依维莫司通过上调TET1蛋白抑制心肌纤维化的研究

发布时间：2018-02-21 02:40

本文关键词： 心肌纤维化依维莫司 TET蛋白　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:心肌梗死目前已经成为威胁整个人类健康的最重要的疾病之一,严重影响患者的寿命和生存质量。心肌纤维化(Myocardial Fibrosis,MF)是心肌梗死后的一个很重要的病理特点,且对疾病的预后有着很大的影响。目前对于心肌梗死的治疗方法比较有限,且只能暂时缓解疾病症状,而不能从根本上缓解甚至逆转心肌纤维化的发生发展。因此,研究缓解心肌纤维化的方法有着很重要的意义。依维莫司是一种抗肿瘤、抗血管增殖的药物,在多种生物学过程中起着重要作用。而研究发现依维莫司还具有抗成纤维细胞增殖的作用。而依维莫司是否具有抗心肌纤维化的作用,还尚不清楚。TET蛋白是一种去甲基化酶,能将5-甲基胞嘧啶转化为5-羟甲基胞嘧啶,从而调节基因的甲基化水平,促进基因表达。既往研究表明TET蛋白在维持胚胎干细胞多能性和肿瘤的发生、发展中起着重要作用。而最近有研究发现,TET蛋白也参与抗心肌纤维化过程,对缓解器官纤维化的发展起着重要作用。目的:建立C57BL/6小鼠心肌梗死模型,并对心梗后小鼠进行依维莫司抗纤维化治疗,观察小鼠心梗术后心肌的病理学变化。通过检测小鼠心肌组织中TET蛋白的m RNA水平,发现TET1的m RNA表达水平具一定的趋势,遂运用Western blot从蛋白水平进一步验证。从而初步探讨依维莫司在心肌纤维化过程中发挥的作用,以及依维莫司是否通过激活TET1蛋白发挥抗心肌纤维化的作用。方法:将心肌梗死术后存活的24只C57BL/6成年小鼠随机分为心肌梗死组、心肌梗死+依维莫司组、正常组各12只。干预组以依维莫司3mg/kg/d灌胃。分别于术后3d、1w、2w取出心肌组织保存于液氮或做脱水处理,做HE染色、Masson染色观察心肌胶原增生的情况,用荧光定量PCR方法检测小鼠心肌梗死后的梗死周边区TET1、TET2、TET3及ColⅠ(CollagenⅠ)和ColⅢ(CollagenⅢ)的m RNA水平,发现TET1的m RNA表达最具趋势,再运用Western blot检测TET1的蛋白水平。结果:1.HE染色和Masson染色显示心肌梗死术后,小鼠心脏梗死及周边区有大量胶原沉积。而干预组的心肌组织胶原含量较心梗组明显减轻。2.依维莫司干预后,小鼠心肌TET1 m RNA水平明显上调,而对TET2和TET3没有影响。之后的western blot检测也验证了这一变化。结论:根据我们的实验结果可以得出,依维莫司具有抗心肌纤维化的作用,且依维莫司可能通过上调TET1发挥抗心肌纤维化的作用。
[Abstract]:Background: myocardial infarction has become one of the most important diseases threatening the health of the whole human being. Myocardial fibrosis is an important pathological feature after myocardial infarction. At present, the treatment of myocardial infarction is limited, and can only temporarily alleviate the symptoms of the disease, but can not fundamentally alleviate or even reverse the occurrence and development of myocardial fibrosis. It is of great significance to study ways to alleviate myocardial fibrosis. Evimostr is an antitumor, antiangiogenic drug. It has been found that Evimostr also has an anti-proliferation effect on fibroblasts. It is not clear whether Evirolimus has an effect on myocardial fibrosis, but it is not clear that the TET protein is a demethylase. It can transform 5-methylcytosine into 5-hydroxymethylcytosine, thereby regulating the methylation level of genes and promoting gene expression. Previous studies have shown that TET protein maintains the pluripotency and tumorigenesis of embryonic stem cells. Development plays an important role. Recently, it has been found that Tet protein is also involved in the process of anti-myocardial fibrosis and plays an important role in alleviating the development of organ fibrosis. Objective: to establish myocardial infarction model of C57BL / 6 mice. The changes of myocardial pathological changes after myocardial infarction were observed, and the expression of m RNA in TET1 was found to be a trend by detecting the level of m RNA of TET protein in myocardial tissue of mice after myocardial infarction. Western blot was used to further verify the protein level. The role of ivermostr in the process of myocardial fibrosis was preliminarily discussed. Methods: 24 C57BL / 6 adult mice survived after myocardial infarction were randomly divided into myocardial infarction group (MI group), myocardial infarction (MI) group (Evirolimus group). In the intervention group, the myocardial tissue was taken out for preservation in liquid nitrogen or dehydrated at 3 mg / kg / d at 3 days after operation, and the proliferation of myocardial collagen was observed by HE staining and Masson staining. The expression of m RNA in the infarct peripheral areas of mice after myocardial infarction was detected by fluorescence quantitative PCR. The results showed that the expression of m RNA in TET1 was the most tendency. Western blot was used to detect the protein level of TET1. Results 1. He staining and Masson staining showed that after myocardial infarction, There was a large amount of collagen deposition in myocardial infarction and peripheral area of mice, but the collagen content in myocardial tissue in the intervention group was significantly decreased compared with that in the myocardial infarction group. The level of TET1 m RNA in the myocardium of the intervention group was significantly up-regulated after the intervention of ivermostr. However, there was no effect on TET2 and TET3. This change was confirmed by subsequent western blot tests. Conclusion: according to our experimental results, Evimostr has the effect of anti-myocardial fibrosis. In addition, Evimostr may play a role in anti-myocardial fibrosis by upregulating TET1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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