本文關(guān)鍵詞：甲基雙加氧酶2在高糖刺激腎小球系膜細胞TGF-β1表達中的作用研究　出處：《第三軍醫(yī)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腎小球系膜細胞 TGF-β1 TET2 甲基化

【摘要】：研究背景糖尿病腎病(DN)是糖尿病最嚴(yán)重的并發(fā)癥之一,目前研究發(fā)現(xiàn)有大約40%以上的糖尿病患者同時患有糖尿病腎病,它同時也是引起終末期腎功能衰竭(ESRF)最主要的病因。大量國內(nèi)外的研究提示,高血糖可以通過多種途徑和機制導(dǎo)致糖尿病腎病的發(fā)生和發(fā)展。近年來,表觀遺傳學(xué)逐漸被人們所認(rèn)識,即在不改變DNA序列的前提下發(fā)生的可遺傳和逆轉(zhuǎn)的改變。表觀遺傳學(xué)的理論能夠解釋高血糖對糖尿病并發(fā)癥風(fēng)險的持續(xù)性效果,目前這種現(xiàn)象被稱為“代謝記憶”現(xiàn)象。DNA甲基化作為表觀遺傳學(xué)的重要機制之一,在基因表觀遺傳修飾及表達調(diào)控中發(fā)揮著關(guān)鍵性的作用,其中甲基轉(zhuǎn)移酶(DNMTs)家族(DNMT1、3A、3B)參與DNA的被動去甲基化,而甲基雙加氧酶(TETs)家族(TET1、TET2、TET3)參與DNA的主動去甲基化。目的本文擬通過動態(tài)觀察在高糖刺激下人腎小球系膜細胞(HMCs),DNA甲基化相關(guān)酶類甲基轉(zhuǎn)移酶(DNMT1、3A、3B)、甲基雙加氧酶(TET1~3)的表達變化,然后通過體內(nèi)和體外實驗分別觀察甲基雙加氧酶TET2與轉(zhuǎn)化生長因子β1(TGF-β1)表達變化及其Cp G島的甲基化狀態(tài)的相互關(guān)系,還包括系膜細胞表型轉(zhuǎn)化相關(guān)分子α-平滑肌肌動蛋白(α-SMA)和細胞增殖情況。隨后,通過sh RNA特異性干擾人腎小球系膜細胞TET2的表達,及通過(多聚ADP核糖聚合酶)PARP抑制劑PJ-34飼喂db/db小鼠抑制TET2的表達觀察相應(yīng)TGF-β1表達變化及其Cp G島的甲基化狀態(tài)以及系膜細胞表型轉(zhuǎn)化相關(guān)分子α-SMA和細胞增殖情況,從而探討TET2在高糖誘導(dǎo)TGF-β1表達激活及高糖誘導(dǎo)系膜細胞表型轉(zhuǎn)化中的作用。方法1.將體外培養(yǎng)的人腎小球系膜細胞(HMCs)按照培養(yǎng)基的葡萄糖濃度隨機分為五組:低糖正常對照組(NG,5.5 mmol/L葡萄糖)、高糖組(HG,30 mmol/L葡萄糖),其中高糖組分別培養(yǎng)12h-72h:高糖12h組(HG-12h)、高糖24h組(HG-24h)、高糖48h組(HG-48h、高糖72h組(HG-72h)。2.通過sh RNA干擾高糖培養(yǎng)下TET2在系膜細胞中的表達,分為空白對照組(control B)、轉(zhuǎn)染試劑對照組(control M)、陰性對照組(control N)、sh RNA干擾組(sh RNA)。3.雌性C57BLKS/J小鼠共20只,其中7周齡db/db小鼠5只、11周齡db/db小鼠及db/m小鼠各5只、15周齡db/db小鼠5只。分別適應(yīng)性喂養(yǎng)一周,隨后進行試驗,分為4組,每組5只小鼠:db/m組:12周齡db/m小鼠、db/db-8w組:8周齡db/db小鼠、db/db-12w組:12周齡db/db小鼠、db/db-16w組:16周齡db/db小鼠。4.4周齡雌性C57BLKS/J小鼠共15只,其中db/db小鼠10只,db/m小鼠5只;分別適應(yīng)性喂養(yǎng)一周,隨后進行試驗,其中db/db小鼠以完全隨機的方法分別分為:db/db對照組和db/db+PJ-34組。將PARP抑制劑PJ-34用蒸餾水溶解至2.4 g/l,并加適量阿斯巴甜便于飼喂,以每天每公斤體重30mg的量定時飼喂db/db+PJ-34組,其余db/m對照組和db/db對照組以等量含阿巴斯甜的蒸餾水飼喂至16周齡。5.分別通過實時熒光定量PCR和Western blotting檢測TGF-β1、DNMT1、3A、3B、TET1~3和α-SMA的m RNA和蛋白表達。6.通過亞硫酸氫鈉測序法(BSP)檢測TGF-β1基因Cp G島的甲基化狀態(tài)。7.腎小球系膜細胞增殖通過噻唑藍比色法(MTT)檢測。8.通過免疫組化檢測小鼠腎臟皮質(zhì)中α-SMA的表達變化。9.通過(過碘酸雪夫染色)PAS染色觀察小鼠腎臟皮質(zhì)中腎小球的病理變化。結(jié)果1.高糖刺激培養(yǎng)人系膜細胞TGF-β1表達上調(diào),同時誘導(dǎo)TGF-β1基因Cp G島出現(xiàn)去甲基化改變。用高糖培養(yǎng)人系膜細胞,觀察TGF-β1表達變化、TGFβ基因表達調(diào)控區(qū)甲基化變化。結(jié)果發(fā)現(xiàn),與對照組相比較,高糖培養(yǎng)24h后,系膜細胞TGF-β1的m RNA及蛋白表達均顯著增加,并存在時間依賴效應(yīng)。同時發(fā)現(xiàn),與對照組比較,高糖培養(yǎng)24h后系膜細胞TGF-β1第一外顯子區(qū)Cp G島的四個CG位點出現(xiàn)明顯的去甲基化改變。2.高糖培養(yǎng)可誘導(dǎo)人系膜細胞TET2表達上調(diào)。我們進一步研究發(fā)現(xiàn),與對照組比較,高糖培養(yǎng)12h后,參與主動去甲基化的TET2出現(xiàn)明顯的表達上調(diào),且存在時間依賴效應(yīng),這與高糖誘導(dǎo)TGF-β1基因Cp G島去甲基化改變相一致。TET1、TET3以及DNMT1、DNMT3A表達未見明顯改變。3.應(yīng)用sh RNA特異性干擾TET2的表達,可逆轉(zhuǎn)高糖誘導(dǎo)的人系膜細胞TGF-β1基因Cp G島所發(fā)生的去甲基化及其高表達。與對照組比較,sh RNA可顯著抑制一般培養(yǎng)條件下系膜細胞TET2的m RNA及蛋白表達。同時發(fā)現(xiàn),sh RNA也可顯著下調(diào)高糖誘導(dǎo)的系膜細胞TGF-β1的基因及蛋白表達。甲基化檢測發(fā)現(xiàn),sh RNA處理后,高糖誘導(dǎo)的TGF-β1基因調(diào)控區(qū)Cp G島去甲基化可被顯著逆轉(zhuǎn)。4.進一步研究證實,應(yīng)用sh RNA特異性干擾TET2的表達后,高糖誘導(dǎo)下的系膜細胞α-SMA表達升高和系膜細胞的顯著增殖也被抑制。5.隨著db/db小鼠糖尿病腎病的發(fā)生和進展,腎皮質(zhì)中TET2和TGFβ1的表達水平均逐步升高,TGF-β1基因調(diào)控區(qū)Cp G島出現(xiàn)明顯的去甲基化改變。我們進一步觀察了不同周齡的db/db小鼠腎臟皮質(zhì)中TGF-β1的表達改變發(fā)現(xiàn):與db/m對照組小鼠比較,腎臟皮質(zhì)TGF-β1的m RNA及蛋白表達均出現(xiàn)明顯地進行性上調(diào)。BSP測序發(fā)現(xiàn),與db/m對照組比較,從8周齡到16周齡的db/db小鼠,TGF-β1啟動子區(qū)及第一外顯子區(qū)的四個位點出現(xiàn)持續(xù)的去甲基化改變。為此,我們進一步研究了DNA甲基化相關(guān)酶在db/db小鼠腎臟皮質(zhì)中的表達變化,我們發(fā)現(xiàn):與db/m對照組比較,隨著周齡的增長,腎臟皮質(zhì)TET2及DNMT3B的m RNA及蛋白表達均出現(xiàn)明顯地上調(diào),但TET1、TET3、DNMT1、DNMT3A未見明顯表達改變。