發(fā)布時(shí)間：2018-07-16 20:16

【摘要】：近年許多研究揭示了 microRNA(miRNA,微小RNA)在糖尿病腎病(diabetic nephropathy,DN)進(jìn)展中的重要作用。已經(jīng)證實(shí)miR-27a在DN表達(dá)上調(diào),但其調(diào)節(jié)腎小管間質(zhì)纖維化(tubulointerstitial fibrosis,TIF)的具體機(jī)制尚不清楚。本研究擬通過(guò)細(xì)胞實(shí)驗(yàn)、動(dòng)物實(shí)驗(yàn)及DN患者研究探討miR-27a/PPARγ軸在其中的作用及可能的分子機(jī)制,為腎纖維化的防治措施提供新的依據(jù)和途徑。第一章高糖條件下NRK-52E細(xì)胞miR-27a表達(dá)的變化及對(duì)纖維化的影響[目的]觀察高糖條件下NRK-52E細(xì)胞miR-27a表達(dá)的變化及對(duì)纖維化的影響。[方法]將NRK-52E分Mannitol、NG、HG三組,分別用含甘露醇30mmol/L和含糖5mmol/L、30mmol/L的DMEM/F12完全培養(yǎng)基培養(yǎng)。qRT-PCR法檢測(cè)各組在不同時(shí)間點(diǎn)(12、36和72h)及含糖組在不同糖濃度(5mmol/L、15mmol/L、30mmol/L)時(shí)miR-27a、PPARγ、TGF-β1、Smad3、CTGF、FN、ColⅠ 的mRNA,Western blot檢測(cè)其蛋白表達(dá),熒光素酶報(bào)告基因檢測(cè)PPARγ-3'UTR的熒光素酶活性。[結(jié)果]HG組miR-27a及TGF-β1、p-Smad3、CTGF、FN、Col Ⅰ 表達(dá)上調(diào),PPARy表達(dá)下調(diào),且為miR-27a的直接靶基因。[結(jié)論]高糖增加miR-27a表達(dá),后者通過(guò)抑制PPARy而誘導(dǎo)NRK-52E纖維化。第二章上調(diào)或下調(diào)表達(dá)miR-27a對(duì)高糖條件下NRK-52E纖維化的影響[目的]探討上調(diào)或下調(diào)表達(dá)miR-27a對(duì)高糖條件下NRK-52E細(xì)胞纖維化的影響。[方法]以HG組NRK-52E為研究對(duì)象,應(yīng)用脂質(zhì)體轉(zhuǎn)染技術(shù)將外源性miR-27a抑制劑(miR-27ai)、miR-27a 類(lèi)似物(miR-27am)、PPARy siRNA 及相應(yīng)對(duì)照物瞬時(shí)轉(zhuǎn)染NEK-52E,PPARγ激動(dòng)劑羅格列酮(Rosi.)及對(duì)照物預(yù)處理細(xì)胞,將其分九組:(1)miR-27ai和相應(yīng)對(duì)照組(miR-iNC);(2)miR-27am和相應(yīng)對(duì)照組(miR-NC);(3)PPARysiRNA 和相應(yīng)對(duì)照組(NTsiRNA);(4)Rosi.和相應(yīng)對(duì)照組(DMSO);(5)PPARy siRNA+miR-27ai 組。qRT-PCR、Western blot 和間接細(xì)胞免疫熒光檢測(cè) PPARγ、TGF-β1、Smad3、CTGF、FN、Col Ⅰ的mRNA和蛋白表達(dá)。[結(jié)果]高糖條件下,miR-27am 下調(diào) PPARy 表達(dá),上調(diào) TGF-β1、p-Smad3、CTGF、FN、ColⅠ表達(dá),miR-27ai則作用相反。PPARγsiRNA和miR-27ai共轉(zhuǎn)染,可增加PPARy表達(dá)。[結(jié)論]PPARγ作為miR-27a的直接靶基因,通過(guò)抑制TGF-β1/Smad3通路而減輕NRK-52E纖維化。第三章STZ糖尿病大鼠模型miR-27a表達(dá)的變化及對(duì)TIF的影響[目的]觀察STZ糖尿病大鼠模型血漿和腎組織中miR-27a表達(dá)的變化及對(duì)TIF的影響。[方法]STZ糖尿病大鼠腹腔注射miR-27ai或miR-27am后分6組:(1)正常對(duì)照組(NC);(2)糖尿病造模組(DM);(3)DM_miR-27ai和相應(yīng)對(duì)照組(DM_miR-iNC):(4)DM_miR-27am 和相應(yīng)對(duì)照組(DM_miR-NC)。持續(xù)干預(yù)至12周后處死。qRT-PCR檢測(cè)大鼠血漿及腎臟miR-27a,Western blot及免疫組化檢測(cè)腎組織 PPARγ、TGF-β1、p-Smad3、CTGF、FN、Col Ⅰ 的表達(dá);Masson三色染色觀察腎組織病理改變。大鼠Scr、BUN、尿NAG、UAER,UACR、Ccr等腎損害相關(guān)指標(biāo)均被分析。[結(jié)果]STZ大鼠血漿及腎組織miR-27a表達(dá)上調(diào),抑制miR-27a能上調(diào)PPARy,減輕TIF及降低腎損害相關(guān)指標(biāo)。激活miR-27a則結(jié)果相反。[結(jié)論]miR-27a/PPARγ軸通過(guò)激活TGF-β1/Smad3信號(hào)啟動(dòng)STZ糖尿病大鼠TIF進(jìn)程。第四章DN患者血漿miR-27a表達(dá)的變化及對(duì)TIF的影響[目的]觀察DN患者血漿miR-27a表達(dá)的變化及對(duì)TIF的影響。[方法]選取行腎活檢的DN患者52例。免疫組化分析其腎組織PPARγ、TGF-β1、p-Smad3、CTGF、FN、Col Ⅰ的表達(dá),Masson三色染色觀察TIF的病理改變。收集DN患者及健康志愿者血標(biāo)本各30例,qRT-PCR法檢測(cè)其血漿miR-27a,并分析其與24h尿蛋白定量、尿NAG、Scr及eGFR等腎損害相關(guān)指標(biāo)的相關(guān)性。[結(jié)果]DN患者血漿miR-27a表達(dá)上調(diào),且與Scr、24h尿蛋白定量及尿NAG呈正相關(guān),與eGFR呈負(fù)相關(guān)。伴隨TIF的加重,PPARγ表達(dá)下調(diào)。[結(jié)論]DN患者血漿miR-27a升高反應(yīng)腎功能惡化及TIF加重,其機(jī)制可能是通過(guò)PPARγ誘導(dǎo)的TGF-β1/Smad3通路的激活。
[Abstract]:In recent years, many studies have revealed the important role of microRNA (miRNA, tiny RNA) in the progression of diabetic nephropathy (diabetic nephropathy, DN). It has been confirmed that the expression of miR-27a in DN is up-regulated, but its specific mechanism for regulating renal tubulointerstitial fibrosis (tubulointerstitial fibrosis, TIF) is not clear. This study is to be conducted through cell experiments and animal experiments. And DN patients to study the role of miR-27a/PPAR gamma axis in it and the possible molecular mechanism to provide a new basis and way for the prevention and treatment of renal fibrosis. Chapter 1: the changes in the expression of miR-27a in NRK-52E cells under high glucose conditions and the effect on fibrosis [Objective] to observe the changes in the expression of miR-27a in NRK-52E cells under high glucose conditions and to the fiber. [Methods] NRK-52E was divided into three groups of Mannitol, NG, HG, and the.QRT-PCR method was used to detect the DMEM/F12 complete medium containing mannitol 30mmol/L and sugar 5mmol/L and 30mmol/L, respectively. MRNA, Western blot detected its protein expression and luciferase reporter gene detected the luciferase activity of PPAR gamma -3'UTR. [results]HG group miR-27a and TGF- beta 1, p-Smad3, CTGF, FN, Col I expression was up-regulated, and the expression was a direct target gene. [Conclusion] high sugar increased the expression, the latter was induced by inhibition of inhibition. 