本文選題：亞溶解型C5b-9(sublytic + C5b-9)�。� 參考：《南京醫(yī)科大學》2010年碩士論文

【摘要】：第一部分體外研究IRF-1在sublytic C5b-9誘導大鼠GMCs凋亡病變中的作用 目的:體外探討亞溶解型C5b-9(sublytic C5b-9)刺激大鼠腎小球系膜細胞(glomerular mesangial cells,GMCs)上調干擾素調節(jié)因子1(interferon regulatory factor 1, IRF-1)基因表達、并發(fā)凋亡現象以及半胱氨酸天冬氨酸特異性蛋白酶(Caspase)的活化情況,同時確定過表達或沉默IRF-1基因對sublytic C5b-9誘發(fā)大鼠GMCs凋亡病變的影響以及沉默IRF-1基因對cleaved Caspase 9、cleaved Caspase 8和cleaved Caspase 3蛋白表達的影響,同時,檢查使用Caspase抑制劑對sublytic C5b-9刺激和過表達IRF-1引起的GMCs凋亡現象的影響。方法:先體外培養(yǎng)大鼠GMCs并給予sublytic C5b-9刺激(實驗設不同處理對照),用RT-PCR和Western blot分別檢查受sublytic C5b-9刺激的GMCs上調IRF-1 mRNA的豐度及蛋白表達的水平,并用流式細胞術(Flow Cytometry, FCM)分析各組GMCs的凋亡情況,同時用Western blot檢查sublytic C5b-9刺激的GMCs中Caspase的活化情況。然后,分別將構建的IRF-1過表達質粒和發(fā)夾式小干涉RNA(short hairpin RNA, shRNA)質粒轉染GMCs,并將GMCs作相應的分組處理,定量分析各組GMCs的凋亡率,同時檢查轉染IRF-1 shRNA質粒的GMCs和對照組GMCs中cleaved Caspase 9、cleaved Caspase 8和cleaved Caspase 3蛋白的表達情況。此外,使用Caspase 8抑制劑(Z-IETD-FMK)和Caspase 3抑制劑(Z-DEVD-FMK)處理大鼠GMCs,觀察其對sublytic C5b-9刺激和過表達IRF-1引起的GMCs凋亡病變的影響。結果:Sublytic C5b-9刺激大鼠GMCs早期可見IRF-1表達上調并伴隨GMCs的凋亡以及Caspase的活化,過表達IRF-1可促進sublytic C5b-9刺激引起的GMCs凋亡病變,而用IRF-1 shRNA不僅可以使sublytic C5b-9刺激的GMCs的凋亡率下降,也可以減少sublytic C5b-9刺激的GMCs中上調cleaved Caspase 8和cleaved Caspase 3的表達量,但對cleaved Caspase 9表達的影響并不顯著。此外,應用Caspase 8抑制劑(Z-IETD-FMK)和Caspase 3抑制劑(Z-DEVD-FMK)亦可顯著減輕由sublytic C5b-9刺激和過表達IRF-1引起的GMCs凋亡數量。結論:Sublytic C5b-9誘導的大鼠GMCs凋亡病變可能與其上調IRF-1表達及激活Caspase有一定的關系。 第二部分體內探討沉默IRF-1基因對Thy-1N大鼠GMCs凋亡以及繼發(fā)增生病變的抑制效應 目的:探討用IRF-1 shRNA沉默大鼠腎組織內IRF-1基因對Thy-1腎炎(Thy-1 nephritis, Thy-1N)大鼠GMCs凋亡和繼發(fā)增生病變的抑制作用。方法:首先,復制大鼠Thy-1N模型,定期采用RT-PCR和Western blot方法檢查其腎組織中IRF-1和cleaved Caspase 9、8和3蛋白的表達情況。然后應用腎動脈灌注加電導入方法,將IRF-1 shRNA質粒導入大鼠腎組織,再經尾靜脈注射注射抗胸腺細胞血清(anti-thymocyte serum,ATS)復制大鼠Thy-1N模型。同時尾靜脈注射Caspase 8抑制劑(Z-IETD-FMK)和Caspase 3抑制劑(Z-DEVD-FMK)后再復制大鼠Thy-1N模型。分別取注射ATS后3h和7d時的大鼠腎組織,應用Western blot檢查3h時腎組織中IRF-1蛋白的表達水平及cleaved Caspase 9、8和3蛋白的表達情況。此外,應用末端轉移酶介導的dUTP缺口末端標記(terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL)技術和電鏡檢查3h時腎組織的病理學改變。另光鏡檢查計數7d時腎小球內的細胞總數,并用生化方法測定各組大鼠24h和7d時尿蛋白總量的變化。結果:大鼠Thy-1N病變3h時,腎組織中IRF-1的表達水平明顯升高。