融合蛋白可以穿過(guò)胞膜并定位于胞漿;遷移和侵襲實(shí)驗(yàn)顯示CTP-NPRL2組腎癌細(xì)胞遷移侵襲能力明顯降低;Real-Time PCR和Western Blot顯示CTP-NPRL2組Raptor、Vimentin與Fibronectin的m RNA、蛋白表達(dá)水平與NPRL2組和BLANK組相比降低(P0.05),而E-cadherin的m RNA、蛋白表達(dá)水平與NPRL2組與BLANK組相比升高(P0.05)。結(jié)論 NPRL2可以影響腎癌原代細(xì)胞遷移能力,可能是通過(guò)對(duì)Raptor表達(dá)的影響,改變m TORC1活性,進(jìn)而調(diào)節(jié)EMT相關(guān)蛋白的表達(dá)量,改變了EMT進(jìn)程,進(jìn)而影響腎癌原代細(xì)胞的遷移侵襲能力。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Adrian Husillos Alonso;Manuel Carbonero García;Carmen González Enguita;;Is there a role for systemic targeted therapy after surgical treatment for metastases of renal cell carcinoma?[J];World Journal of Nephrology;2015年02期

2 Minal Garg;;Epithelial-mesenchymal transition-activating transcription factors-multifunctional regulators in cancer[J];World Journal of Stem Cells;2013年04期

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本文編號(hào)：1619477