本文選題：系膜增殖性腎炎 + microRNA　； 參考：《南開大學》2014年博士論文

【摘要】：背景：系膜增殖性腎炎(mesangial proliferative glomerulonephritis, MsPGN)是我國發(fā)病率最高的腎臟疾病之一。其主要的病理變化是彌漫性腎小球系膜細胞(glomerular mesangial cell, GMC)增生,以及不同程度的細胞外基質(extracellular matrix, ECM)增多。同時,MsPGN是導致慢性腎臟病(chronic kidney disease, CKD)的首要原因,可引起大量蛋白尿、高血壓腎損害等一系列并發(fā)癥,如果病情繼續(xù)進展惡化,還可引起腎小球硬化及腎間質纖維化,最終成為導致終末期腎臟病(end-stage renal disease, ESRD)的主要病因。抑制系膜細胞增殖是治療系膜增殖性腎小球疾病重要的治療策略。近年來生命科學研究領域的一個重大突破就是在真核生物體內發(fā)現(xiàn)了具有調節(jié)功能的非編碼RNA——microRNA (miRNA)。這些小RNA廣泛參與調控生物增殖／凋亡、發(fā)育/衰老等病理生理過程,與人類疾病的發(fā)生發(fā)展密切相關。目前研究表明,miR-34a可能參與調控多個信號通路而影響細胞的增殖。RNA干擾(RNAi)在實驗室中是一種強大的實驗工具,利用具有同源性的雙鏈RNA (dsRNA)誘導序列特異的目標基因的沉寂,可以迅速阻斷基因活性。siRNA在RNA沉默中起中心作用,可對特定信使RNA (mRNA)進行特異性的降解。siRNA是RNAi途徑中的中間產(chǎn)物,是RNAi發(fā)揮效應所必需的元件。siRNA與miRNA具有眾多相似性,所以利用siRNA模擬miRNA實驗以證明miRNA對于靶基因作用的特異性。胎牛血清(fetal bovine serum, FBS)是細胞培養(yǎng)中最常用的營養(yǎng)物質,其中含有幾十種蛋白質、微量元素、生長因子等。在培養(yǎng)細胞時通常采用10%-15%FBS進行,實驗中我們可以利用高濃度(20%)FBS培養(yǎng)細胞,以刺激細胞進行過度增殖,與以往單一利用某種刺激因子比較,高濃度FBS更能模擬生物體內病理情況復雜的環(huán)境。如果我們可以利用miR-34a可以拮抗這一現(xiàn)象,可以推測其在體內也可以有相似作用。 目的：制備抗Thyl抗體并制備抗Thyl系膜增殖性腎炎大鼠模型。觀察miR-34a在各病理時間點變化趨勢。之后,利用siPDGFR-β證明miR-34a對于已經(jīng)證明的靶基因PDGFR-β及Ras/MAPK信號通路的調控作用,并在體外證實miR-34a可以拮抗外源性刺激引起的細胞過度增殖現(xiàn)象。 方法： 1.利用OX-7雜交瘤細胞株產(chǎn)生抗Thy1抗體,純化后經(jīng)尾靜脈注射Wistar大鼠建立抗Thyl系膜增殖性腎炎大鼠模型。檢測不同病理時間點(第0、3、5、7、10、14天)腎功能和24小時尿蛋白定量； 2.利用免疫組化方法,檢測各病理時間點Ki67及細胞周期蛋白cyclin D1的表達變化趨勢； 3.留取各病理時間點腎臟組織并提取腎小球,Western Blot檢測G0/G1期細胞周期相關蛋白cyclin D1、cyclin E、CDK2、CDK4、CDK6、p27kip1表達變化趨勢； 4.利用TaqMan Real-time PCR檢測各病理時間點miR-34a表達的變化趨勢； 5.將siPDGFR-β或siRNA negative control轉染大鼠系膜細胞系(RMC),利用Cell Counting Kit-8方法檢測不同時間點(24h,48h,72h)各組細胞增殖速率的變化程度； 6.將siPDGFR-β或siRNA negative control轉染大鼠系膜細胞系(RMC)48h后,利用流式細胞儀檢測RMC細胞周期變化； 7.