6.PJ-34抑制TET2的表達,不僅使db/db小鼠腎皮質(zhì)TGFβ1基因調(diào)控區(qū)Cp G島去甲基化被逆轉(zhuǎn),也使TGF-β1基因表達水平下調(diào)。為了進一步證明在體情況下TET2-TGFβ基因調(diào)控區(qū)Cp G島甲基化變化-TGFβ表達變化三者之間的調(diào)控關(guān)系,我們應(yīng)用PARP抑制劑PJ-34飼喂抑制TET2的表達后,與db/db對照組比較,發(fā)現(xiàn)db/db+PJ-34組TGFβ1啟動子區(qū)及第一外顯子區(qū)上四個位點的甲基化率明顯升高,與此同時,TGF-β1的m RNA及蛋白表達也出現(xiàn)明顯地下調(diào)。7.抑制db/db小鼠腎皮質(zhì)中TET2的表達后,db/db小鼠腎小球系膜細胞增殖及α-SMA的合成也被顯著抑制。我們通過PAS染色觀察腎臟的病理改變,結(jié)果發(fā)現(xiàn),與db/db對照組相比較,db/db+PJ-34組腎小球肥大、系膜基質(zhì)、系膜區(qū)面積、毛細血管基底膜厚度等病理改變均明顯減輕。通過免疫組化觀察α-SMA在腎臟組織中的沉積,結(jié)果發(fā)現(xiàn):與db/db對照組相比較,db/db+PJ-34組小鼠腎皮質(zhì)α-SMA的表達量明顯減少。結(jié)論TET2表達激活介導(dǎo)了高糖誘導(dǎo)的TGF-β1基因表達調(diào)控區(qū)Cp G島去甲基化和TGF-β1的表達上調(diào),干預(yù)TET2表達可抑制高糖誘導(dǎo)的系膜細胞表型轉(zhuǎn)化和糖尿病腎病的發(fā)生發(fā)展。
[Abstract]:Background: diabetic nephropathy (DN) is one of the most serious complications of diabetes, the study found that patients with diabetes more than about 40% of the patients with diabetes nephropathy, it is also the cause of end-stage renal failure (ESRF). The main cause of a large number of domestic and foreign studies suggested that high blood sugar can lead to the occurrence and development of diabetic nephropathy through a variety of ways and mechanism. In recent years, epigenetics is gradually recognized that without changing the DNA sequence occurred under genetic and reverse change. To explain the effect of persistent hyperglycemia on the risk of diabetes complications of epigenetics theory at present, this phenomenon is called "metabolic memory" the phenomenon of.DNA methylation is one of the important mechanisms of epigenetics in epigenetic changes and plays a key role in the regulation of expression, including methyl transfer The enzyme (DNMTs) family (DNMT1,3A, 3B) passive demethylation in DNA, and methyl dioxygenase (TETs) family (TET1, TET2, TET3) active demethylation in DNA. The purpose of this is to observe in human mesangial cells stimulated by high glucose dynamics (HMCs), DNA methylation methyltransferase enzymes (DNMT1,3A, 3B), methyl dioxygenase (TET1~3) expression, and then through the in vitro and in vivo were observed with TET2 and methyl double oxygenase transformation growth factor beta 1 (TGF- beta 1) the relationship between Cp expression and methylation status of G Island, including phenotype transformation of mesangial cells related molecules of alpha smooth muscle actin (alpha -SMA) and cell proliferation. Then, through the SH RNA specific expression of TET2 in human mesangial cells, and the (poly ADP ribose polymerase) expression of PARP inhibitor PJ-34 db/db mice inhibited TET2 on T GF- beta 1 expression and Cp G island methylation status and phenotype transformation of mesangial cells and related molecular alpha -SMA cell proliferation, and to explore the role of TET2 in high glucose induced TGF- beta 1 expression and activation of glucose induced phenotype transformation of mesangial cells in 1.. Methods human glomerular mesangial cells cultured in vitro (HMCs) according to the concentration of glucose in culture medium were randomly divided into five groups: low glucose control group (NG, 5.5 mmol/L glucose), high glucose group (HG, 30 mmol/L glucose), high glucose group which were cultured in 12h-72h: high glucose group 12h (HG-12h) 24h (HG-24h), high glucose group, high glucose group (HG-48h glucose, 