本文選題：心肌梗死 + 心肌纖維化��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:Hedgehog(HH)通路是一條經(jīng)典的信號通路,在從低等動物到高等動物中普遍存在。正常表達的HH信號在人類生長發(fā)育過程中發(fā)揮了關(guān)鍵作用,如參與諸如血管、神經(jīng)管、末梢組織等的形成。但持續(xù)異常激活的HH信號可導(dǎo)致不同類型增殖性疾病的發(fā)生,包括各種腫瘤。此外,活化的HH信號也參與了組織的纖維化。而Gli1是HH信號通路中最重要的下游核轉(zhuǎn)錄因子,目前許多研究已經(jīng)證實Gli1在纖維化的形成和發(fā)展中扮演著重要的角色,但Gli1在心血管系統(tǒng)疾病研究甚少,特別是在心肌梗死方面。因此通過檢測成年小鼠心肌梗死后Gli1在缺血心肌組織中的表達特點,來探討Gli小分子抑制劑GANT61減輕心肌梗死后心肌纖維化的分子機制意義重大。第一部分:探討Gli1在心肌梗死后心肌組織的表達特點目的:通過建立小鼠心肌梗死動物模型,探討Gli1在心肌梗死后心肌組織的表達特點。方法:在27只C57BL/6雄性小鼠中,運用隨機數(shù)字法選擇15只小鼠作為心肌梗死組(心梗組)建立心肌梗死模型,并分組為心梗組1周、心梗組2周、心梗組3周。在余下的12只小鼠中再次運用隨機數(shù)字法選擇9只作為假手術(shù)組,分組為假手術(shù)組1周、假手術(shù)組2周、假手術(shù)組3周。最后的3只作為正常組。運用實時熒光定量PCR和Western blot檢測心梗組1周、2周、3周梗死周圍區(qū)缺血心肌組織Gli1m RNA及蛋白的表達,并與正常組及假手術(shù)組相比較。運用免疫熒光檢驗心肌梗死和周邊區(qū)缺血心肌組織Gli1的表達。結(jié)果:與心梗組2周相比較,心梗組3周Gli1 m RNA的表達量明顯升高(P0.05),差異有統(tǒng)計學(xué)意義;假手術(shù)組與正常組心肌組織中Gli1 m RNA表達無明顯變化(P0.05),差異無統(tǒng)計學(xué)意義;與心梗組2周相比,心肌梗死后第3周Gli1蛋白表達量明顯升高,其結(jié)果與q RT-PCR相似;免疫熒光結(jié)果提示:在正常組的心肌組織,Gli1不表達;在心肌梗死后2周的表達量少于心肌梗死后3周。結(jié)論:Gli1在正常組、假手術(shù)組及心梗組1周不表達,而在心梗組2周Gli1存在激活的現(xiàn)象,并表現(xiàn)為隨著時間的延長,Gli1 m RNA及蛋白的表達量有逐漸升高的趨勢。第二部分:探討GANT61改善成年小鼠實驗性心肌梗死后心肌纖維化的分子機制目的:探討GANT61改善成年小鼠實驗性心肌梗死后心肌纖維化的分子機制。方法:在80只C57BL/6雄性小鼠中,隨機取60只小鼠建立心肌梗死動物模型。心梗術(shù)后死亡18只。將存活的小鼠按照隨機數(shù)字表法分為心梗組、生理鹽水組、干預(yù)組。干預(yù)組于手術(shù)當(dāng)天起以50mg/kg/d的劑量皮下注射GANT61,生理鹽水組皮下注射與GANT61相等體積的生理鹽水。此外隨機取10只小鼠則作為假手術(shù)組,假手術(shù)組打開胸腔后,縫線只穿過左前降支血管,不拉緊。剩下的10只小鼠則作為正常組。分別運用實時熒光定量PCR和Western blot檢測心梗組1周、2周、3周,生理鹽水組1周、2周、3周及干預(yù)組1周、2周、3周梗死和周圍區(qū)缺血心肌組織Gli1、TGF-β1、Ⅰ型及Ⅲ型膠原m RNA及蛋白表達。并與正常組及假手術(shù)組相比較.運用Masson染色觀察心臟梗死周邊區(qū)組織形態(tài)及心肌纖維化程度。結(jié)果:Masson染色顯示,心梗模型小鼠術(shù)后心肌纖維化程度隨著時間延長而加重,GANT61干預(yù)后能減輕心肌纖維化;q RT-PCR和Western blot結(jié)果顯示,與心梗組3周相比較,GANT61干預(yù)3周后,可以明顯下調(diào)組織中Gli1 m RNA和蛋白的表達水平(P0.05),差異有統(tǒng)計學(xué)意義;與正常組相比,心梗后2周TGF-β1、Ⅰ型及Ⅲ型膠原m RNA及蛋白表達升高(P0.05),差異有統(tǒng)計學(xué)意義;與心梗組3周相比,經(jīng)過GANT61干預(yù)3周后TGF-β1、Ⅰ型及Ⅲ型膠原m RNA及蛋白表達均有下降(P0.05),差異有統(tǒng)計學(xué)意義。結(jié)論:GANT61可以通過抑制HH信號通路中Gli1的表達來減輕心肌纖維化。預(yù)測Gli1可能成為改善心肌纖維化的新的目標(biāo)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R542.22

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