IRF-1 shRNA處理的Thy-1N大鼠(IRF-1 shRNA+Thy-1N組),其腎組織中IRF-1基因的表達以及Caspase 8和Caspase 3的活化水平顯著低于Thy-1N模型組。另IRF-1 shRNA+Thy-1N組大鼠以及經Caspase抑制劑處理的大鼠(Z-IETD-FMK+Thy-1N組和Z-DEVD-FMK+Thy-1N組),3h時腎小球內GMCs凋亡數量顯著減少。實驗7d時,腎小球細胞的增生程度有所減輕,并且,24h尿蛋白的分泌量也明顯減少。結論:沉默大鼠腎組織內IRF-1基因以及使用Caspase抑制劑均能明顯減輕Thy-1N大鼠GMCs的凋亡病變及繼發(fā)增生現象,包括尿蛋白的分泌。 第三部分體外研究sublytic C5b-9刺激大鼠GMCs啟動IRF-1轉錄的作用及其信號通路 目的:研究sublytic C5b-9刺激大鼠GMCs啟動IRF-1轉錄的作用,同時,探討sublytic C5b-9刺激GMCs上調IRF-1表達可能涉及的信號轉導通路。方法:以大鼠IRF-1啟動子全長1700bp熒光素酶報告基因為模板,用DNA重組技術將大鼠IRF-1基因轉錄起始位點上游13000bp、600bp、200bp和100bp的啟動子片段分別克隆入pGL3-Basic熒光素酶報告基因載體中,分別命名為:pGL3-Basic/IRF-1 promoter 1300bp、pGL3-Basic/IRF-1 promoter 600bp、pGL3-Basic/IRF-1 promoter 200bp和pGL3-Basic/IRF-1 promoter 100bp,酶切分析及序列測定正確后,用GenEscortTMIII轉染試劑將上述各質粒以及pGL3-Basic/3×GAS熒光素酶報告基因分別與對照質粒PRL-SV40共轉染大鼠GMCs細胞,轉染后45h行sublytic C5b-9刺激,測定Sublytic C5b-9刺激組與其他各處理組的相對熒光活性。此外,利用Western blot檢查Sublytic C5b-9刺激組與其他各對照組GMCs中一些信號通路相關蛋白,如信號傳導蛋白和轉錄激活物(signal transducers and activators of transcription, STAT)、p-STAT1、p38、p-p38、C-Jun基末端激酶(c-Jun N-terminal kinase, JNK)、p-JNK、細胞外信號調節(jié)蛋白激酶(extra cellular signal-regulated protein kinase, ERK)和p-ERK的表達情況,同時分別應用JAK抑制劑(AG490)、p38抑制劑(SB203580)、JNK抑制劑(SP600125)和ERK抑制劑(PD98059)處理大鼠GMCs,然后檢查上述抑制劑對sublytic C5b-9誘導大鼠GMCs表達p-STAT1和IRF-1蛋白的影響。結果:熒光素酶報告實驗表明,轉染pGL3-Basic/IRF-1 promoter 1700bp、pGL3-Basic/IRF-1 promoter 1300bp、pGL3-Basic/IRF-1 promoter 600bp和pGL3-Basic/IRF-1 promoter 200bp熒光素酶報告基因后再行sublytic C5b-9刺激的GMCs中的熒光素酶活性明顯高于其他各對照組,轉染pGL3-Basic/3×GAS熒光素酶報告基因的GMCs行sublytic C5b-9刺激后其熒光素酶活性升高較為顯著,而轉染pGL3-Basic/IRF-1 promoter 100bp熒光素酶報告基因的GMCs經sublytic C5b-9刺激后熒光素酶活性較其余對照組無明顯差別。另Sublytic C5b-9刺激組GMCs中的p-STAT1、p-p38、p-JNK和p-ERK表達均較其余對照組明顯上調。JAK抑制劑(AG490)可抑制sublytic C5b-9刺激GMCs后p-STAT1和IRF-1的表達量,p38抑制劑(SB203580)亦可使sublytic C5b-9刺激GMCs后p-STAT1和IRF-1的表達減少,而JNK抑制劑和ERK抑制劑對sublytic C5b-9刺激GMCs后IRF-1蛋白的表達量影響不顯著。結論:Sublytic C5b-9刺激大鼠GMCs后可上調IRF-1基因啟動子的活性,其中干擾素活化序列(IFN-gamma activated sequence, GAS)在sublytic C5b-9激活IRF-1基因轉錄中起著較大的作用,此外,sublytic C5b-9上調GMCs IRF-1基因表達可能與p38MAPK和JAK-STAT信號通路的活化存在一定的關系。
[Abstract]:The first part is to study the role of IRF-1 in sublytic C5b-9 induced apoptosis of GMCs in vitro.