48h group 72h (HG-72h).2. by SH interference RNA expression of TET2 in high glucose cultured mesangial cells, divided into control group (control B), transfection reagent control group (control M), negative control group (control N), sh RNA (SH RNA).3. interference group, a total of 20 female C57BLKS/J mice among them, 7 5 week old db/db mice, 11 weeks old db/db mice and db/m mice of each 5, 15 week old db/db mice 5. Were fed for one week, then were divided into 4 groups, 5 mice in each group: db/m group: 12 week old db/m mice, db/db-8w group: 8 week old db/db mice, db/db-12w group: 12 week old db/db mice, db/db-16w group: 16 week old db/db mice of.4.4 weeks old female C57BLKS/J mice were 15, of which 10 db/db mice and 5 db/m mice; were fed for one week, followed by a test, in which db/db mice with completely random method were divided into: db/db control group and db/db+PJ-34 group. The PARP inhibitor PJ-34 dissolved in distilled water to 2.4 g/l, and a proper amount of aspartame for feeding, with timing feeding group db/db+PJ-34 per kilogram of body weight per day of 30mg, the db/m control group and db/db control group were injected with distilled water containing Abbas sweet feeding until 16 weeks of age respectively by.5. Real time fluorescence quantitative PCR and Western blotting detection of TGF- beta 1, DNMT1,3A, 3B, RNA and TET1~3 protein expression of M and alpha -SMA.6. by Sodium Bisulfite sequencing (BSP) proliferation and methylation of.7. in glomerular mesangial cells and detection of TGF- beta 1 gene Cp G island by MTT colorimetric assay (MTT) expression of.9. detection of.8. by immunohistochemistry in renal cortex were detected by alpha -SMA (PAS) to observe the pathological changes of glomeruli in the mouse kidney cortex PAS staining. Results 1. glucose stimulated cultured human mesangial cells TGF- beta 1 expression and induction of TGF- beta 1 gene Cp G Island demethylation culture change. Human mesangial cells in high glucose and observe TGF- beta 1 expression changes of methylation regulation of the expression of TGF gene. The results showed that compared with the control group, high glucose 24h, mesangial cell TGF- beta m and RNA 1 protein expression were significantly increased, the coexistence of In the time dependent effect. At the same time, compared with the control group, four CG loci of 24h in mesangial cells cultured in high glucose TGF- beta 1 in exon 1 of Cp G Island appeared to change the methylation of.2. in high glucose induced human mesangial cell TET2 expression. We found that, compared with the control group compared with high glucose culture 12h, active participation in the demethylation of TET2 expression was significantly up-regulated, and time dependent effects, and the high glucose induced TGF- beta 1 gene Cp G island methylation changes consistent with.TET1, TET3 and DNMT1, DNMT3A expression had no obvious change in.3. application sh RNA specific TET2 interference that can be reversed in high glucose induced human mesangial cell TGF- beta 1 gene Cp G island methylation and its high expression. Compared with control group, sh RNA could significantly inhibit the expression of M RNA protein and incubation conditions of mesangial cells of TET2. At the same time found, SH RNA can down regulate the expression of gene and protein in mesangial