2E fibrosis. The second chapter up-regulated or downregulated the effect of miR-27a on NRK-52E fibrosis in high glucose conditions. [Objective] to investigate the effect of up or down expression of miR-27a on NRK-52E cell fibrosis under high glucose conditions. [Methods] HG group NRK-52E was used as the research object. Exogenous miR-27a inhibitor (miR-27ai), miR-27a was used in liposome transfection. Analogues (miR-27am), PPARy siRNA and corresponding controls were transiently transfected to NEK-52E, PPAR gamma agonist rosiglitazone (Rosi.) and control cells, which were divided into nine groups: (1) miR-27ai and corresponding control group (miR-iNC); (2) miR-27am and corresponding control group (miR-NC); (3) PPARysiRNA and corresponding control group (NTsiRNA); (4) and corresponding control group (4) (5) PPARy siRNA+miR-27ai group.QRT-PCR, Western blot and indirect cell immunofluorescence detection of PPAR gamma, TGF- beta 1, Smad3, CTGF, FN, Col I expression of mRNA and protein. Increase the expression of PPARy. [conclusion]PPAR gamma, as a direct target gene for miR-27a, reduces NRK-52E fibrosis by inhibiting the TGF- beta 1/Smad3 pathway. The changes in miR-27a expression in the third chapter STZ diabetic rat model and the effect on TIF [Objective] to observe the changes in miR-27a expression in plasma and renal tissues of STZ diabetic rats and the effect on TIF. After intraperitoneal injection of miR-27ai or miR-27am in]STZ diabetic rats, 6 groups were divided into two groups: (1) normal control group (NC); (2) diabetic model group (DM); (3) DM_miR-27ai and corresponding control group (DM_miR-iNC): (4) DM_miR-27am and corresponding control group (DM_miR-NC). After continuous intervention to 12 weeks, the rats were executed by.QRT-PCR to detect the miR-27a of the kidney and the kidneys and the immune group The expression of renal tissue PPAR gamma, TGF- beta 1, p-Smad3, CTGF, FN, Col I was detected, and the renal tissue pathological changes were observed by Masson tricolor staining. The renal damage related indexes such as Scr, BUN, NAG, UAER, UACR, and kidney tissue were analyzed. The result of activation of miR-27a is opposite. [conclusion]miR-27a/PPAR gamma axis activates the TIF process of STZ diabetic rats by activating TGF- beta 1/Smad3 signal. The changes in plasma miR-27a expression in fourth chapter DN patients and the effect on TIF [Objective] observe the changes in miR-27a expression of plasma in DN patients and the effect on TIF. [Methods] select 5 patients with renal biopsy. 2 cases. Immunohistochemical analysis of the expression of PPAR gamma, TGF- beta 1, p-Smad3, CTGF, FN, Col I, Masson tricolor staining was used to observe the pathological changes of TIF. 30 cases of DN patients and healthy volunteers were collected, and miR-27a in plasma was detected by qRT-PCR. Results the expression of plasma miR-27a in]DN patients was up-regulated, and it was positively correlated with Scr, 24h urine protein quantitative and urine NAG, and negative correlation with eGFR. With the aggravation of TIF, the expression of PPAR gamma was downregulated. [conclusion]DN patients' plasma miR-27a increased reaction to renal function deterioration and TIF aggravation, the mechanism may be activated by PPAR gamma induced beta pathway.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R587.2;R692.9

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