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[Abstract]:The role of the first part in the study of the expression of XAF1 gene in the apoptosis of rat GMC induced by sublytic C5b-9 Objective: To study the effect of sublytic C5b-9 complex on the apoptosis of rat mesangial cells (GMC) in rat mesangial cells (GMC). Effect of the expression of XAF1 gene on the expression of XAF1 gene in the apoptosis of rat GMC induced by sublytic C5b-9 Methods: The expression of XAF1 protein in GMC with different time was first stimulated by Western blot, and the table of XAF1 protein in GMC treated with different groups was determined. And then, constructing the XAF1 eukaryotic expression plasmid (pEGFP-N1/ XAF1) and the XAF1 hairpin-shaped small interference RNA (shRNA) expression plasmid, respectively transfecting the plasmid into the GMC, wherein the XAF1 shRNA (shXAF1) is transfected for 48 hours, then the sublytic C5b-9 is stimulated for 6 hours, and the table of the XAF1 protein in each group of GMC is checked by the Western blot method. Up to date, and the level of GMC was determined by flow cytometry. Results: The expression of XAF1 protein could be up-regulated after the stimulation of GMC by Sublastic C5b-9 (6 h after stimulation) The pEGFP-N1/ XAF1 plasmid could significantly increase the expression of the XAF1 protein and increase the GMC significantly. The expression of the XAF1 protein induced by the GMC was significantly reduced by the transfection of the shXAF1 plasmid, and the sublytic C5b-9 was effectively inhibited by the transfection of the shXAF1 plasmid. Conclusion: The expression of XAF1 gene can be promoted by up-regulation of the expression of XAF1 gene after the stimulation of GMC by Sublastic C5b-9. In the second part, the expression of XAF1 in rat GMC by IRF-1 induced by sublytic C5b-9 was discussed in the second part. Objective: To study the regulation and mechanism of interferon regulatory factor 1 (IRF-1) induced by sublytic C5b-9 to induce up-regulation of GMC in rats, and to investigate the effect of regulatory factor 1 (IRF-1) on XAF1 gene. The regulation and mechanism of transcription and the further study of the expression of IRF-1 on sublytic C5b-9 Methods: The full-length sequence (-1490 ~ + 157nt) of the rat XAF1 gene promoter was amplified by PCR and inserted into the luciferase reporter gene vector pGL3-basic to get the results. The GMC was then co-transfected with IRF-1 eukaryotic expression vector (pcDNA3.1/ IRF-1) or IRF-1 shRNA (shIRF-1), and then stimulated by sublytic C5b-9 for 6 hours to determine the G of different treatment groups. In addition, a luciferase reporter plasmid (that is, pGL3-XAF1-1, pGL3-XAF1-2, pGL3-XAF1-3, pGL3-XAF1-3, pGL3-XAF1-3, pGL3-XAF1-2, pGL3-XAF1-3, And pGL3-XAF1-4). The total length of the XAF1 gene promoter and the truncated luciferase reporter plasmids and the pcDNA3.1/ IRF-1 were co-transfected with GMC, and the luciferase activity was measured again. After screening the binding region of the IRF-1, the primer was designed for the possible binding site of the region IRF-1, and the XAF1 was further confirmed by the chromatin immunoprecipitation (ChIP) experiment. In addition, pcDNA3.1/ IRF-1 and shIRF-1 were transfected into GMC and 48h, and then sublytic C5b-9 was stimulated for 6 h. The expression of XAF1 protein in GMC was detected by Western blot. Results: After the GMC was transfected with pcDNA3.1/ IRF-1 vector, the promoter activity of the XAF1 gene could be significantly enhanced, while the SHIRF-1 could significantly inhibit the stimulation of GMC induced by sublytic C5b-9. up-regulation of the promoter activity of the XAF1 gene. The further XAF1 gene promoter truncation and the results of the ChIP test show that in the rat XAF1 gene promoter-337--47n In the t region, the binding site of the IRF-1 is present. Furthermore, the transfected GMC of the pcDNA3.1/ IRF-1 vector can significantly increase the table of the XAF1 protein. The expression of XAF1 induced by sublytic C5b-9 was significantly inhibited after GMC was treated with shIRF-1. Conclusion: Sublastic C5b-9 can induce up-regulated IRF-1 induced by GMC in rats. In order to promote the apoptosis of GMC, the third part is to study the induced GM of CBP/ p300B-modified IRF-1 to the sublytic C5b-9. The purpose of the regulation and control of C-expression of XAF1 gene and GMC apoptosis is to explore the role of sublytic C5b-9 in the stimulation of GMC-induced cAMP response element-binding protein (CBP) and its homolog p300. IRF-1 binding and its ethylation modification to IRF-1, and The expression of XAF1 and the apoptosis of GMC were further studied. In addition, the expression of IRF-1 and XAF1 gene in GMC induced by sublytic C5b-9 was examined by Western blot after the expression of p300 shRNA (shp300) was transfected into GMC-silent p300 gene. The results showed that Sublastic C5b-9 stimulated the combination of p300 with IRF-1 and up-regulate the level of IRF-1, while the silent p300 gene significantly inhibited the IRF-1 protein induced by Sublastic C5b-9. Conclusion: Sublastic C5b-9 stimulates the expression of XAF1 protein and the apoptosis of GMC. Conclusion: Sublastic C5b-9 stimulates the expression of p300 after GMC, which can promote the ethylation of IRF-1, which can significantly enhance the initiation of IRF-1 and XAF1 gene.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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