SHH信号通路在新生血管形成中的作用

发布时间：2018-05-02 07:49

本文选题：缺血再灌注损伤 + 血管再生　；参考：《青岛大学》2017年博士论文

【摘要】：目的:近年研究发现,SHH信号通路参与了多种动物缺血模型中的血管再生,包括脑缺血,骨骼肌缺血、角膜及肢体缺血等。然而,SHH信号通路在心肌缺血再灌注中的研究却很少,并且给予重组人SHH蛋白干预是否对心脏微血管内皮细胞具有保护作用及其潜在作用机制是否与激活SHH-Patched-SMO-Gli信号通路有关尚无文献报道。而且SHH在急性心肌梗死后冠状动脉侧枝循环的形成中发挥何种作用及其机制尚无相关研究。因此,我们将从以下方面探讨:1.研究重组人SHH蛋白对CMECs细胞凋亡与增殖的影响,探讨SHH信号通路激活对大鼠CMECs缺血再灌注损伤后促血管再生的作用及其潜在机制,为临床上心肌缺血再灌注损伤的防治提供理论依据。2.探讨急性心肌梗死患者冠状动脉侧枝循环建立与血浆SHH水平的相关性,了解SHH在急性心肌梗死后冠状动脉侧枝循环形成中的作用,为临床上急性心肌梗死治疗提供新的治疗手段。方法:原代培养SD雄性大鼠心脏微血管内皮细胞(Cardiac microvascular endothelial cells,CMECs),建立氧糖剥夺/复氧(Oxygen-glucose deprivation/reoxygenation,OGD/R)模拟体外缺血再灌注损伤模型,给予外源性重组人SHH蛋白、Cyclopamine处理,RT-PCR检测对照组和OGD组中SHH的mRNA水平;用MTT和Annexin-V-FITC/PI双标记流式细胞术分别检测细胞活力和细胞凋亡;应用ELISA和RT-PCR方法分别检测血管新生因子(VEGF,FGF,Ang-1)的血清水平和mRNA表达水平;应用RT-PCR和Western blotting方法分别检测Sonic hedgehog(SHH)信号通路靶分子(SHH,SMO,Patched-1,Gli-1,Gli-2)的mRNA和蛋白表达水平。在临床试验中,选择2014年12月-2016年6月于济宁市第一人民医院心血管诊疗中心收治的行急诊PCI术的急性心肌梗死患者298例为研究对象,懫用盲法进行Rentrop评分,分为无侧枝循环组,侧支循环较差组和侧性循环较好组3组。并且采集患者年龄、性别等基本临床资料以及实验室检查的结果,确定心肌梗死相关动脉,并记录梗死发病的时间。留取患者的术前血浆标本,并采用ELISA法测定血浆SHH水平。懫用SPSS20.0软件进行统计学分析,从而探讨急性心肌梗死患者冠状动脉侧枝循环建立与血浆SHH水平的相关性。结果:1.与正常对照组相比,OGD组SHH的mRNA表达水平明显升高,差异有统计学意义(p0.05),上述结果表明氧糖剥夺/复氧cmecs缺血再灌注损伤可以激活shh信号通路,促使shhmrna表达水平升高。2.与正常对照组相比,加入外源性重组人shh蛋白后,正常氧糖条件下cmecs的细胞活力显著增加,但其影响可以被抑制剂cyclopamine显著消减(p0.05),上述结果表明shh可以促进正常氧糖条件下cmecs的细胞活力增强。相反,当细胞在ogd/r条件下,细胞活力与正常对照组相比显著减弱(p0.05),表明氧糖剥夺/复氧会导致细胞活力受到抑制;而在shh处理后与ogd组相比细胞活力显著增加(p0.05);但其影响也可以被抑制剂cyclopamine显著消减(p0.05),上述结果表明shh可以促进氧糖剥夺/复氧cmecs的细胞活力增强。3.与正常对照组相比,加入外源性重组人shh蛋白后,正常氧糖条件下cmecs的细胞凋亡仅稍减少(4.09%),差异无统计学意义(p0.05);当给予抑制剂cyclopamine处理后cmecs的细胞凋亡较正常对照组相比仅稍增加(6.43%),差异无统计学意义(p0.05)。在ogd/r条件下,cmecs细胞凋亡较正常对照组增加(12.4%),差异有统计学意义(p0.05),表明ogd/r导致了cmecs的细胞凋亡显著增加;当我们给予激活剂shh蛋白处理后cmecs的细胞凋亡出现显著减少(5.23%),差异有统计学意义(p0.05);给予抑制剂cyclopamine处理后发现cmecs细胞凋亡与ogd组相比显著增加(16.7%),差异有统计学意义(p0.01),上述结果表明shh可以抑制氧糖剥夺/复氧cmecs的细胞凋亡。4.与正常对照组相比,加入外源性重组人shh蛋白后,正常氧糖条件下血管新生因子vegf,fgf和ang-1的血清水平和mrna表达显著增加,差异有统计学意义(p0.05),但是shh的生物学效应可以被shh信号通路特异性抑制剂cyclopamine所抑制(p0.01),上述结果表明shh可以促进正常氧糖条件下cmecs的血管新生。在ogd/r条件下,血管新生因子vegf,fgf和ang-1的血清水平和mrna表达显著增加,差异有统计学意义(p0.01),同样shh的生物学效应可以被shh信号通路特异性抑制剂cyclopamine所抑制(p0.01),上述结果表明shh可以促进氧糖剥夺/复氧cmecs的血管新生。5.加入外源性重组人shh蛋白后,在ogd/r条件下,shh信号通路中靶分子shh,smo,patched-1,gli-1和gli-2的mrna表达水平明显升高,与ogd组相比,差异有统计学意义(p0.05),但是shh的生物学效应可以被shh信号通路特异性抑制剂cyclopamine所抑制(p0.01)。同样,在ogd/r条件下,shh信号通路中靶分子shh,smo,patched-1,gli-1和gli-2的蛋白表达水平明显升高,与ogd组相比,差异有统计学意义(p0.05),但shh的生物学效应也可以被shh信号通路特异性抑制剂Cyclopamine所抑制(p0.01)。以上结果表明重组人SHH蛋白在激活SHH信号通路的下游信号蛋白表达过程中扮演重要的角色。6.急性心肌梗死患者冠脉侧枝循环较差组血浆SHH水平与对照组相比差异无统计学意义(p0.05);并且冠脉侧枝循环较好组中血浆SHH水平显著高于冠脉侧枝循环较差组以及对照组(p0.01),提示在急性心肌梗死患者冠脉侧枝循环评分较高的患者中其血浆SHH含量也比较高(p0.01),并且其相关性独立于年龄、性别、BMI、高血压、糖尿病家族史、心肌梗死类型、吸烟史、饮酒史、空腹血糖水平、血肌酐、血尿素氮、总胆固醇、甘油三酯、HDL-C、BNP等危险因素单独存在。结论:1.SHH对缺血再灌注损伤的心脏微血管内皮细胞具有保护作用;CMECs缺血再灌注损伤存在着SHH信号通路的激活;外源性加入重组人SHH蛋白可以增强细胞活力,减轻细胞凋亡,上调血管新生因子VEGF,FGF和Ang-1的表达,促进新生血管形成,提示SHH信号通路激活在CMECs缺血再灌注损伤后血管再生过程中发挥着举足轻重的作用,可能成为心肌缺血再灌注损伤的潜在治疗靶点。2.急性心肌梗死患者冠状动脉侧支循环建立与血浆SHH水平明显相关,且血浆SHH水平独立于年龄、性别、BMI、高血压、糖尿病家族史、心肌梗死类型、吸烟史、饮酒史、空腹血糖水平、血肌酐、血尿素氮、总胆固醇、甘油三酯、HDL-C、BNP等危险因素作用于急性心肌梗死患者缺血心肌区域侧枝循环的形成过程。提示可以将SHH血清水平作为急性心肌梗死患者冠状动脉侧枝循环形成状况好坏的预测因子。
