本文選題：白細(xì)胞介素-15(IL-15) + 腎癌　； 參考：《南京醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：考察一種新型膜結(jié)合IL-15分子介導(dǎo)人腎癌細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化的分子機(jī)理和信號(hào)通路的改變。方法：1.流式細(xì)胞表面染色和免疫共沉淀檢測(cè)原代人腎癌細(xì)胞(ACHN、786-0)上膜結(jié)合型IL-15的表達(dá)；2.利用可溶性IL-15受體α鏈刺激細(xì)胞(786-0)膜上新型膜結(jié)合IL-15,免疫熒光法觀察細(xì)胞形態(tài)改變,免疫印跡法和熒光定量PCR檢測(cè)上皮、間質(zhì)表型的蛋白和mRNA的含量變化；遷移實(shí)驗(yàn)和侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞遷移和侵襲能力的變化；3.免疫印跡法檢測(cè)可溶性IL-15受體α鏈在不同作用時(shí)間以及不同劑量下對(duì)Src、Erk和Akt激酶蛋白表達(dá)水平的影響；4.免疫熒光法和免疫共沉淀檢測(cè)GSK-3β和β-catenin蛋白的變化,免疫共沉淀檢測(cè)Akt激酶和GSK-3β相互作用的關(guān)系；5.免疫印跡法檢測(cè)FAK激酶與Src激酶的作用關(guān)系,遷移實(shí)驗(yàn)和侵襲實(shí)驗(yàn)觀察FAK激酶對(duì)細(xì)胞遷移和侵襲能力的影響。6.收集一定數(shù)量的正常腎組織標(biāo)本和腎癌組織標(biāo)本,對(duì)其進(jìn)行熒光定量PCR和免疫組化,比較IL-15分子的mRNA水平和蛋白表達(dá)水平。結(jié)果：1.腎癌細(xì)胞株上可高表達(dá)分子量為27 kDa的膜結(jié)合型IL-15分子2.新型膜結(jié)合型IL-15分子在可溶性IL-15受體α鏈(1OOng/ml,4天)的刺激下可促使細(xì)胞形態(tài)由鵝卵石樣變成成纖維樣；同時(shí)上皮表型E-Cadherin、ZO-1的蛋白和mRNA水平發(fā)生下調(diào),間質(zhì)表型vimentin、N-Cadherin的蛋白和mRNA水平發(fā)生上調(diào)；細(xì)胞的遷移和侵襲能力也在刺激后明顯增強(qiáng)。3. Src、Erk和Akt激酶在IL-15受體α鏈的刺激下均可發(fā)生明顯的磷酸化,而TGF-p通路不發(fā)生明顯改變。運(yùn)用Src特異性抑制劑PP2抑制上皮間質(zhì)過(guò)程可發(fā)現(xiàn)Erk和Akt具有Src依賴(lài)性,即Src是PI3K/AKT通路和Ras/ERK通路的上游調(diào)控分子。運(yùn)用Erk特異性抑制劑PD58059和Akt特異性抑制劑MK2206可發(fā)現(xiàn)Akt激酶,而非Erk激酶,是真正介導(dǎo)上皮間質(zhì)轉(zhuǎn)化的分子。4.AKT激酶與GSK-3β可直接作用結(jié)合,而GSK-3β則引起β-catenin的變化,使β-catenin在刺激后發(fā)生入核。5.在siRNA-FAK干擾FAK激酶表達(dá)的情況下,細(xì)胞遷移和侵襲能力能大大下降；并且發(fā)現(xiàn)Src激酶作用于FAK激酶上游,二者可能形成復(fù)合物調(diào)控細(xì)胞運(yùn)動(dòng)、粘附的功能。6.IL-15的mRNA和蛋白表達(dá)水平在腎癌組織中較正常腎組織中明顯增高。結(jié)論：1.腎腎癌細(xì)胞上高表達(dá)新型膜結(jié)合型IL-15。2.新型膜結(jié)合型IL-15在可溶性IL-15受體α鏈刺激下,可通過(guò)Src激酶介導(dǎo)PI3K/AKT通路發(fā)生磷酸化,并誘導(dǎo)GSK-3β/β-catenin通路變化介導(dǎo)β-catenin入核,從而使細(xì)胞發(fā)生上皮間質(zhì)轉(zhuǎn)化,最終促進(jìn)細(xì)胞遷移和侵襲的進(jìn)展。3.FAK激酶在Src激酶介導(dǎo)下,對(duì)細(xì)胞遷移和侵襲能力起到不可或缺的調(diào)控作用。4.IL-15的表達(dá)水平在腎癌組織中高于正常腎組織,IL-15可能有助于腎癌的發(fā)展。
[Abstract]:Aim: to investigate the molecular mechanism and signal pathway of epithelial interstitial transformation of human renal cancer cells mediated by a novel membrane binding IL-15 molecule. Method 1: 1. Flow cytometry and co-immunoprecipitation were used to detect the expression of supermembrane binding IL-15 in primary human renal cell carcinoma cell line ACHN 786-0. A novel membrane of soluble IL-15 receptor 偽 chain stimulating cells was used to bind IL-15. The morphological changes of cells were observed by immunofluorescence, the epithelial cells were detected by immunoblotting and fluorescence quantitative PCR, and the contents of protein and mRNA in interstitial phenotypes were detected. Migration assay and invasion assay were used to detect the change of cell migration and invasion ability. The effect of soluble IL-15 receptor 偽 chain on the expression of SRC Erk and Akt kinase protein at different time and dose was detected by Western blot. The changes of GSK-3 尾 and 尾 -catenin protein were detected by immunofluorescence and co-immunoprecipitation, and the