將siPDGFR-β或siRNA negative control轉染大鼠系膜細胞系(RMC)48h后,檢測靶基因PDGFR-β. Ras/MAPK信號通路的各個靶點K-ras、Rafl、 p-Rafl、MEK1、p-MEK1、ERK1/2、p-ERK1/2及細胞周期相關蛋白cyclin D1、 cyclin E、CDK2、CDK4、CDK6、p27kip1的蛋白表達變化趨勢； 8.利用雙熒光素酶報告質粒系統(tǒng)檢測miR-34a對可能靶基因CDK2、cyclin E表達的調控作用； 9.利用高濃度胎牛血清(20%FBS)刺激轉染miR-34a mimics的大鼠系膜細胞,利用Cell Counting Kit-8方法檢測不同時間點(24h,48h,72h)各組細胞增殖速率的變化程度； 10.利用高濃度胎牛血清(20%FBS)刺激轉染miR-34a mimics的大鼠系膜細胞,利用流式細胞儀檢測RMC細胞周期變化； 11.利用高濃度胎牛血清(20%FBS)刺激轉染miR-34a mimics的大鼠系膜細胞,檢測靶基因PDGFR-β、Ras/MAPK信號通路的各個靶點K-ras、Raf1、p-Raf1、 MEK1、p-MEK1、ERK1/2、p-ERK1/2及細胞周期相關蛋白cyclin D1、cyclin E、CDK2、CDK4、CDK6、p27kipl的蛋白表達變化趨勢。 結果： 1.成功制備抗Thy1抗體,并利用該抗體制備了抗Thyl系膜增殖性腎炎大鼠模型。與對照組相比,病理組大鼠第5天和第7天24h尿蛋白定量顯著升高(p0.05),第10天后24h尿蛋白定量開始下降。 2.各病理時間點Ki67表達隨病理變化成正關系。與對照組相比,病理組第7天Ki67表達量顯著增加(p0.05),并隨病理轉歸而下降。細胞周期蛋白cyclinD1的表達與Ki67趨勢相同,在病理最終的第7天表達量最高(p0.05),隨病理轉歸而回到正常。 3. Western Blot檢測各病理組細胞周期相關蛋白表達。結果表明,與對照組相比,病理組第7天,cyclin D1、cyclin E、CDK2、CDK4、CDK6的表達最高(p0.05),而p27kiP1表大量最低(P0.05)。 4. TaqMan Real-time PCR檢測各病理組mIR-34a表達量變化,結果表明,與對照組相比,miR-34a表達與病理變化成負相關,病理組第7天表達量最低(p0.05),隨病理轉歸回到正常。 5.用siPDGFR-β或siRNA negative control轉染大鼠系膜細胞系(RMC)。與對照組相比,siPDGFR-β組增殖活性從48h開始明顯下降(p0.05),至72h更加明顯(p0.05)。同時通過流式細胞技術檢測細胞周期變化,siPDGFR-β組細胞周期主要是阻滯在G0/G1期(p0.05)(使得G0/G1期延長)； 6.用siPDGFR-β或siRNA negative control轉染大鼠系膜細胞系(RMC),Western Blot檢測對PDGFR-β與p-PDGFR-β的影響。與對照組相比,siPR-β處理組的PDGFR-β、p-PDGFR-β蛋白表達量明顯下降(p0.05)。 7.利用siPDGFR-β或siCON轉染RMCs,檢測其對RAS/MAPK信號通路關鍵靶點的改變。結果表明,與對照組相比,K-Ras的總蛋白水平下調(p0.05),Rafl、MEK1、ERK1/2的總蛋白水平不變(p0.05)。p-Rafl、p-MEK1、 p-ERK1/2蛋白水平下調(p0.05)； 8.利用siPDGFR-β或siCON轉染RMC后,檢測其對細胞周期蛋白cyclin D1、 cyclin E, CDK蛋白CDK2、CDK4、CDK6, CKI蛋白,p27kiPl的蛋白影響。結果表明,siPDGFR-β可以下調cyclin D1, cyclin E, CDK2, CDK4, CDK6的蛋白水平(p0.05),p27kip1表達量明顯上調(p0.05)； 9.