甲基双加氧酶2在高糖刺激肾小球系膜细胞TGF-β1表达中的作用研究

发布时间：2018-01-04 22:33

本文关键词：甲基双加氧酶2在高糖刺激肾小球系膜细胞TGF-β1表达中的作用研究　出处：《第三军医大学》2016年硕士论文　论文类型：学位论文

更多相关文章： 肾小球系膜细胞 TGF-β1 TET2 甲基化

【摘要】：研究背景糖尿病肾病(DN)是糖尿病最严重的并发症之一,目前研究发现有大约40%以上的糖尿病患者同时患有糖尿病肾病,它同时也是引起终末期肾功能衰竭(ESRF)最主要的病因。大量国内外的研究提示,高血糖可以通过多种途径和机制导致糖尿病肾病的发生和发展。近年来,表观遗传学逐渐被人们所认识,即在不改变DNA序列的前提下发生的可遗传和逆转的改变。表观遗传学的理论能够解释高血糖对糖尿病并发症风险的持续性效果,目前这种现象被称为“代谢记忆”现象。DNA甲基化作为表观遗传学的重要机制之一,在基因表观遗传修饰及表达调控中发挥着关键性的作用,其中甲基转移酶(DNMTs)家族(DNMT1、3A、3B)参与DNA的被动去甲基化,而甲基双加氧酶(TETs)家族(TET1、TET2、TET3)参与DNA的主动去甲基化。目的本文拟通过动态观察在高糖刺激下人肾小球系膜细胞(HMCs),DNA甲基化相关酶类甲基转移酶(DNMT1、3A、3B)、甲基双加氧酶(TET1~3)的表达变化,然后通过体内和体外实验分别观察甲基双加氧酶TET2与转化生长因子β1(TGF-β1)表达变化及其Cp G岛的甲基化状态的相互关系,还包括系膜细胞表型转化相关分子α-平滑肌肌动蛋白(α-SMA)和细胞增殖情况。随后,通过sh RNA特异性干扰人肾小球系膜细胞TET2的表达,及通过(多聚ADP核糖聚合酶)PARP抑制剂PJ-34饲喂db/db小鼠抑制TET2的表达观察相应TGF-β1表达变化及其Cp G岛的甲基化状态以及系膜细胞表型转化相关分子α-SMA和细胞增殖情况,从而探讨TET2在高糖诱导TGF-β1表达激活及高糖诱导系膜细胞表型转化中的作用。方法1.将体外培养的人肾小球系膜细胞(HMCs)按照培养基的葡萄糖浓度随机分为五组:低糖正常对照组(NG,5.5 mmol/L葡萄糖)、高糖组(HG,30 mmol/L葡萄糖),其中高糖组分别培养12h-72h:高糖12h组(HG-12h)、高糖24h组(HG-24h)、高糖48h组(HG-48h、高糖72h组(HG-72h)。2.通过sh RNA干扰高糖培养下TET2在系膜细胞中的表达,分为空白对照组(control B)、转染试剂对照组(control M)、阴性对照组(control N)、sh RNA干扰组(sh RNA)。3.雌性C57BLKS/J小鼠共20只,其中7周龄db/db小鼠5只、11周龄db/db小鼠及db/m小鼠各5只、15周龄db/db小鼠5只。分别适应性喂养一周,随后进行试验,分为4组,每组5只小鼠:db/m组:12周龄db/m小鼠、db/db-8w组:8周龄db/db小鼠、db/db-12w组:12周龄db/db小鼠、db/db-16w组:16周龄db/db小鼠。4.4周龄雌性C57BLKS/J小鼠共15只,其中db/db小鼠10只,db/m小鼠5只;分别适应性喂养一周,随后进行试验,其中db/db小鼠以完全随机的方法分别分为:db/db对照组和db/db+PJ-34组。将PARP抑制剂PJ-34用蒸馏水溶解至2.4 g/l,并加适量阿斯巴甜便于饲喂,以每天每公斤体重30mg的量定时饲喂db/db+PJ-34组,其余db/m对照组和db/db对照组以等量含阿巴斯甜的蒸馏水饲喂至16周龄。5.分别通过实时荧光定量PCR和Western blotting检测TGF-β1、DNMT1、3A、3B、TET1~3和α-SMA的m RNA和蛋白表达。6.通过亚硫酸氢钠测序法(BSP)检测TGF-β1基因Cp G岛的甲基化状态。7.