MicroRNA-27a在糖尿病肾病肾小管间质纤维化中的作用及机制研究

发布时间：2018-07-16 20:16

【摘要】：近年许多研究揭示了 microRNA(miRNA,微小RNA)在糖尿病肾病(diabetic nephropathy,DN)进展中的重要作用。已经证实miR-27a在DN表达上调,但其调节肾小管间质纤维化(tubulointerstitial fibrosis,TIF)的具体机制尚不清楚。本研究拟通过细胞实验、动物实验及DN患者研究探讨miR-27a/PPARγ轴在其中的作用及可能的分子机制,为肾纤维化的防治措施提供新的依据和途径。第一章高糖条件下NRK-52E细胞miR-27a表达的变化及对纤维化的影响[目的]观察高糖条件下NRK-52E细胞miR-27a表达的变化及对纤维化的影响。[方法]将NRK-52E分Mannitol、NG、HG三组,分别用含甘露醇30mmol/L和含糖5mmol/L、30mmol/L的DMEM/F12完全培养基培养。qRT-PCR法检测各组在不同时间点(12、36和72h)及含糖组在不同糖浓度(5mmol/L、15mmol/L、30mmol/L)时miR-27a、PPARγ、TGF-β1、Smad3、CTGF、FN、ColⅠ 的mRNA,Western blot检测其蛋白表达,荧光素酶报告基因检测PPARγ-3'UTR的荧光素酶活性。[结果]HG组miR-27a及TGF-β1、p-Smad3、CTGF、FN、Col Ⅰ 表达上调,PPARy表达下调,且为miR-27a的直接靶基因。[结论]高糖增加miR-27a表达,后者通过抑制PPARy而诱导NRK-52E纤维化。第二章上调或下调表达miR-27a对高糖条件下NRK-52E纤维化的影响[目的]探讨上调或下调表达miR-27a对高糖条件下NRK-52E细胞纤维化的影响。[方法]以HG组NRK-52E为研究对象,应用脂质体转染技术将外源性miR-27a抑制剂(miR-27ai)、miR-27a 类似物(miR-27am)、PPARy siRNA 及相应对照物瞬时转染NEK-52E,PPARγ激动剂罗格列酮(Rosi.)及对照物预处理细胞,将其分九组:(1)miR-27ai和相应对照组(miR-iNC);(2)miR-27am和相应对照组(miR-NC);(3)PPARysiRNA 和相应对照组(NTsiRNA);(4)Rosi.和相应对照组(DMSO);(5)PPARy siRNA+miR-27ai 组。qRT-PCR、Western blot 和间接细胞免疫荧光检测 PPARγ、TGF-β1、Smad3、CTGF、FN、Col Ⅰ的mRNA和蛋白表达。[结果]高糖条件下,miR-27am 下调 PPARy 表达,上调 TGF-β1、p-Smad3、CTGF、FN、ColⅠ表达,miR-27ai则作用相反。PPARγsiRNA和miR-27ai共转染,可增加PPARy表达。[结论]PPARγ作为miR-27a的直接靶基因,通过抑制TGF-β1/Smad3通路而减轻NRK-52E纤维化。第三章STZ糖尿病大鼠模型miR-27a表达的变化及对TIF的影响[目的]观察STZ糖尿病大鼠模型血浆和肾组织中miR-27a表达的变化及对TIF的影响。[方法]STZ糖尿病大鼠腹腔注射miR-27ai或miR-27am后分6组:(1)正常对照组(NC);(2)糖尿病造模组(DM);(3)DM_miR-27ai和相应对照组(DM_miR-iNC):(4)DM_miR-27am 和相应对照组(DM_miR-NC)。持续干预至12周后处死。qRT-PCR检测大鼠血浆及肾脏miR-27a,Western blot及免疫组化检测肾组织 PPARγ、TGF-β1、p-Smad3、CTGF、FN、Col Ⅰ 的表达;Masson三色染色观察肾组织病理改变。大鼠Scr、BUN、尿NAG、UAER,UACR、Ccr等肾损害相关指标均被分析。[结果]STZ大鼠血浆及肾组织miR-27a表达上调,抑制miR-27a能上调PPARy,减轻TIF及降低肾损害相关指标。激活miR-27a则结果相反。[结论]miR-27a/PPARγ轴通过激活TGF-β1/Smad3信号启动STZ糖尿病大鼠TIF进程。第四章DN患者血浆miR-27a表达的变化及对TIF的影响[目的]观察DN患者血浆miR-27a表达的变化及对TIF的影响。[方法]选取行肾活检的DN患者52例。免疫组化分析其肾组织PPARγ、TGF-β1、p-Smad3、CTGF、FN、Col Ⅰ的表达,Masson三色染色观察TIF的病理改变。收集DN患者及健康志愿者血标本各30例,qRT-PCR法检测其血浆miR-27a,并分析其与24h尿蛋白定量、尿NAG、Scr及eGFR等肾损害相关指标的相关性。[结果]DN患者血浆miR-27a表达上调,且与Scr、24h尿蛋白定量及尿NAG呈正相关,与eGFR呈负相关。伴随TIF的加重,PPARγ表达下调。[结论]DN患者血浆miR-27a升高反应肾功能恶化及TIF加重,其机制可能是通过PPARγ诱导的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.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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