SublyticC5b-9诱导IRF-1表达对Thy-1肾炎大鼠GMCs凋亡病变的影响

发布时间：2018-06-18 16:46

  本文选题：亚溶解型C5b-9(sublytic + C5b-9)　； 参考：《南京医科大学》2010年硕士论文

【摘要】：第一部分体外研究IRF-1在sublytic C5b-9诱导大鼠GMCs凋亡病变中的作用 目的:体外探讨亚溶解型C5b-9(sublytic C5b-9)刺激大鼠肾小球系膜细胞(glomerular mesangial cells,GMCs)上调干扰素调节因子1(interferon regulatory factor 1, IRF-1)基因表达、并发凋亡现象以及半胱氨酸天冬氨酸特异性蛋白酶(Caspase)的活化情况,同时确定过表达或沉默IRF-1基因对sublytic C5b-9诱发大鼠GMCs凋亡病变的影响以及沉默IRF-1基因对cleaved Caspase 9、cleaved Caspase 8和cleaved Caspase 3蛋白表达的影响,同时,检查使用Caspase抑制剂对sublytic C5b-9刺激和过表达IRF-1引起的GMCs凋亡现象的影响。方法:先体外培养大鼠GMCs并给予sublytic C5b-9刺激(实验设不同处理对照),用RT-PCR和Western blot分别检查受sublytic C5b-9刺激的GMCs上调IRF-1 mRNA的丰度及蛋白表达的水平,并用流式细胞术(Flow Cytometry, FCM)分析各组GMCs的凋亡情况,同时用Western blot检查sublytic C5b-9刺激的GMCs中Caspase的活化情况。然后,分别将构建的IRF-1过表达质粒和发夹式小干涉RNA(short hairpin RNA, shRNA)质粒转染GMCs,并将GMCs作相应的分组处理,定量分析各组GMCs的凋亡率,同时检查转染IRF-1 shRNA质粒的GMCs和对照组GMCs中cleaved Caspase 9、cleaved Caspase 8和cleaved Caspase 3蛋白的表达情况。此外,使用Caspase 8抑制剂(Z-IETD-FMK)和Caspase 3抑制剂(Z-DEVD-FMK)处理大鼠GMCs,观察其对sublytic C5b-9刺激和过表达IRF-1引起的GMCs凋亡病变的影响。结果:Sublytic C5b-9刺激大鼠GMCs早期可见IRF-1表达上调并伴随GMCs的凋亡以及Caspase的活化,过表达IRF-1可促进sublytic C5b-9刺激引起的GMCs凋亡病变,而用IRF-1 shRNA不仅可以使sublytic C5b-9刺激的GMCs的凋亡率下降,也可以减少sublytic C5b-9刺激的GMCs中上调cleaved Caspase 8和cleaved Caspase 3的表达量,但对cleaved Caspase 9表达的影响并不显著。此外,应用Caspase 8抑制剂(Z-IETD-FMK)和Caspase 3抑制剂(Z-DEVD-FMK)亦可显著减轻由sublytic C5b-9刺激和过表达IRF-1引起的GMCs凋亡数量。结论:Sublytic C5b-9诱导的大鼠GMCs凋亡病变可能与其上调IRF-1表达及激活Caspase有一定的关系。 第二部分体内探讨沉默IRF-1基因对Thy-1N大鼠GMCs凋亡以及继发增生病变的抑制效应 目的:探讨用IRF-1 shRNA沉默大鼠肾组织内IRF-1基因对Thy-1肾炎(Thy-1 nephritis, Thy-1N)大鼠GMCs凋亡和继发增生病变的抑制作用。方法:首先,复制大鼠Thy-1N模型,定期采用RT-PCR和Western blot方法检查其肾组织中IRF-1和cleaved Caspase 9、8和3蛋白的表达情况。然后应用肾动脉灌注加电导入方法,将IRF-1 shRNA质粒导入大鼠肾组织,再经尾静脉注射注射抗胸腺细胞血清(anti-thymocyte serum,ATS)复制大鼠Thy-1N模型。同时尾静脉注射Caspase 8抑制剂(Z-IETD-FMK)和Caspase 3抑制剂(Z-DEVD-FMK)后再复制大鼠Thy-1N模型。分别取注射ATS后3h和7d时的大鼠肾组织,应用Western blot检查3h时肾组织中IRF-1蛋白的表达水平及cleaved Caspase 9、8和3蛋白的表达情况。此外,应用末端转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL)技术和电镜检查3h时肾组织的病理学改变。