relationship between Akt kinase and GSK-3 尾 was detected by immunoprecipitation. The relationship between FAK kinase and Src kinase was detected by Western blot. The effect of FAK kinase on cell migration and invasion was observed by migration assay and invasion assay. A certain number of normal renal tissue specimens and renal cell carcinoma tissue specimens were collected, and the fluorescence quantitative PCR and immunohistochemistry were used to compare the mRNA level and protein expression level of IL-15 molecule. The result is 1: 1. The membrane-binding IL-15 molecule with molecular weight of 27 kDa can be highly expressed in the cell line of renal cell carcinoma (RCC). A novel membrane-binding IL-15 molecule stimulated by soluble IL-15 receptor 偽 1 O O Ong / ml for 4 days could induce cell morphology from cobblestone to fibroid, and down-regulate the protein and mRNA levels of E-Cadherinine ZO-1 in epithelial phenotype. The level of protein and mRNA in mesenchymal phenotype of N-Cadherin was up-regulated, and the ability of migration and invasion of cells was also significantly increased after stimulation. Both SRC Erk and Akt kinase were significantly phosphorylated under the stimulation of 偽 chain of IL-15 receptor, but the TGF-p pathway did not change significantly. It was found that Erk and Akt were Src dependent by PP2, a specific inhibitor of Src. Src is the upstream regulator of PI3K/AKT and Ras/ERK pathways. Using PD58059, a specific inhibitor of Erk, and MK2206, a specific inhibitor of Akt, can find that Akt kinase, rather than Erk kinase, is a molecule that really mediates epithelial mesenchymal transformation. AK T kinase binds directly to GSK-3 尾, while GSK-3 尾 induces the change of 尾 -catenin. Make 尾 -catenin enter nucleus after stimulation. When siRNA-FAK interfered with the expression of FAK kinase, the ability of cell migration and invasion was significantly decreased, and it was found that Src kinase acted on the upstream of FAK kinase, which might form a complex to regulate cell movement. The expression of mRNA and protein in adhesion function. 6. IL-15 was significantly higher in RCC than in normal renal tissue. Conclusion 1. A novel membrane binding type IL-15.2is highly expressed on renal cancer cells. Under the stimulation of soluble IL-15 receptor 偽 chain, novel membrane-binding IL-15 can mediate the phosphorylation of PI3K/AKT pathway through Src kinase, and induce the change of GSK-3 尾 / 尾 -catenin pathway to induce 尾 -catenin to enter the nucleus, resulting in the epithelial interstitial transformation of cells. Finally promote the progress of cell migration and invasion. 3. FAK kinase, mediated by Src kinase, plays an indispensable role in regulating cell migration and invasion. 4. IL-15 expression level in renal cell carcinoma is higher than that in normal renal tissue, which may contribute to the development of renal cell carcinoma.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R737.11

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