肾小球系膜细胞增殖通过噻唑蓝比色法(MTT)检测。8.通过免疫组化检测小鼠肾脏皮质中α-SMA的表达变化。9.通过(过碘酸雪夫染色)PAS染色观察小鼠肾脏皮质中肾小球的病理变化。结果1.高糖刺激培养人系膜细胞TGF-β1表达上调,同时诱导TGF-β1基因Cp G岛出现去甲基化改变。用高糖培养人系膜细胞,观察TGF-β1表达变化、TGFβ基因表达调控区甲基化变化。结果发现,与对照组相比较,高糖培养24h后,系膜细胞TGF-β1的m RNA及蛋白表达均显著增加,并存在时间依赖效应。同时发现,与对照组比较,高糖培养24h后系膜细胞TGF-β1第一外显子区Cp G岛的四个CG位点出现明显的去甲基化改变。2.高糖培养可诱导人系膜细胞TET2表达上调。我们进一步研究发现,与对照组比较,高糖培养12h后,参与主动去甲基化的TET2出现明显的表达上调,且存在时间依赖效应,这与高糖诱导TGF-β1基因Cp G岛去甲基化改变相一致。TET1、TET3以及DNMT1、DNMT3A表达未见明显改变。3.应用sh RNA特异性干扰TET2的表达,可逆转高糖诱导的人系膜细胞TGF-β1基因Cp G岛所发生的去甲基化及其高表达。与对照组比较,sh RNA可显著抑制一般培养条件下系膜细胞TET2的m RNA及蛋白表达。同时发现,sh RNA也可显著下调高糖诱导的系膜细胞TGF-β1的基因及蛋白表达。甲基化检测发现,sh RNA处理后,高糖诱导的TGF-β1基因调控区Cp G岛去甲基化可被显著逆转。4.进一步研究证实,应用sh RNA特异性干扰TET2的表达后,高糖诱导下的系膜细胞α-SMA表达升高和系膜细胞的显著增殖也被抑制。5.随着db/db小鼠糖尿病肾病的发生和进展,肾皮质中TET2和TGFβ1的表达水平均逐步升高,TGF-β1基因调控区Cp G岛出现明显的去甲基化改变。我们进一步观察了不同周龄的db/db小鼠肾脏皮质中TGF-β1的表达改变发现:与db/m对照组小鼠比较,肾脏皮质TGF-β1的m RNA及蛋白表达均出现明显地进行性上调。BSP测序发现,与db/m对照组比较,从8周龄到16周龄的db/db小鼠,TGF-β1启动子区及第一外显子区的四个位点出现持续的去甲基化改变。为此,我们进一步研究了DNA甲基化相关酶在db/db小鼠肾脏皮质中的表达变化,我们发现:与db/m对照组比较,随着周龄的增长,肾脏皮质TET2及DNMT3B的m RNA及蛋白表达均出现明显地上调,但TET1、TET3、DNMT1、DNMT3A未见明显表达改变。6.PJ-34抑制TET2的表达,不仅使db/db小鼠肾皮质TGFβ1基因调控区Cp G岛去甲基化被逆转,也使TGF-β1基因表达水平下调。为了进一步证明在体情况下TET2-TGFβ基因调控区Cp G岛甲基化变化-TGFβ表达变化三者之间的调控关系,我们应用PARP抑制剂PJ-34饲喂抑制TET2的表达后,与db/db对照组比较,发现db/db+PJ-34组TGFβ1启动子区及第一外显子区上四个位点的甲基化率明显升高,与此同时,TGF-β1的m RNA及蛋白表达也出现明显地下调。7.抑制db/db小鼠肾皮质中TET2的表达后,db/db小鼠肾小球系膜细胞增殖及α-SMA的合成也被显著抑制。我们通过PAS染色观察肾脏的病理改变,结果发现,与db/db对照组相比较,db/db+PJ-34组肾小球肥大、系膜基质、系膜区面积、毛细血管基底膜厚度等病理改变均明显减轻。通过免疫组化观察α-SMA在肾脏组织中的沉积,结果发现:与db/db对照组相比较,db/db+PJ-34组小鼠肾皮质α-SMA的表达量明显减少。结论TET2表达激活介导了高糖诱导的TGF-β1基因表达调控区Cp G岛去甲基化和TGF-β1的表达上调,干预TET2表达可抑制高糖诱导的系膜细胞表型转化和糖尿病肾病的发生发展。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.2;R692.9

【参考文献】