DDAH2基因甲基化在勃起功能障碍发病中的作用和机制研究

发布时间：2018-07-20 18:50

【摘要】：背景与目的：ED是指阴茎不能达到和(或)维持足够的勃起以完成满意的性生活,且病程持续6个月以上。阴茎勃起是一个由神经发动的动脉供血、海绵体储血的综合过程,由NOS以左旋精氨酸为底物催化生成的NO,是导致血管扩张、海绵体舒张的主要信使,参与勃起的诱导和维持。DDAH2蛋白是维持血管内皮功能的重要因子,广泛表达于内皮细胞、血管平滑肌细胞以及血管外膜组织等多种血管壁成分中。通过有丝分裂或是减数分裂来传递非DNA序列信息的现象称为表观遗传,DNA甲基化是表观遗传的重要组成部分。研究证实,DDAH2基因甲基化的修饰参与了血管内皮细胞功能的维持。因此,本研究拟研究高同型半胱氨酸血症ED大鼠中DDAH2的表达及其启动子区域甲基化水平,并通过给予甲基化抑制剂5-aza处理ED大鼠,对DDAH2甲基化与ED的关系进行研究,了解DDAH2基因甲基化在ED中的作用及其机制,探索去甲基化剂治疗ED的可能性和方法。第一部分实验：高同型半胱氨酸勃起功能障碍大鼠模型的建立及表观遗传学机制探讨实验方法：1、SD大鼠分组：正常饲料喂养组(对照组),正常饲料+4%蛋氨酸喂养(蛋氨酸组)。喂养6周后,测定ICP、MAP后,取材,保存。2、勃起功能评价：利用电刺激大鼠盆腔星状神经节法测定阴茎海绵体内压测定(ICP),颈动脉穿刺测定平均动脉压(MAP), ICP/MAP作为勃起功能的评价指标；3、组织学改变：免疫组化观察各组大鼠阴茎海绵体内皮细胞、平滑肌细胞数量结构变化,马松染色观察胶原纤维变化；3、 Hey:Elisa法测定不同组别大鼠血清中Hcy、 ADMA水平；4、 NO/cGMP通路改变：利用NO、cGMP试剂盒测定阴茎海绵体中NO、 cGMP含量；5、焦磷酸盐测序法检测各组大鼠血液和阴茎海绵体DDAH2基因启动子区CpG岛甲基化状态。实验结果：SD大鼠高蛋氨酸饮食6周后,血清同型半胱氨酸明显高于对照组,且大鼠勃起功能(ICP/MAP)降低。NO、cGMP含量变化与勃起功能一致。较之正常对照组,蛋氨酸组平滑肌及内皮含量减少,并且胶原纤维增多。蛋氨酸组大鼠血液和阴茎组织DNA DDAH2基因启动子区域CpG岛甲基化程度高于对照组。结论：高蛋氨酸饮食可造成高同型半胱氨酸血症ED模型。NO/cGMP通路活性降低可能是导致高同型半胱氨酸ED的原因。作为一种血管内皮保护因子,DDAH2与其调控的NO/cGMP通路改变与勃起功能一致。推测DDAH2启动子区域甲基化状态改变与高同型半胱氨酸ED的发病有关。第二部分实验：甲基化调控对高同型半胱氨酸氨酸大鼠勃起功能影响研究实验方法1.实验分组：正常饲料喂养组(对照组),正常饲料+4%蛋氨酸喂养(蛋氨酸组),正常饲料+4%蛋氨酸喂养+5-氮杂脱氧胞苷(5-aza组)。5-aza组除给予正常饲料+4%蛋氨酸组喂养外,每隔3天给予5-aza 1.5mg/kg腹腔注射；喂养6周后,测定ICP、 MAP后,取材,保存。2.疗效观测：1)、勃起功能测定：利用电刺激大鼠盆腔星状神经节法测定阴茎海绵体内压测定(ICP),颈动脉穿刺测定平均动脉压(MAP), ICP/MAP作为勃起功能的评价指标；2)、组织学观察：免疫组化观察各组大鼠阴茎海绵体内皮细胞、平滑肌细胞数量结构变化；马松染色观察胶原纤维变化；3.机制探索：1)、各组大鼠阴茎海绵体Hey、 ADMA;2)、各组大鼠甲基化供体SAM含量；3)、各组大鼠阴茎海绵体中DDAH2-mRNA变化；4)、各组大鼠阴茎海绵体中DDAH2表达；5)、NO-cGMP通路：测定各组大鼠阴茎海绵体中NO、 cGMP含量；6)、测定各组大鼠血液和阴茎海绵体DDAH2基因启动子区域甲基化状态的变化；实验结果：甲基化抑制剂5-aza治疗能够改善勃起功能(ICP/MAP),提高阴茎组织中NO、 cGMP含量,提高平滑肌及内皮细胞含量,5-aza同样可以逆转DDAH2启动子区域甲基化改变,并且与DDAH2-mRNA和DDAH2蛋白表达改变一致。DDAH2启动子区域甲基化参与了高同型半胱氨酸ED的发生,甲基化抑制剂5-aza可改善高同型半胱氨酸ED大鼠的勃起功能。结论：DDAH2基因甲基化在高同型半胱氨酸ED发病中具有重要作用。DDAH2基因启动子区域甲基化通过调控DDAH2-mRNA和DDAH2蛋白以及NO/cGMP活性的途径参与高同型半胱氨酸ED的发生、进展。由于甲基化的可逆转性,为临床治疗ED提供了新的靶点。
[Abstract]:Background and purpose: ED means that the penis can not achieve and (or) maintain a sufficient erection to complete a satisfactory sexual life, and the course of the disease lasts more than 6 months. The erection of the penis is a blood supply from the nerves waging, the synthesis process of the cavernous body, the NO catalyzed by NOS with levoarginine as the base, which leads to vasodilatation and corpus cavernosus The main messenger of Zhang, which participates in the erectile induction and maintenance of.DDAH2 protein, is an important factor in the maintenance of vascular endothelial function, widely expressed in various vascular wall components such as endothelial cells, vascular smooth muscle cells and epicardial tissue. The phenomenon of transmission of non DNA sequence information through mitosis or meiosis is called epigenetic, DNA Methylation is an important part of epigenetics. Studies have shown that the modification of DDAH2 gene methylation participates in the maintenance of vascular endothelial cell function. Therefore, this study intends to study the expression of DDAH2 in ED rats with hyperhomocysteinemia and the level of methylation in the promoter region, and to treat ED rats by the administration of methylation inhibitor 5-aza. Study the relationship between DDAH2 methylation and ED, understand the role and mechanism of DDAH2 gene methylation in ED, explore the possibility and method of treating ED with demethylation agent. First part experiment: establishment of Hyperhomocysteine erectile dysfunction rat model and epigenetic mechanism study methods: 1, SD rats group: positive The normal feed group (control group), normal feed +4% methionine feeding (methionine group). After feeding for 6 weeks, ICP, MAP, ICP, preservation, erectile function evaluation: using electrical stimulation of the pelvic stellate ganglion method for the determination of penile corpus cavernosum internal pressure (ICP), carotid artery puncture mean arterial pressure (MAP), ICP/MAP as an erectile function 3, histological changes: immunohistochemical observation of the cavernous endothelial cells in the corpus cavernosum, the changes