Objective: To investigate in vitro C5b-9 (sublytic C5b-9) stimulated rat glomerular mesangial cells (glomerular mesangial cells, GMCs) up-regulated the expression of interferon regulatory factor 1 (interferon regulatory factor 1, IRF-1), apoptosis and the activation of cysteine aspartate specific protease (Caspase). The effects of the expression or silence of IRF-1 gene on the apoptosis of GMCs apoptosis induced by sublytic C5b-9 and the effect of silent IRF-1 gene on the expression of cleaved Caspase 9, cleaved Caspase 8 and cleaved Caspase 3 protein were determined. Methods: the rat GMCs was cultured in vitro and the sublytic C5b-9 stimulation was given. The abundance of GMCs and the protein expression level of IRF-1 stimulated by sublytic C5b-9 were examined by RT-PCR and Western blot, and the apoptosis of each group was analyzed by flow cytometry. Lot was used to examine the activation of Caspase in GMCs stimulated by sublytic C5b-9. Then, the constructed IRF-1 overexpressed plasmid and RNA (short hairpin RNA, shRNA) plasmid were transfected into GMCs. The expression of cleaved Caspase 9, cleaved Caspase 8 and cleaved Caspase 3 protein in MCs. In addition, the rat GMCs was treated with Caspase 8 inhibitor (Z-IETD-FMK) and Caspase 3 inhibitor (Z-DEVD-FMK). In the early stage, the expression of IRF-1 was up up and accompanied by the apoptosis of GMCs and the activation of Caspase. Overexpression of IRF-1 could promote the apoptosis of GMCs induced by sublytic C5b-9 stimulation, while IRF-1 shRNA not only reduced the apoptosis rate of GMCs, but also reduced the apoptosis rate of sublytic C5b-9 stimulated. The expression of Caspase 3 was not significantly affected by the expression of cleaved Caspase 9. In addition, the application of Caspase 8 inhibitor (Z-IETD-FMK) and Caspase 3 inhibitor (Z-DEVD-FMK) could also significantly reduce the number of GMCs apoptosis caused by sublytic C5b-9 stimulation and overexpression IRF-1. Up regulation of IRF-1 expression and activation of Caspase have a certain relationship.
The second part is to investigate the inhibitory effect of silencing IRF-1 gene on GMCs apoptosis and secondary proliferative lesions in Thy-1N rats.