cells induced by high glucose TGF- beta 1. It was found that methylation detection, sh RNA after treatment with high glucose induced TGF- beta 1 gene Cp G Island demethylation can be significantly reversed.4. further study confirmed that sh expression by specific RNA interference TET2 after high glucose induced mesangial cell proliferation significantly increased expression of -SMA alpha and mesangial cells was also inhibited by.5. with the occurrence and development of diabetic nephropathy in db/db mice, the expression of TET2 and TGF beta 1 in renal cortex were gradually increased, the TGF- beta 1 gene regulatory region of Cp G island is obvious to methyl change. We observed the expression of TGF- beta 1 different week old db/db mice were found in renal cortex change: groups of mice were compared with db/m control, the expression of renal cortex TGF- beta M protein and RNA 1 were apparent to upregulation of.BSP and db/m was found. Compared to the control group from 8 weeks old to 16 weeks old db/db mice, TGF- beta 1 promoter and exon four site sub region of the persistent demethylation change. Therefore, we further study the expression changes of DNA methylation related enzymes in the renal cortex of db/db mice, we found compared with db/m control, with the growth of age, the expression of M protein and RNA in renal cortex of TET2 and DNMT3B were significantly up-regulated, but TET1, TET3, DNMT1, expression of DNMT3A was not altered.6.PJ-34 expression inhibition of TET2, not only the db/db mice renal cortical TGF beta 1 gene regulatory region of Cp G Island demethylation is reversed, the TGF- beta 1 gene expression. In order to control the relationship between the expression of further proof of the regulatory region of TET2-TGF beta gene Cp G island methylation changes of -TGF beta in vivo under the changes of the three expression, we used PARP inhibitor PJ-34 inhibited TET2 feeding After compared with the db/db control group, db/db+PJ-34 group TGF beta 1 promoter and the first exon methylation of four loci was significantly increased, at the same time, the expression of TGF- beta M protein and RNA 1 expression was significantly downregulated.7. also inhibition of TET2 in renal cortex of db/db mice after db/db synthesis mouse glomerular mesangial cells proliferation and alpha -SMA was significantly inhibited by PAS staining. We kidney pathological changes were observed, compared with the db/db control group, db/db+PJ-34 group, glomerular hypertrophy, mesangial matrix and mesangial area, capillary basement membrane thickness and other pathological changes were significantly reduced. By immunohistochemistry study on deposition of alpha -SMA in kidney tissue showed that: compared with db/db control group, db/db+PJ-34 group expression in renal cortex of mice with alpha -SMA decreased significantly. Conclusion the expression of TET2 is mediated by activation of TGF- induced by high glucose beta 1 gene expression Up regulation of Cp G Island demethylation and TGF- beta 1 expression were upregulated. Intervention of TET2 expression can inhibit the phenotype transformation of high glucose induced mesangial cells and the occurrence and development of diabetic nephropathy.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R587.2;R692.9

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