of the number and structure of smooth muscle cells, the changes of collagen fiber by Masson staining, and the determination of Hcy, ADMA in the serum of different groups of rats by the method of Hey:Elisa; 4, the change of the NO/cGMP pathway: the use of NO and cGMP kit to determine the penis sponge. The content of NO and cGMP in the body; 5, pyrophosphate sequencing method was used to detect the methylation status of CpG island in the promoter region of the DDAH2 gene of the cavernous corpus cavernosum of the rats. The results were as follows: after 6 weeks of high methionine diet in SD rats, the serum homocysteine was significantly higher than that of the control group, and the erectile function (ICP/MAP) decreased the.NO, cGMP content and erectile function in the rat. Consistent. Compared with normal control group, the content of smooth muscle and endothelium in methionine group decreased and collagen fiber increased. Methionine group DNA DDAH2 gene promoter region CpG island methylation level was higher than that of control group. Conclusion: high methionine diet can cause the.NO/cGMP pathway of hyperhomocysteinemia ED model to decrease the activity of.NO/cGMP pathway. Low may be the cause of high homocysteine ED. As a vascular endothelial protective factor, DDAH2 and its regulated NO/cGMP pathway change with erectile function. It is speculated that the change of methylation status in the DDAH2 promoter region is related to the pathogenesis of high homocysteine ED. The second part is verified by methylation regulation of homocysteine Experimental methods 1. groups: normal feed feeding group (control group), normal feed +4% methionine feeding (methionine group), normal feed +4% methionine feeding +5- n-hetero deoxycytidine (group 5-aza).5-aza group fed with normal feed +4% methionine group and 5-aza 1.5mg/kg peritoneal injection every 3 days after feeding in normal feed group (control group) Shoot, after feeding for 6 weeks, after measuring ICP, MAP, materials, preservation of.2. efficacy observation: 1), erectile function determination: using electrical stimulation of rat pelvic stellate ganglion method to determine the internal pressure of corpus cavernosum (ICP), carotid artery puncture mean arterial pressure (MAP), ICP/MAP as the evaluation index of erectile function; 2) histological observation: immunohistochemical observation The number and structure of smooth muscle cells in the cavernous corpus cavernosum and the changes of the smooth muscle cells were observed in each group. The changes of collagen fibers were observed by Masson staining; 3. mechanism was explored: 1), the penis corpus cavernosum Hey, ADMA; 2), the SAM content of the methylation donor in the rats in each group, 3), the changes of DDAH2-mRNA in the cavernous corpus cavernosum in each group, 4), 4), each group of the cavernous corpus cavernosum DDAH2 expression; 5), NO-cGMP pathway: Determination of NO, cGMP content in the corpus cavernosum of rats and the changes in the methylation status of the DDAH2 gene promoter region of the cavernous corpus cavernosum in each group; experimental results: the methylation inhibitor 5-aza can improve the erectile function (ICP/MAP), improve the NO, cGMP content in the penis tissue. Quantity, increase the content of smooth muscle and endothelial cells, 5-aza can also reverse the methylation of DDAH2 promoter region, and the methylation of.DDAH2 promoter region is involved in the occurrence of high homocysteine ED, and the methylation inhibitor 5-aza can improve the erectile function of high homocysteine ED rats. Conclusion: methylation of DDAH2 gene plays an important role in the pathogenesis of high homocysteine ED. Methylation of.DDAH2 gene promoter region is involved in the development of high homocysteine ED through the regulation of DDAH2-mRNA and DDAH2 protein and NO/cGMP activity. The reversible transfer of methylation provides a new target for the clinical treatment of ED.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698

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