miR-34a调控系膜增殖性肾炎的机制研究

发布时间：2018-05-24 09:37

本文选题：系膜增殖性肾炎 + microRNA　；参考：《南开大学》2014年博士论文

【摘要】：背景：系膜增殖性肾炎(mesangial proliferative glomerulonephritis, MsPGN)是我国发病率最高的肾脏疾病之一。其主要的病理变化是弥漫性肾小球系膜细胞(glomerular mesangial cell, GMC)增生,以及不同程度的细胞外基质(extracellular matrix, ECM)增多。同时,MsPGN是导致慢性肾脏病(chronic kidney disease, CKD)的首要原因,可引起大量蛋白尿、高血压肾损害等一系列并发症,如果病情继续进展恶化,还可引起肾小球硬化及肾间质纤维化,最终成为导致终末期肾脏病(end-stage renal disease, ESRD)的主要病因。抑制系膜细胞增殖是治疗系膜增殖性肾小球疾病重要的治疗策略。近年来生命科学研究领域的一个重大突破就是在真核生物体内发现了具有调节功能的非编码RNA——microRNA (miRNA)。这些小RNA广泛参与调控生物增殖／凋亡、发育/衰老等病理生理过程,与人类疾病的发生发展密切相关。目前研究表明,miR-34a可能参与调控多个信号通路而影响细胞的增殖。RNA干扰(RNAi)在实验室中是一种强大的实验工具,利用具有同源性的双链RNA (dsRNA)诱导序列特异的目标基因的沉寂,可以迅速阻断基因活性。siRNA在RNA沉默中起中心作用,可对特定信使RNA (mRNA)进行特异性的降解。siRNA是RNAi途径中的中间产物,是RNAi发挥效应所必需的元件。siRNA与miRNA具有众多相似性,所以利用siRNA模拟miRNA实验以证明miRNA对于靶基因作用的特异性。胎牛血清(fetal bovine serum, FBS)是细胞培养中最常用的营养物质,其中含有几十种蛋白质、微量元素、生长因子等。在培养细胞时通常采用10%-15%FBS进行,实验中我们可以利用高浓度(20%)FBS培养细胞,以刺激细胞进行过度增殖,与以往单一利用某种刺激因子比较,高浓度FBS更能模拟生物体内病理情况复杂的环境。如果我们可以利用miR-34a可以拮抗这一现象,可以推测其在体内也可以有相似作用。目的：制备抗Thyl抗体并制备抗Thyl系膜增殖性肾炎大鼠模型。观察miR-34a在各病理时间点变化趋势。之后,利用siPDGFR-β证明miR-34a对于已经证明的靶基因PDGFR-β及Ras/MAPK信号通路的调控作用,并在体外证实miR-34a可以拮抗外源性刺激引起的细胞过度增殖现象。方法： 1.利用OX-7杂交瘤细胞株产生抗Thy1抗体,纯化后经尾静脉注射Wistar大鼠建立抗Thyl系膜增殖性肾炎大鼠模型。检测不同病理时间点(第0、3、5、7、10、14天)肾功能和24小时尿蛋白定量； 2.利用免疫组化方法,检测各病理时间点Ki67及细胞周期蛋白cyclin D1的表达变化趋势； 3.留取各病理时间点肾脏组织并提取肾小球,Western Blot检测G0/G1期细胞周期相关蛋白cyclin D1、cyclin E、CDK2、CDK4、CDK6、p27kip1表达变化趋势； 4.利用TaqMan Real-time PCR检测各病理时间点miR-34a表达的变化趋势； 5.将siPDGFR-β或siRNA negative control转染大鼠系膜细胞系(RMC),利用Cell Counting Kit-8方法检测不同时间点(24h,48h,72h)各组细胞增殖速率的变化程度； 6.将siPDGFR-β或siRNA negative control转染大鼠系膜细胞系(RMC)48h后,利用流式细胞仪检测RMC细胞周期变化； 7.