IRF-1乙酰化调控亚溶解型C5b-9复合物诱导的大鼠肾小球系膜细胞凋亡病变的机制研究

发布时间：2019-06-14 05:49

【摘要】：第一部分研究XAF1基因表达在sublytic C5b-9诱导大鼠GMC凋亡中的作用目的：检查亚溶解型C5b-9（sublytic C5b-9）复合物刺激大鼠肾小球系膜细胞（glomerular mesangial cells, GMC）后对其X染色体连锁的凋亡抑制蛋白相关因子1（X-linked inhibitor of apoptosis associated factor1, XAF1）表达的影响，并探讨XAF1基因表达在sublytic C5b-9诱导大鼠GMC凋亡中的作用。方法：首先通过Western blot检查sublytic C5b-9刺激不同时间的GMC中XAF1蛋白的表达情况，并测定不同分组处理的GMC中XAF1蛋白的表达水平。然后，构建XAF1真核表达质粒（pEGFP-N1/XAF1）和XAF1发夹状小干涉RNA（shorthairpin RNA, shRNA）表达质粒，并将上述质粒分别转染入GMC，其中XAF1shRNA（shXAF1）转染48h后再给予sublytic C5b-9刺激6h，用Western blot方法检查各组GMC中XAF1蛋白的表达情况，并通过流式细胞术测定GMC的凋亡数量。结果：Sublytic C5b-9刺激GMC后可明显上调XAF1蛋白的表达（刺激后6h达到高峰）。pEGFP-N1/XAF1质粒转染GMC能显著上调XAF1蛋白的表达，并明显增加GMC的凋亡数量。而shXAF1质粒的转染可明显下调sublytic C5b-9刺激GMC诱导的XAF1蛋白的表达，并有效抑制sublytic C5b-9引发的GMC凋亡。结论：Sublytic C5b-9刺激大鼠GMC后可通过上调XAF1基因的表达促进GMC的凋亡病变。第二部分探讨sublytic C5b-9诱导上调的IRF-1对大鼠GMC表达XAF1基因的调控作用及其机制目的：研究sublytic C5b-9刺激大鼠GMC后诱导上调的干扰素调节因子1（interferon regulatory factor1, IRF-1）对XAF1基因转录的调控作用及其机制，并进一步探讨IRF-1表达对sublytic C5b-9刺激诱导的GMC凋亡的影响。方法：采用PCR技术，扩增出大鼠XAF1基因启动子全长序列（-1490~+157nt），并将其插入到荧光素酶报告基因载体pGL3-basic中，得到pGL3-XAF1报告质粒。随后将pGL3-XAF1分别与IRF-1真核表达载体（pcDNA3.1/IRF-1）或IRF-1shRNA（shIRF-1）共转染GMC，再用sublytic C5b-9刺激6h，测定不同处理组的GMC中其相应的荧光素酶活性。同时，应用生物信息学软件（TFsearch）预测XAF1基因启动子上IRF-1可能的结合位点，并据此构建XAF1基因启动子截断的荧光素酶报告质粒（即pGL3-XAF1-1、pGL3-XAF1-2、pGL3-XAF1-3和pGL3-XAF1-4）。将上述XAF1基因启动子全长和各截断的荧光素酶报告质粒和pcDNA3.1/IRF-1共转染GMC，再行荧光素酶活性测定，筛选IRF-1的结合区域。之后，针对此区域IRF-1可能的结合位点设计引物，通过染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）实验进一步确证XAF1启动子区IRF-1的结合位点。此外，将pcDNA3.1/IRF-1和shIRF-1分别转染GMC，48h后再给予sublytic C5b-9刺激6h，通过Western blot检测各组GMC中XAF1蛋白的表达，并用流式细胞术检测GMC的凋亡情况。结果：pcDNA3.1/IRF-1载体转染GMC后可显著增强XAF1基因启动子活性，而shIRF-1能明显抑制由sublytic C5b-9刺激GMC诱导的XAF1基因启动子活性的上调。进一步的XAF1基因启动子截断和ChIP实验结果均表明，在大鼠XAF1基因启动子的-337~-47nt区域存在IRF-1的结合位点。再者，pcDNA3.1/IRF-1载体转染GMC能显著上调XAF1蛋白的表达，并使GMC的凋亡率显著增加。而用shIRF-1处理GMC后则能明显抑制由sublytic C5b-9刺激诱导的XAF1蛋白的表达以及GMC的凋亡反应。结论：Sublytic C5b-9刺激大鼠GMC后诱导上调的IRF-1可启动XAF1基因的转录，，进而促进GMC的凋亡反应。第三部分：研究CBP/p300乙酰化修饰IRF-1对sublytic C5b-9诱导GMC表达XAF1基因及GMC凋亡的调控作用目的：探讨sublytic C5b-9刺激GMC诱导的cAMP反应元件结合蛋白（cAMPresponse element-binding protein, CBP）和它的同源物p300与IRF-1结合及其对IRF-1的乙酰化修饰，并进一步研究XAF1的表达和GMC的凋亡情况。方法：首先通过免疫共沉淀（co-immunoprecipitation, Co-IP）实验检查sublyticC5b-9刺激GMC所诱导的CBP/p300与IRF-1结合以及IRF-1的乙酰化情况。此外，通过将p300shRNA（shp300）转染入GMC沉默p300基因的表达后，再用Western blot检查其对sublytic C5b-9诱导的GMC中IRF-1、XAF1基因表达的影响，并通过Co-IP实验检查对IRF-1乙酰化的影响，同时用流式细胞术测定GMC的凋亡情况。结果：Sublytic C5b-9刺激GMC能够诱导p300与IRF-1结合以及上调IRF-1乙酰化水平，而沉默p300基因可明显抑制由Sublytic C5b-9诱导的IRF-1蛋白的乙酰化修饰、XAF1蛋白的表达及GMC的凋亡反应。结论：Sublytic C5b-9刺激GMC后诱导表达的p300能促进IRF-1的乙酰化，而此乙酰化修饰可显著增强IRF-1与XAF1基因启动子区的结合，从而促进XAF1基因的转录和GMC的凋亡。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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