為驗證CDK2或cyclin E是否為miR-34a的靶基因,我們將CDK2/cyclin E mRNA的3'UTR序列克隆至雙熒光素酶報告載體質粒psiCHECKTM-2上。結果顯示,miR-34a mimics可以下調RMC細胞psiCHECK-CDK2/cyclin E的熒光素酶活性(p0.05)； 10.利用miR-34a mimics或者niRNA negative control轉染細胞后,并用20%FBS血清刺激細胞。結果表明,從48小時開始,20%FBS組與20%FBS+Con組增殖能力高于10%FBS組及20%FBS+miR34a組(p0.05),但是10%FBS與20%FBS+miR34a組無差別(p0.05)； 11.利用niR-34a mimics或者miRNA negative control轉染細胞后,并用20%FBS血清刺激細胞。結果表明,在48小時開始,20%FBS+Con組的細胞周期G0/G1期與10%FBS組相比明顯縮短(p0.05),而20%FBS+miR34a組的G0/G1期與20%FBS+Con相比明顯延長(p0.05)。每次檢測的CV(%)均小于10%,表明結果可信； 12.利用miR-34a mimics或者miRNA negative control轉染細胞后,并用20%FBS血清刺激細胞。結果表明,在刺激48小時后,與10%FBS組與20%FBS+miR34a組相比,20%FBS+Con組PDGFR-β與p-PDGFR-β的蛋白表達明顯增高(p0.05),20%FBS+miR34a組與10%組無差別(p0.05)； 13.利用miR-34a mimics或者miRNA negative control轉染細胞后,并用20%FBS血清刺激細胞。結果表明,在刺激48小時后,與10%FBS組與20%FBS+miR34a組相比,20%FBS+Con組的Raf1、ERK1/2的總蛋白無明顯差異((P0.05), K-Ras的總蛋白水平上調(p0.05)。p-Raf1、p-MEK1、 p-ERK1/2蛋白水平上調(p0.05)。與20%FBS+Con組相比,20%FBS+miR34a組的MEK1蛋白明顯降低(p0.05)； 14.利用miR-34a mimics或者miRNA negative control轉染細胞后,并用20%FBS血清刺激細胞。我們檢測其對細胞周期蛋白cyclin D1、cyclin E, CDK蛋白CDK2、CDK4、CDK6, CKI蛋白,p27kiP;的蛋白影響。結果表明,與20%FBS+miR34組相比,20%FBS+Con組的cyclin D1, cyclin E, CDK2,CDK4, CDK6的蛋白水平明顯上調(p0.05), p27kip1表達量明顯下調p0.05)。 結論： 1.在抗Thy1系膜增殖性腎炎大鼠模型中,miR-34a隨著病理變化而變化,呈負相關； 2. miR-34a可以通過直接作用于靶基因PDGFR-β,進而作用于Ras/MAPK信號通路活性,影響細胞周期G0/G1期,從而抑制系膜細胞增殖； 3. siPDGFR-β可以通過抑制PDGFR-β而作用于Ras/MAPK信號通路,延長G0/G1期,從而使RMC細胞增殖活性下降,此結果有力證實miR-34a是通過作用于靶基因PDGFR-β從而影響大鼠系膜細胞增殖； 4.miR-34a可以拮抗外源性刺激引起的細胞過度增殖現(xiàn)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 .
【學位授予單位】：南開大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R692.3

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