将siPDGFR-β或siRNA negative control转染大鼠系膜细胞系(RMC)48h后,检测靶基因PDGFR-β. Ras/MAPK信号通路的各个靶点K-ras、Rafl、 p-Rafl、MEK1、p-MEK1、ERK1/2、p-ERK1/2及细胞周期相关蛋白cyclin D1、 cyclin E、CDK2、CDK4、CDK6、p27kip1的蛋白表达变化趋势； 8.利用双荧光素酶报告质粒系统检测miR-34a对可能靶基因CDK2、cyclin E表达的调控作用； 9.利用高浓度胎牛血清(20%FBS)刺激转染miR-34a mimics的大鼠系膜细胞,利用Cell Counting Kit-8方法检测不同时间点(24h,48h,72h)各组细胞增殖速率的变化程度； 10.利用高浓度胎牛血清(20%FBS)刺激转染miR-34a mimics的大鼠系膜细胞,利用流式细胞仪检测RMC细胞周期变化； 11.利用高浓度胎牛血清(20%FBS)刺激转染miR-34a mimics的大鼠系膜细胞,检测靶基因PDGFR-β、Ras/MAPK信号通路的各个靶点K-ras、Raf1、p-Raf1、 MEK1、p-MEK1、ERK1/2、p-ERK1/2及细胞周期相关蛋白cyclin D1、cyclin E、CDK2、CDK4、CDK6、p27kipl的蛋白表达变化趋势。结果： 1.成功制备抗Thy1抗体,并利用该抗体制备了抗Thyl系膜增殖性肾炎大鼠模型。与对照组相比,病理组大鼠第5天和第7天24h尿蛋白定量显著升高(p0.05),第10天后24h尿蛋白定量开始下降。 2.各病理时间点Ki67表达随病理变化成正关系。与对照组相比,病理组第7天Ki67表达量显著增加(p0.05),并随病理转归而下降。细胞周期蛋白cyclinD1的表达与Ki67趋势相同,在病理最终的第7天表达量最高(p0.05),随病理转归而回到正常。 3. Western Blot检测各病理组细胞周期相关蛋白表达。结果表明,与对照组相比,病理组第7天,cyclin D1、cyclin E、CDK2、CDK4、CDK6的表达最高(p0.05),而p27kiP1表大量最低(P0.05)。 4. TaqMan Real-time PCR检测各病理组mIR-34a表达量变化,结果表明,与对照组相比,miR-34a表达与病理变化成负相关,病理组第7天表达量最低(p0.05),随病理转归回到正常。 5.用siPDGFR-β或siRNA negative control转染大鼠系膜细胞系(RMC)。与对照组相比,siPDGFR-β组增殖活性从48h开始明显下降(p0.05),至72h更加明显(p0.05)。同时通过流式细胞技术检测细胞周期变化,siPDGFR-β组细胞周期主要是阻滞在G0/G1期(p0.05)(使得G0/G1期延长)； 6.用siPDGFR-β或siRNA negative control转染大鼠系膜细胞系(RMC),Western Blot检测对PDGFR-β与p-PDGFR-β的影响。与对照组相比,siPR-β处理组的PDGFR-β、p-PDGFR-β蛋白表达量明显下降(p0.05)。 7.利用siPDGFR-β或siCON转染RMCs,检测其对RAS/MAPK信号通路关键靶点的改变。结果表明,与对照组相比,K-Ras的总蛋白水平下调(p0.05),Rafl、MEK1、ERK1/2的总蛋白水平不变(p0.05)。p-Rafl、p-MEK1、 p-ERK1/2蛋白水平下调(p0.05)； 8.利用siPDGFR-β或siCON转染RMC后,检测其对细胞周期蛋白cyclin D1、 cyclin E, CDK蛋白CDK2、CDK4、CDK6, CKI蛋白,p27kiPl的蛋白影响。结果表明,siPDGFR-β可以下调cyclin D1, cyclin E, CDK2, CDK4, CDK6的蛋白水平(p0.05),p27kip1表达量明显上调(p0.05)； 9.