[Abstract]:Objective: in recent years, SHH signaling pathway has been found to be involved in vascular regeneration in a variety of animal ischemic models, including cerebral ischemia, skeletal muscle ischemia, corneal and limb ischemia. However, there are few studies on the SHH signaling pathway in myocardial ischemia reperfusion, and the intervention of recombinant human SHH protein to the cardiac microvascular endothelial cells is guaranteed. There is no literature about whether the protective effect and its potential mechanism are related to the activation of the SHH-Patched-SMO-Gli signaling pathway. And what role and mechanism of SHH in the formation of coronary collateral circulation after acute myocardial infarction has not yet been studied. Therefore, we will discuss the following aspects: 1. the study of recombinant human SHH protein to CMECs The effect of apoptosis and proliferation, the effect of SHH signaling pathway activation on vascular regeneration after CMECs ischemia-reperfusion injury in rats and its potential mechanism, provide a theoretical basis for the prevention and treatment of myocardial ischemia reperfusion injury in clinical,.2. study on the correlation between the establishment of coronary artery lateral branch circulation and plasma SHH level in patients with acute myocardial infarction, The role of SHH in the formation of coronary collateral circulation after acute myocardial infarction provides a new treatment for the treatment of acute myocardial infarction. Methods: the primary culture of SD male rat cardiac microvascular endothelial cells (Cardiac microvascular endothelial cells, CMECs), and the establishment of oxygen glucose deprivation / reoxygenation (Oxygen-glucose deprivation/reox). Ygenation, OGD/R) simulated in vitro ischemia-reperfusion injury model, gave exogenous recombinant human SHH protein, Cyclopamine treatment, RT-PCR detected the mRNA level of SHH in the control group and OGD group. MTT and Annexin-V-FITC/PI double standard flow cytometry were used to detect cell viability and apoptosis respectively; ELISA and RT-PCR methods were used to detect angiogenesis respectively. The serum level of factor (VEGF, FGF, Ang-1) and the level of mRNA expression; the RT-PCR and Western blotting methods were used to detect the protein expression level of Sonic hedgehog (SHH) signaling pathway target molecule (SHH, SMO, Ang-1). In the clinical trial, the cardiovascular diagnosis of the first people's Hospital of Jining was selected in June. 298 patients with acute myocardial infarction treated with emergency PCI were treated with blind method of Rentrop. They were divided into no collateral circulation group, poor collateral circulation group and 3 group with better lateral circulation. The basic clinical data of age, sex and laboratory examination were collected, and the correlation of myocardial infarction was determined. Blood plasma samples were recorded and plasma SHH levels were measured by ELISA method. The correlation between coronary artery collateral circulation establishment and plasma SHH level was investigated by SPSS20.0 software. Results: 1. compared with the normal control group, the mRNA of SHH in the OGD group was mRNA. The expression level was significantly increased (P0.05). The results showed that oxygen glucose deprivation / reoxygenation CMECs ischemia-reperfusion injury could activate the Shh signaling pathway, promote the increase of shhmrna expression level and.2. compared with the normal control group. After adding exogenous recombinant human Shh protein, the cell viability of CMECs increased significantly under normal oxygen glucose condition. But the effect could be significantly reduced by the inhibitor cyclopamine (P0.05). The results showed that Shh could promote the cell viability of CMECs under the condition of normal oxygen glucose. On the contrary, the cell viability was significantly decreased compared with the normal control group (P0.05) under ogd/r conditions (P0.05), indicating that the oxygen glucose deprivation / reoxygenation could lead to the inhibition of cell viability; and in SH, the cell viability was inhibited. After h treatment, the cell viability was significantly increased (P0.05) compared with the OGD group, but the effect could also be significantly reduced by the inhibitor cyclopamine (P0.05). The results indicated that Shh could promote the cell viability of the oxygen glucose deprivation / reoxygenation CMECs as compared with the normal control group. After adding the exogenous recombinant human Shh protein, the CMECs cells in the normal oxygen glucose CMECs were the cells. There was only a slight decrease in apoptosis (4.09%), the difference was not statistically significant (P0.05). When the inhibitor cyclopamine was treated, the apoptosis of CMECs was only slightly increased (6.43%) compared with that of the normal control group (P0.05). In ogd/r, the apoptosis of CMECs cells was increased (12.4%), and the difference was statistically significant (P0.05), indicating og D/r resulted in a significant increase in apoptosis in CMECs; the apoptosis of CMECs cells decreased significantly (5.23%) when we treated the activator Shh protein (P0.05), and the apoptosis of CMECs cells was significantly increased (16.7%) compared with that of OGD group (P0.01), and the difference was statistically significant (P0.01). The results showed that Shh could inhibit the apoptosis of oxygen glucose deprivation / reoxygenation CMECs cell apoptosis.4. compared with the normal control group. After adding exogenous recombinant human Shh protein, the serum level of angiogenic factor VEGF, FGF and Ang-1 increased significantly under normal oxygen glucose conditions, and the difference was statistically significant (P0.05), but the biological effect of Shh could be carried out by SHH letter. The signal pathway specific inhibitor cyclopamine inhibited (P0.01). The results showed that Shh could promote the angiogenesis of CMECs under the condition of normal oxygen glucose. The serum level and mRNA expression of the angiogenesis factor VEGF, FGF and Ang-1 increased significantly under ogd/r conditions, and the difference was statistically significant (P0.01), and the biological effect of the same Shh could be expressed in Shh letter. The signal transduction pathway specific inhibitor cyclopamine inhibits (P0.01). The results show that Shh can promote the addition of recombinant human Shh protein to the angiogenic.5. of oxygen glucose deprivation / reoxygenation CMECs. In ogd/r, the