GANT61通过下调Gli1改善成年小鼠实验性心肌梗死后心肌纤维化的研究

发布时间：2018-05-29 05:11

本文选题：心肌梗死 + 心肌纤维化　；参考：《重庆医科大学》2017年硕士论文

【摘要】：背景:Hedgehog(HH)通路是一条经典的信号通路,在从低等动物到高等动物中普遍存在。正常表达的HH信号在人类生长发育过程中发挥了关键作用,如参与诸如血管、神经管、末梢组织等的形成。但持续异常激活的HH信号可导致不同类型增殖性疾病的发生,包括各种肿瘤。此外,活化的HH信号也参与了组织的纤维化。而Gli1是HH信号通路中最重要的下游核转录因子,目前许多研究已经证实Gli1在纤维化的形成和发展中扮演着重要的角色,但Gli1在心血管系统疾病研究甚少,特别是在心肌梗死方面。因此通过检测成年小鼠心肌梗死后Gli1在缺血心肌组织中的表达特点,来探讨Gli小分子抑制剂GANT61减轻心肌梗死后心肌纤维化的分子机制意义重大。第一部分:探讨Gli1在心肌梗死后心肌组织的表达特点目的:通过建立小鼠心肌梗死动物模型,探讨Gli1在心肌梗死后心肌组织的表达特点。方法:在27只C57BL/6雄性小鼠中,运用随机数字法选择15只小鼠作为心肌梗死组(心梗组)建立心肌梗死模型,并分组为心梗组1周、心梗组2周、心梗组3周。在余下的12只小鼠中再次运用随机数字法选择9只作为假手术组,分组为假手术组1周、假手术组2周、假手术组3周。最后的3只作为正常组。运用实时荧光定量PCR和Western blot检测心梗组1周、2周、3周梗死周围区缺血心肌组织Gli1m RNA及蛋白的表达,并与正常组及假手术组相比较。运用免疫荧光检验心肌梗死和周边区缺血心肌组织Gli1的表达。结果:与心梗组2周相比较,心梗组3周Gli1 m RNA的表达量明显升高(P0.05),差异有统计学意义;假手术组与正常组心肌组织中Gli1 m RNA表达无明显变化(P0.05),差异无统计学意义;与心梗组2周相比,心肌梗死后第3周Gli1蛋白表达量明显升高,其结果与q RT-PCR相似;免疫荧光结果提示:在正常组的心肌组织,Gli1不表达;在心肌梗死后2周的表达量少于心肌梗死后3周。结论:Gli1在正常组、假手术组及心梗组1周不表达,而在心梗组2周Gli1存在激活的现象,并表现为随着时间的延长,Gli1 m RNA及蛋白的表达量有逐渐升高的趋势。第二部分:探讨GANT61改善成年小鼠实验性心肌梗死后心肌纤维化的分子机制目的:探讨GANT61改善成年小鼠实验性心肌梗死后心肌纤维化的分子机制。方法:在80只C57BL/6雄性小鼠中,随机取60只小鼠建立心肌梗死动物模型。心梗术后死亡18只。将存活的小鼠按照随机数字表法分为心梗组、生理盐水组、干预组。干预组于手术当天起以50mg/kg/d的剂量皮下注射GANT61,生理盐水组皮下注射与GANT61相等体积的生理盐水。此外随机取10只小鼠则作为假手术组,假手术组打开胸腔后,缝线只穿过左前降支血管,不拉紧。剩下的10只小鼠则作为正常组。分别运用实时荧光定量PCR和Western blot检测心梗组1周、2周、3周,生理盐水组1周、2周、3周及干预组1周、2周、3周梗死和周围区缺血心肌组织Gli1、TGF-β1、Ⅰ型及Ⅲ型胶原m RNA及蛋白表达。并与正常组及假手术组相比较.运用Masson染色观察心脏梗死周边区组织形态及心肌纤维化程度。结果:Masson染色显示,心梗模型小鼠术后心肌纤维化程度随着时间延长而加重,GANT61干预后能减轻心肌纤维化;q RT-PCR和Western blot结果显示,与心梗组3周相比较,GANT61干预3周后,可以明显下调组织中Gli1 m RNA和蛋白的表达水平(P0.05),差异有统计学意义;与正常组相比,心梗后2周TGF-β1、Ⅰ型及Ⅲ型胶原m RNA及蛋白表达升高(P0.05),差异有统计学意义;与心梗组3周相比,经过GANT61干预3周后TGF-β1、Ⅰ型及Ⅲ型胶原m RNA及蛋白表达均有下降(P0.05),差异有统计学意义。结论:GANT61可以通过抑制HH信号通路中Gli1的表达来减轻心肌纤维化。预测Gli1可能成为改善心肌纤维化的新的目标因子。
[Abstract]:Background: the Hedgehog (HH) pathway is a classic signaling pathway, which is common in lower animals and higher animals. Normal HH signals play a key role in human growth, such as the formation of blood vessels, nerve tubes, and end tissue, but the continuous abnormal activation of HH signals can lead to different types of proliferation. The occurrence of disease includes various kinds of tumors. In addition, activated HH signals are also involved in tissue fibrosis. Gli1 is the most important downstream nuclear transcription factor in the HH signaling pathway. Many studies have shown that Gli1 plays an important role in the formation and development of fibrosis, but the study of Gli1 in cardiovascular system diseases is very rare, especially in the case of cardiovascular disease. In the field of myocardial infarction, the molecular mechanism of the Gli small molecule inhibitor GANT61 to reduce myocardial fibrosis after myocardial infarction is investigated by detecting the expression of Gli1 in the ischemic myocardium after myocardial infarction in adult mice. Part 1: To explore the purpose of Gli1 expression in myocardial tissue after myocardial infarction: by establishing a small group of myocardium The rat model of myocardial infarction was used to investigate the expression of Gli1 in myocardial tissue after myocardial infarction. Methods: in 27 male C57BL/6 mice, 15 mice were selected as myocardial infarction group (MI group) to establish myocardial infarction model, and were divided into myocardial infarction group for 1 weeks, myocardial infarction group for 2 weeks and myocardial infarction group for 3 weeks. In the remaining 12 mice, the myocardial infarction group was divided into two groups. The random number method was used to select 9 sham operation groups, 1 weeks in sham operation group, 2 weeks in sham operation group and 3 weeks in sham operation group. The last 3 were in normal group. The expression of Gli1m RNA and protein in ischemic myocardium around infarct area was detected by real time fluorescence quantitative PCR and Western blot, and the expression of Gli1m RNA and protein in ischemic myocardium around infarct area, and with normal group and false group. The expression of Gli1 in