为验证CDK2或cyclin E是否为miR-34a的靶基因,我们将CDK2/cyclin E mRNA的3'UTR序列克隆至双荧光素酶报告载体质粒psiCHECKTM-2上。结果显示,miR-34a mimics可以下调RMC细胞psiCHECK-CDK2/cyclin E的荧光素酶活性(p0.05)； 10.利用miR-34a mimics或者niRNA negative control转染细胞后,并用20%FBS血清刺激细胞。结果表明,从48小时开始,20%FBS组与20%FBS+Con组增殖能力高于10%FBS组及20%FBS+miR34a组(p0.05),但是10%FBS与20%FBS+miR34a组无差别(p0.05)； 11.利用niR-34a mimics或者miRNA negative control转染细胞后,并用20%FBS血清刺激细胞。结果表明,在48小时开始,20%FBS+Con组的细胞周期G0/G1期与10%FBS组相比明显缩短(p0.05),而20%FBS+miR34a组的G0/G1期与20%FBS+Con相比明显延长(p0.05)。每次检测的CV(%)均小于10%,表明结果可信； 12.利用miR-34a mimics或者miRNA negative control转染细胞后,并用20%FBS血清刺激细胞。结果表明,在刺激48小时后,与10%FBS组与20%FBS+miR34a组相比,20%FBS+Con组PDGFR-β与p-PDGFR-β的蛋白表达明显增高(p0.05),20%FBS+miR34a组与10%组无差别(p0.05)； 13.利用miR-34a mimics或者miRNA negative control转染细胞后,并用20%FBS血清刺激细胞。结果表明,在刺激48小时后,与10%FBS组与20%FBS+miR34a组相比,20%FBS+Con组的Raf1、ERK1/2的总蛋白无明显差异((P0.05), K-Ras的总蛋白水平上调(p0.05)。p-Raf1、p-MEK1、 p-ERK1/2蛋白水平上调(p0.05)。与20%FBS+Con组相比,20%FBS+miR34a组的MEK1蛋白明显降低(p0.05)； 14.利用miR-34a mimics或者miRNA negative control转染细胞后,并用20%FBS血清刺激细胞。我们检测其对细胞周期蛋白cyclin D1、cyclin E, CDK蛋白CDK2、CDK4、CDK6, CKI蛋白,p27kiP;的蛋白影响。结果表明,与20%FBS+miR34组相比,20%FBS+Con组的cyclin D1, cyclin E, CDK2,CDK4, CDK6的蛋白水平明显上调(p0.05), p27kip1表达量明显下调p0.05)。结论： 1.在抗Thy1系膜增殖性肾炎大鼠模型中,miR-34a随着病理变化而变化,呈负相关； 2. miR-34a可以通过直接作用于靶基因PDGFR-β,进而作用于Ras/MAPK信号通路活性,影响细胞周期G0/G1期,从而抑制系膜细胞增殖； 3. siPDGFR-β可以通过抑制PDGFR-β而作用于Ras/MAPK信号通路,延长G0/G1期,从而使RMC细胞增殖活性下降,此结果有力证实miR-34a是通过作用于靶基因PDGFR-β从而影响大鼠系膜细胞增殖； 4.miR-34a可以拮抗外源性刺激引起的细胞过度增殖现象。
[Abstract]:BACKGROUND : mesangial proliferative glomerulonephritis ( MsPGN ) is one of the most common renal diseases in China . It can be speculated that it can also have a similar effect in vivo .