target molecules Shh, SMO, patched-1, Gli-1, and the expression level of the Shh signaling pathway are significantly higher than those of the shh. There were statistical significance (P0.05), but the biological effects of Shh could be inhibited by the Shh signaling pathway specific inhibitor cyclopamine (P0.01). Similarly, the protein expression levels of the target molecules Shh, SMO, patched-1, Gli-1 and gli-2 were significantly higher in the Shh signaling pathway under the ogd/r condition, and the difference was statistically significant compared with those of the group. Biological effects can also be inhibited by the Shh signaling pathway specific inhibitor Cyclopamine (P0.01). The results show that the recombinant human SHH protein plays an important role in the process of activating the downstream signal protein in the SHH signaling pathway. The plasma SHH level of the coronary collateral circulation group in patients with acute myocardial infarction is different from that of the control group. There was no statistical significance (P0.05), and the plasma SHH level in the better coronary collateral circulation group was significantly higher than that of the poor coronary collateral circulation group and the control group (P0.01), suggesting that the plasma SHH content was higher in patients with higher coronary collateral circulation score (P0.01) in patients with acute myocardial infarction (P0.01), and the correlation was independent of age, sex, BMI, Hypertension, diabetes family history, myocardial infarction type, smoking history, drinking history, fasting blood glucose level, blood creatinine, blood urea nitrogen, total cholesterol, triglyceride, HDL-C, BNP and other risk factors exist alone. Conclusion: 1.SHH has protective effect on cardiac microvascular intravascular cells of ischemia-reperfusion injury; CMECs ischemia reperfusion injury has SHH letter. The extraneous addition of recombinant human SHH protein can enhance cell vitality, reduce cell apoptosis, increase the expression of angiogenic factor VEGF, FGF and Ang-1, and promote the formation of neovascularization, suggesting that the activation of SHH signaling pathway plays an important role in the process of vascular regeneration after CMECs ischemia reperfusion injury, and may become the heart. The potential therapeutic target of myocardial ischemia reperfusion injury in.2. patients with acute myocardial infarction is closely related to the level of plasma SHH, and the level of plasma SHH is independent of age, sex, BMI, hypertension, family history of diabetes, myocardial infarction type, smoking history, drinking wine history, fasting blood glucose level, serum creatinine, blood urea nitrogen, total gallbladder. Risk factors such as alcohols, triglycerides, HDL-C, BNP and other risk factors are used in the formation of collateral circulation in the ischemic myocardium of patients with acute myocardial infarction. It is suggested that the serum level of SHH can be used as a predictor of the condition of the formation of coronary collateral circulation in patients with acute myocardial infarction.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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