發(fā)布時間：2019-06-14 05:49

【摘要】：第一部分研究XAF1基因表達在sublytic C5b-9誘導大鼠GMC凋亡中的作用 目的：檢查亞溶解型C5b-9（sublytic C5b-9）復合物刺激大鼠腎小球系膜細胞（glomerular mesangial cells, GMC）后對其X染色體連鎖的凋亡抑制蛋白相關(guān)因子1（X-linked inhibitor of apoptosis associated factor1, XAF1）表達的影響，并探討XAF1基因表達在sublytic C5b-9誘導大鼠GMC凋亡中的作用。 方法：首先通過Western blot檢查sublytic C5b-9刺激不同時間的GMC中XAF1蛋白的表達情況，并測定不同分組處理的GMC中XAF1蛋白的表達水平。然后，構(gòu)建XAF1真核表達質(zhì)粒（pEGFP-N1/XAF1）和XAF1發(fā)夾狀小干涉RNA（shorthairpin RNA, shRNA）表達質(zhì)粒，并將上述質(zhì)粒分別轉(zhuǎn)染入GMC，其中XAF1shRNA（shXAF1）轉(zhuǎn)染48h后再給予sublytic C5b-9刺激6h，用Western blot方法檢查各組GMC中XAF1蛋白的表達情況，并通過流式細胞術(shù)測定GMC的凋亡數(shù)量。 結(jié)果：Sublytic C5b-9刺激GMC后可明顯上調(diào)XAF1蛋白的表達（刺激后6h達到高峰）。pEGFP-N1/XAF1質(zhì)粒轉(zhuǎn)染GMC能顯著上調(diào)XAF1蛋白的表達，并明顯增加GMC的凋亡數(shù)量。而shXAF1質(zhì)粒的轉(zhuǎn)染可明顯下調(diào)sublytic C5b-9刺激GMC誘導的XAF1蛋白的表達，并有效抑制sublytic C5b-9引發(fā)的GMC凋亡。 結(jié)論：Sublytic C5b-9刺激大鼠GMC后可通過上調(diào)XAF1基因的表達促進GMC的凋亡病變。 第二部分探討sublytic C5b-9誘導上調(diào)的IRF-1對大鼠GMC表達XAF1基因的調(diào)控作用及其機制 目的：研究sublytic C5b-9刺激大鼠GMC后誘導上調(diào)的干擾素調(diào)節(jié)因子1（interferon regulatory factor1, IRF-1）對XAF1基因轉(zhuǎn)錄的調(diào)控作用及其機制，并進一步探討IRF-1表達對sublytic C5b-9刺激誘導的GMC凋亡的影響。 方法：采用PCR技術(shù)，擴增出大鼠XAF1基因啟動子全長序列（-1490~+157nt），并將其插入到熒光素酶報告基因載體pGL3-basic中，得到pGL3-XAF1報告質(zhì)粒。隨后將pGL3-XAF1分別與IRF-1真核表達載體（pcDNA3.1/IRF-1）或IRF-1shRNA（shIRF-1）共轉(zhuǎn)染GMC，再用sublytic C5b-9刺激6h，測定不同處理組的GMC中其相應的熒光素酶活性。同時，應用生物信息學軟件（TFsearch）預測XAF1基因啟動子上IRF-1可能的結(jié)合位點，并據(jù)此構(gòu)建XAF1基因啟動子截斷的熒光素酶報告質(zhì)粒（即pGL3-XAF1-1、pGL3-XAF1-2、pGL3-XAF1-3和pGL3-XAF1-4）。將上述XAF1基因啟動子全長和各截斷的熒光素酶報告質(zhì)粒和pcDNA3.1/IRF-1共轉(zhuǎn)染GMC，再行熒光素酶活性測定，篩選IRF-1的結(jié)合區(qū)域。之后，針對此區(qū)域IRF-1可能的結(jié)合位點設計引物，通過染色質(zhì)免疫共沉淀（chromatin immunoprecipitation, ChIP）實驗進一步確證XAF1啟動子區(qū)IRF-1的結(jié)合位點。此外，將pcDNA3.1/IRF-1和shIRF-1分別轉(zhuǎn)染GMC，48h后再給予sublytic C5b-9刺激6h，通過Western blot檢測各組GMC中XAF1蛋白的表達，并用流式細胞術(shù)檢測GMC的凋亡情況。 結(jié)果：pcDNA3.1/IRF-1載體轉(zhuǎn)染GMC后可顯著增強XAF1基因啟動子活性，而shIRF-1能明顯抑制由sublytic C5b-9刺激GMC誘導的XAF1基因啟動子活性的上調(diào)。進一步的XAF1基因啟動子截斷和ChIP實驗結(jié)果均表明，在大鼠XAF1基因啟動子的-337~-47nt區(qū)域存在IRF-1的結(jié)合位點。再者，pcDNA3.1/IRF-1載體轉(zhuǎn)染GMC能顯著上調(diào)XAF1蛋白的表達，并使GMC的凋亡率顯著增加。而用shIRF-1處理GMC后則能明顯抑制由sublytic C5b-9刺激誘導的XAF1蛋白的表達以及GMC的凋亡反應。 結(jié)論：Sublytic C5b-9刺激大鼠GMC后誘導上調(diào)的IRF-1可啟動XAF1基因的轉(zhuǎn)錄，，進而促進GMC的凋亡反應。 第三部分：研究CBP/p300乙�；揎桰RF-1對sublytic C5b-9誘導GMC表達XAF1基因及GMC凋亡的調(diào)控作用 目的：探討sublytic C5b-9刺激GMC誘導的cAMP反應元件結(jié)合蛋白（cAMPresponse element-binding protein, CBP）和它的同源物p300與IRF-1結(jié)合及其對IRF-1的乙�；揎�，并進一步研究XAF1的表達和GMC的凋亡情況。 方法：首先通過免疫共沉淀（co-immunoprecipitation, Co-IP）實驗檢查sublyticC5b-9刺激GMC所誘導的CBP/p300與IRF-1結(jié)合以及IRF-1的乙�；闆r。此外，通過將p300shRNA（shp300）轉(zhuǎn)染入GMC沉默p300基因的表達后，再用Western blot檢查其對sublytic C5b-9誘導的GMC中IRF-1、XAF1基因表達的影響，并通過Co-IP實驗檢查對IRF-1乙�；挠绊�，同時用流式細胞術(shù)測定GMC的凋亡情況。 結(jié)果：Sublytic C5b-9刺激GMC能夠誘導p300與IRF-1結(jié)合以及上調(diào)IRF-1乙�；�，而沉默p300基因可明顯抑制由Sublytic C5b-9誘導的IRF-1蛋白的乙�；揎�、XAF1蛋白的表達及GMC的凋亡反應。 結(jié)論：Sublytic C5b-9刺激GMC后誘導表達的p300能促進IRF-1的乙�；�，而此乙�；揎椏娠@著增強IRF-1與XAF1基因啟動子區(qū)的結(jié)合，從而促進XAF1基因的轉(zhuǎn)錄和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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392

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