Objective : To prepare an anti - Thyl antibody and to prepare a model of anti - Thyl - mesangial proliferative glomerulonephritis in rats .

Method :

1 . Using OX - 7 hybridoma cell line to produce anti - Thy1 antibody , the rat model of anti - Thyl series membrane proliferative glomerulonephritis was established by tail vein injection of Wistar rats . The renal function and 24 hour urinary protein quantification were detected at different pathological time points ( 0 , 3 , 5 , 7 , 10 , 14 days ) ;

2 . The expression of cyclin D1 was detected by immunohistochemistry .

3 . The expression of cyclin D1 , cyclin E , CDK2 , cd4 , CDk6 and p27kip1 in G0 / G1 phase was detected by Western Blot .

4 . detecting the change trend of miR - 34a expression in each pathological time point by using the real - time PCR ;

5 . Cells were transfected with siPDGFR - 尾 or siRNA negative control ( siRNA negative control ) , and the cell proliferation rate was measured by Cell Transplantation Kit - 8 method at different time points ( 24h , 48h , 72h ) .

6 . After 48h , siPDGFR - 尾 or siRNA negative control was transfected into the rat mesangial cell line .

7 . After 48h , siPDGFR - 尾 or siRNA negative control was transfected into the rat mesangial cell line , PDGFR - 尾 was detected . The expression of K - ras , Rafl , p - Rafl , MEK1 , p - MEK1 , ERK 1 / 2 , p - 1 / 2 and cyclin D1 , cyclin E , CDK2 , cd6 , p27kip1 in Ras / MAPK signaling pathway showed a tendency to change .

8 . Using the dual luciferase reporter plasmid system to detect the expression of miR - 34a on the possible target gene CDK2 and cyclin E ;

9 . The rat mesangial cells transfected with miR - 34a were stimulated with high concentration fetal bovine serum ( 20 % FBS ) . Cell proliferation Kit - 8 method was used to detect the change of cell proliferation rate at different time points ( 24h , 48h , 72h ) .

10 . The rat mesangial cells transfected with miR - 34a cells were stimulated with high concentration fetal bovine serum ( 20 % FBS ) , and the cell cycle changes were detected by flow cytometry .

11 . Using high concentration fetal bovine serum ( 20 % FBS ) to stimulate the rat mesangial cells transfected with miR - 34a , the expression of K - ras , Raf1 , p - Raf1 , MEK1 , p - MEK1 , ERK / 2 , p - 1 / 2 and cyclin D1 , cyclin E , CDK2 , cd4 , CDk6 and p27kipl in the target gene PDGFR - 尾 , Ras / MAPK signaling pathway were detected .

Results :

1 . Anti - Thy1 antibody was prepared successfully , and the model of anti - Thyl - mesangial proliferative glomerulonephritis was prepared by using the antibody . Compared with the control group , the quantity of urinary protein increased significantly ( p < 0.05 ) on the 5th and 7th day of the pathological group , and the quantity of urinary protein began to decrease after 10 days .

2 . The expression of Ki - 67 in the pathological group was significantly increased ( p < 0.05 ) , and the expression of cyclin D1 was the same as that of the control group . The expression of cyclin D1 was the same as that in the control group , and the expression level of cyclin D1 was highest ( p < 0.05 ) at the 7th day of pathology , and returned to normal with pathological outcome .

3 . Western Blot was used to detect the expression of cyclin D1 , cyclin E , CDK2 , cd4 and CDk6 in the pathological group . The results showed that the expression of cyclin D1 , cyclin E , CDK2 , cd4 and CDk6 was the highest in the pathological group ( p < 0.05 ) , while p27kip1 was the lowest ( P0.05 ) .

4 . The expression of mIR - 34a in each pathological group was detected by real - time PCR . The results showed that the expression of miR - 34a was negatively correlated with the pathological changes compared with the control group , and the expression of miR - 34a was the lowest in the pathological group ( p < 0.05 ) , and the expression level of mIR - 34a was returned to normal .

5 . Compared with the control group , the proliferation activity of siPDGFR - 尾 group was significantly decreased from 48 h ( p < 0.05 ) to 72 h ( p < 0.05 ) , and the cell cycle of siPDGFR - 尾 group was mainly blocked in G0 / G1 phase ( P 0.05 ) ( making G0 / G1 phase prolonged ) .

