新型膜结合IL-15分子介导人肾癌细胞上皮间质转化的分子机理

发布时间：2018-05-02 11:58

本文选题：白细胞介素-15(IL-15) + 肾癌　；参考：《南京医科大学》2014年硕士论文

【摘要】：目的：考察一种新型膜结合IL-15分子介导人肾癌细胞发生上皮间质转化的分子机理和信号通路的改变。方法：1.流式细胞表面染色和免疫共沉淀检测原代人肾癌细胞(ACHN、786-0)上膜结合型IL-15的表达；2.利用可溶性IL-15受体α链刺激细胞(786-0)膜上新型膜结合IL-15,免疫荧光法观察细胞形态改变,免疫印迹法和荧光定量PCR检测上皮、间质表型的蛋白和mRNA的含量变化；迁移实验和侵袭实验检测细胞迁移和侵袭能力的变化；3.免疫印迹法检测可溶性IL-15受体α链在不同作用时间以及不同剂量下对Src、Erk和Akt激酶蛋白表达水平的影响；4.免疫荧光法和免疫共沉淀检测GSK-3β和β-catenin蛋白的变化,免疫共沉淀检测Akt激酶和GSK-3β相互作用的关系；5.免疫印迹法检测FAK激酶与Src激酶的作用关系,迁移实验和侵袭实验观察FAK激酶对细胞迁移和侵袭能力的影响。6.收集一定数量的正常肾组织标本和肾癌组织标本,对其进行荧光定量PCR和免疫组化,比较IL-15分子的mRNA水平和蛋白表达水平。结果：1.肾癌细胞株上可高表达分子量为27 kDa的膜结合型IL-15分子2.新型膜结合型IL-15分子在可溶性IL-15受体α链(1OOng/ml,4天)的刺激下可促使细胞形态由鹅卵石样变成成纤维样；同时上皮表型E-Cadherin、ZO-1的蛋白和mRNA水平发生下调,间质表型vimentin、N-Cadherin的蛋白和mRNA水平发生上调；细胞的迁移和侵袭能力也在刺激后明显增强。3. Src、Erk和Akt激酶在IL-15受体α链的刺激下均可发生明显的磷酸化,而TGF-p通路不发生明显改变。运用Src特异性抑制剂PP2抑制上皮间质过程可发现Erk和Akt具有Src依赖性,即Src是PI3K/AKT通路和Ras/ERK通路的上游调控分子。运用Erk特异性抑制剂PD58059和Akt特异性抑制剂MK2206可发现Akt激酶,而非Erk激酶,是真正介导上皮间质转化的分子。4.AKT激酶与GSK-3β可直接作用结合,而GSK-3β则引起β-catenin的变化,使β-catenin在刺激后发生入核。5.在siRNA-FAK干扰FAK激酶表达的情况下,细胞迁移和侵袭能力能大大下降；并且发现Src激酶作用于FAK激酶上游,二者可能形成复合物调控细胞运动、粘附的功能。6.IL-15的mRNA和蛋白表达水平在肾癌组织中较正常肾组织中明显增高。结论：1.肾肾癌细胞上高表达新型膜结合型IL-15。2.新型膜结合型IL-15在可溶性IL-15受体α链刺激下,可通过Src激酶介导PI3K/AKT通路发生磷酸化,并诱导GSK-3β/β-catenin通路变化介导β-catenin入核,从而使细胞发生上皮间质转化,最终促进细胞迁移和侵袭的进展。3.FAK激酶在Src激酶介导下,对细胞迁移和侵袭能力起到不可或缺的调控作用。4.IL-15的表达水平在肾癌组织中高于正常肾组织,IL-15可能有助于肾癌的发展。
[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 伪 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 伪 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 伪 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 伪 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 伪 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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.11

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