Objective: To investigate the inhibition effect of IRF-1 gene in IRF-1 shRNA on the apoptosis and secondary proliferation of GMCs (Thy-1 nephritis, Thy-1N) rats with Thy-1 nephritis (Thy-1 nephritis, Thy-1N). Methods: first, the rat Thy-1N model was replicated, and RT-PCR and Western blot were used to examine the expression of the renal tissue and the expression of 3 protein in the renal tissue. Then the IRF-1 shRNA plasmid was introduced into the rat kidney tissue and the rat Thy-1N model was replicated by injection of anti thymic cell sera (anti-thymocyte serum, ATS) by the tail vein injection, and the tail vein was injected with Caspase 8 inhibitor (Z-IETD-FMK) and Caspase 3 inhibitor (Z-DEVD-FMK) to replicate the Thy rat Thy. -1N model. The rat kidney tissues of 3H and 7d were taken after the injection of ATS respectively. The expression of IRF-1 protein in the renal tissue and the expression of cleaved Caspase 9,8 and 3 protein were examined by Western blot, and the expression of the cleaved Caspase 9,8 and the protein was also used. NEL) the pathological changes of renal tissue during 3H were examined by technology and electron microscopy. The total number of cells in the glomeruli was counted by light microscopy, and the changes in the total amount of urine protein were measured by biochemical methods. Results: the expression of IRF-1 in the renal tissue was obviously elevated in Thy-1N rats (IRF-1) treated with.IRF-1 shRNA (IRF-1) when the Thy-1N lesion was 3H in rats (IRF-1) (IRF-1) (IRF-1) was significantly increased by.IRF-1 shRNA (IRF-1). The expression of IRF-1 gene in the renal tissue and the activation level of Caspase 8 and Caspase 3 were significantly lower than that of the Thy-1N model group. The rats in the other IRF-1 shRNA+Thy-1N group and the rats treated with Caspase inhibitors (group Z-IETD-FMK+Thy-1N and Z-DEVD-FMK+Thy-1N) were significantly reduced in the number of apoptotic glomerular GMCs. The proliferation of small ball cells was reduced, and the secretion of 24h urine protein decreased significantly. Conclusion: the silence of IRF-1 gene and the use of Caspase inhibitors in rat kidney can significantly reduce the apoptosis and secondary proliferation of GMCs in Thy-1N rats, including the secretion of urine protein.
The third part is to study the role of sublytic C5b-9 in stimulating IRF-1 transcription in rat GMCs and its signaling pathway in vitro.
Aim: To study the effect of sublytic C5b-9 on the activation of IRF-1 transcription by GMCs in rats, and to explore the signal transduction pathway that sublytic C5b-9 stimulates GMCs to regulate the expression of IRF-1. Methods: the full length 1700bp luciferase reporter gene of the rat IRF-1 promoter was used as a template, and the upstream transcriptional starting site of rat IRF-1 gene was 130 upstream. The promoter fragments of 00bp, 600bp, 200bp and 100bp were cloned into the pGL3-Basic luciferase reporter gene vector respectively. They were named pGL3-Basic/IRF-1 promoter 1300bp, pGL3-Basic/IRF-1 promoter 600bp, pGL3-Basic/IRF-1 digestion and sequencing were correct. The III transfection reagent transfected the above-mentioned plasmids and the pGL3-Basic/3 x GAS luciferase reporter gene with the control plasmid PRL-SV40 to co transfect the GMCs cells with the control plasmid PRL-SV40 respectively. After the transfection, 45h was stimulated by sublytic C5b-9, and the relative fluorescence activity of the Sublytic C5b-9 stimulation group and the other treatment groups was measured. Some of the signal pathway related proteins in GMCs and other control groups, such as signal transduction protein and transcriptional activator (signal transducers and activators of transcription, STAT), p-STAT1, p38, p-p38, C-Jun based terminal kinase, and extracellular signal regulated protein kinase The expression of rotein kinase, ERK) and p-ERK, and the effects of the JAK inhibitor (AG490), the p38 inhibitor (SB203580), JNK inhibitor (SP600125) and ERK inhibitor (PD98059), and the effect of the inhibitor on the induced expression and protein of the rat. Results: the luciferase report test table After transfection of pGL3-Basic/IRF-1 promoter 1700bp, pGL3-Basic/IRF-1 promoter 1300bp, pGL3-Basic/IRF-1 promoter 600bp and pGL3-Basic/IRF-1 promoter 200bp luciferase reporter gene, the luciferase activity was significantly higher than that of other groups. The activity of luciferase increased significantly after the sublytic C5b-9 stimulation of the gene GMCs, but the luciferase activity of the pGL3-Basic/IRF-1 promoter 100bp luciferase reporter gene transfected by sublytic C5b-9 stimulation was not significantly different from that of the other controls. Compared with the other control groups,.JAK inhibitor (AG490) inhibited the expression of p-STAT1 and IRF-1 after sublytic C5b-9 stimulated GMCs, and p38 inhibitor (SB203580) also reduced the expression of sublytic C5b-9 after GMCs. Conclusion: Sublytic C5b-9 stimulates GMCs in rats to increase the activity of IRF-1 gene promoter, in which the activation sequence of interferon (IFN-gamma activated sequence, GAS) plays a major role in the activation of IRF-1 gene in sublytic C5b-9. There is a certain relationship between the activation of the road.
【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】：2010
【分類號】：R392

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