myocardial infarction and peripheral ischemic myocardium was examined by immunofluorescence. Results: the expression of Gli1 m RNA in myocardial infarction group was significantly higher than that of 2 weeks in myocardial infarction group (P0.05), and the difference was statistically significant. There was no significant change in the expression of Gli1 m RNA in the sham operation group and the normal group (P0.05), and there was no difference between the myocardial infarction group and the normal group (P0.05). Statistical significance; compared with the 2 weeks of myocardial infarction, the expression of Gli1 protein increased significantly at third weeks after myocardial infarction, and the results were similar to that of Q RT-PCR. The immunofluorescence results suggested that Gli1 was not expressed in the normal group of myocardium, and the expression in 2 weeks after myocardial infarction was less than 3 weeks after myocardial infarction. Conclusion: Gli1 was in the normal group, the sham operation group and the myocardial infarction group for 1 weeks. No expression, but in the 2 week Gli1 of the myocardial infarction group, the expression of Gli1 m RNA and protein has a tendency to increase gradually. The second part: To explore the molecular mechanism of GANT61 to improve myocardial fibrosis after myocardial infarction in adult mice: To explore GANT61 to improve experimental myocardial infarction in adult mice. The molecular mechanism of myocardial fibrosis after death. Methods: in 80 C57BL/6 male mice, 60 mice were randomly selected to establish an animal model of myocardial infarction. After myocardial infarction, 18 died. The surviving mice were divided into myocardial infarction group, saline group and intervention group according to the random number table method. The intervention group injected GANT6 subcutaneously at the dose of 50mg/kg/d from the day of operation. 1, the physiological saline group was subcutaneously injected with the same volume of normal saline with GANT61. In addition, 10 mice were taken as the sham operation group. The sham group opened the thoracic cavity and the suture only passed through the left anterior descending vessel and did not tighten. The remaining 10 mice were used as the normal group. The real-time fluorescein PCR and Western blot were used to detect the myocardial infarction group for 1 weeks, 2 weeks, 3, respectively. The Gli1, TGF- beta 1, type I and type III collagen m RNA and protein expression of Gli1, TGF- beta 1, type I and type III collagen in the infarcted and peripheral ischemic myocardium in the 1 weeks, 3 weeks, and the 3 weeks of the intervention group were compared with the normal group and the sham group. Masson staining was used to observe the tissue morphology and the degree of myocardial fibrosis by Masson staining. Results: Masson staining showed that Myocardial fibrosis in myocardial infarction model mice increased with time, and GANT61 intervention could reduce myocardial fibrosis. The results of Q RT-PCR and Western blot showed that the expression level of Gli1 m RNA and protein in the tissue could be significantly reduced after 3 weeks of GANT61 intervention (P0.05), and the difference was statistically significant; and the difference was statistically significant. Compared with the 2 weeks after myocardial infarction, TGF- beta 1, type I and type III collagen m RNA and protein expression increased (P0.05), and the difference was statistically significant. Compared with the 3 weeks of myocardial infarction group, TGF- beta 1 after GANT61 intervention, the m RNA and protein expression of type I and type III collagen decreased (P0.05), and the difference was statistically significant. Conclusion: GANT61 can inhibit HH signaling pathway. The expression of Gli1 can alleviate myocardial fibrosis. Prediction of Gli1 may be a new target factor for improving myocardial fibrosis.
【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】：R542.22

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