另光镜检查计数7d时肾小球内的细胞总数,并用生化方法测定各组大鼠24h和7d时尿蛋白总量的变化。结果:大鼠Thy-1N病变3h时,肾组织中IRF-1的表达水平明显升高。IRF-1 shRNA处理的Thy-1N大鼠(IRF-1 shRNA+Thy-1N组),其肾组织中IRF-1基因的表达以及Caspase 8和Caspase 3的活化水平显著低于Thy-1N模型组。另IRF-1 shRNA+Thy-1N组大鼠以及经Caspase抑制剂处理的大鼠(Z-IETD-FMK+Thy-1N组和Z-DEVD-FMK+Thy-1N组),3h时肾小球内GMCs凋亡数量显著减少。实验7d时,肾小球细胞的增生程度有所减轻,并且,24h尿蛋白的分泌量也明显减少。结论:沉默大鼠肾组织内IRF-1基因以及使用Caspase抑制剂均能明显减轻Thy-1N大鼠GMCs的凋亡病变及继发增生现象,包括尿蛋白的分泌。 第三部分体外研究sublytic C5b-9刺激大鼠GMCs启动IRF-1转录的作用及其信号通路 目的:研究sublytic C5b-9刺激大鼠GMCs启动IRF-1转录的作用,同时,探讨sublytic C5b-9刺激GMCs上调IRF-1表达可能涉及的信号转导通路。方法:以大鼠IRF-1启动子全长1700bp荧光素酶报告基因为模板,用DNA重组技术将大鼠IRF-1基因转录起始位点上游13000bp、600bp、200bp和100bp的启动子片段分别克隆入pGL3-Basic荧光素酶报告基因载体中,分别命名为: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,酶切分析及序列测定正确后,用GenEscortTMIII转染试剂将上述各质粒以及pGL3-Basic/3×GAS荧光素酶报告基因分别与对照质粒PRL-SV40共转染大鼠GMCs细胞,转染后45h行sublytic C5b-9刺激,测定Sublytic C5b-9刺激组与其他各处理组的相对荧光活性。此外,利用Western blot检查Sublytic C5b-9刺激组与其他各对照组GMCs中一些信号通路相关蛋白,如信号传导蛋白和转录激活物(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、细胞外信号调节蛋白激酶(extra cellular signal-regulated protein kinase, ERK)和p-ERK的表达情况,同时分别应用JAK抑制剂(AG490)、p38抑制剂(SB203580)、JNK抑制剂(SP600125)和ERK抑制剂(PD98059)处理大鼠GMCs,然后检查上述抑制剂对sublytic C5b-9诱导大鼠GMCs表达p-STAT1和IRF-1蛋白的影响。结果:荧光素酶报告实验表明,转染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荧光素酶报告基因后再行sublytic C5b-9刺激的GMCs中的荧光素酶活性明显高于其他各对照组,转染pGL3-Basic/3×GAS荧光素酶报告基因的GMCs行sublytic C5b-9刺激后其荧光素酶活性升高较为显著,而转染pGL3-Basic/IRF-1 promoter 100bp荧光素酶报告基因的GMCs经sublytic C5b-9刺激后荧光素酶活性较其余对照组无明显差别。另Sublytic C5b-9刺激组GMCs中的p-STAT1、p-p38、p-JNK和p-ERK表达均较其余对照组明显上调。JAK抑制剂(AG490)可抑制sublytic C5b-9刺激GMCs后p-STAT1和IRF-1的表达量,p38抑制剂(SB203580)亦可使sublytic C5b-9刺激GMCs后p-STAT1和IRF-1的表达减少,而JNK抑制剂和ERK抑制剂对sublytic C5b-9刺激GMCs后IRF-1蛋白的表达量影响不显著。结论:Sublytic C5b-9刺激大鼠GMCs后可上调IRF-1基因启动子的活性,其中干扰素活化序列(IFN-gamma activated sequence, GAS)在sublytic C5b-9激活IRF-1基因转录中起着较大的作用,此外,sublytic C5b-9上调GMCs IRF-1基因表达可能与p38MAPK和JAK-STAT信号通路的活化存在一定的关系。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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