6 . The effects of siPDGFR - 尾 or siRNA negative control on PDGFR - 尾 and p - PDGFR - 尾 were detected by Western Blot . Compared with the control group , the expression of PDGFR - 尾 , p - PDGFR - 尾 protein in siPR - 尾treated group was significantly decreased ( p < 0.05 ) .

7 . Using siPDGFR - 尾 or siCON - transfected RMCs , the changes of the key target sites of RAS / MAPK signal pathway were detected . The results showed that the total protein level of K - Ras was down - regulated ( p . 05 ) , Rafl , MEK1 and ERK / 2 were unchanged ( p . 05 ) . p - Rafl , p - MEK1 , p - 1 / 2 protein levels were down - regulated ( p . 05 ) .

8 . After transfected with siPDGFR - 尾 or siCON , the protein level of cyclin D1 , cyclin E , CDK1 and p27kip1 was detected by siPDGFR - 尾 . The results showed that siPDGFR - 尾 could down - regulate the level of cyclin D1 , cyclin E , CDK2 , cd4 , CDk6 ( p0.05 ) , and the expression of p27kip1 was up - regulated ( p < 0.05 ) .

9 . To verify whether CDK2 or cyclin E is the target gene of miR - 34a , we cloned the 3 ' untranslated region of CDK2 / cyclin E mRNA to the double luciferase reporter plasmid psi3TM - 2 . The results showed that miR - 34a could downregulate luciferase activity of psiCHECK - CDK2 / cyclin E ( p0.05 ) ;

10 . After transfected with miR - 34a transcript or niRNA negative control , the cells were stimulated with 20 % FBS . The results showed that the proliferative ability of 20 % FBS group and 20 % FBS + Con group was higher than that in 10 % FBS group and 20 % FBS + miR34a group ( p < 0.05 ) , but there was no difference between 10 % FBS and 20 % FBS + miR34a group ( p < 0.05 ) .

11 . After transfection of cells with niR - 34a or miRNA negative control , the cells were stimulated with 20 % FBS . The results showed that the G0 / G1 phase of the 20 % FBS + Con group was significantly shorter than that of the 10 % FBS group ( p < 0.05 ) at 48 hours , while the G0 / G1 phase of 20 % FBS + miR34a group was significantly prolonged compared with 20 % FBS + Con ( p < 0.05 ) . The CV ( % ) of each test was less than 10 % , indicating that the results were credible ;

12 . The expression of PDGFR - 尾 and p - PDGFR - 尾 in 20 % FBS + Con group was significantly higher than that of 20 % FBS + miR34a group ( p < 0.05 ) , and 20 % FBS + miR34a group had no difference compared with 10 % FBS + miR34a group ( p < 0.05 ) .

Compared with 20 % FBS + miR34a group , the total protein level of 20 % FBS + Con group was up - regulated ( P0.05 ) .

14 . The protein level of cyclin D1 , cyclin E , CDK2 , cd4 , CDk6 , CKI and p27kiP in 20 % FBS + Con group was significantly higher than that of 20 % FBS + miR34 group ( p < 0.05 ) , and p27kip1 expression was down - regulated ( p < 0.05 ) .

Conclusion :

1 . In the rat model of anti - Thy1 mesangial proliferative glomerulonephritis , miR - 34a was negatively correlated with pathological changes ;

2 . miR - 34a can inhibit the G0 / G1 phase of the cell cycle by directly acting on the target gene PDGFR - beta , thereby inhibiting the cell cycle G0 / G1 phase , thereby inhibiting the proliferation of mesangial cells ;

3 . siPDGFR - 尾 can act on Ras / MAPK signaling pathway by inhibiting PDGFR - 尾 and prolong G0 / G1 phase , so as to decrease the proliferation activity of the cells . This results strongly confirm that miR - 34a is the target gene PDGFR - 尾 , which affects the proliferation of rat mesangial cells ;

4 . miR - 34a can inhibit the phenomenon of excessive proliferation of cells caused by exogenous stimuli .
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.3

【共引文献】