以阻抑内质网应激为策略的小分子化合物的发现及其对心衰大鼠保护作用的研究

发布时间：2018-07-21 20:59

【摘要】：目的：内质网稳态对于维持细胞内生理活动稳定有序进行具有重要的意义。心肌梗塞及心衰等许多疾病都与内质网应激相关。此研究的目的是(1)明确衣霉素(TM)诱导心肌细胞发生内质网应激的时间依赖效应。(2)筛选抑制内质网应激的小分子化合物。(3)在大鼠心肌细胞缺氧模型、大鼠急性心梗模型和大鼠慢性心衰模型3个模型中观察小分子化合物PP1-14是否具有保护作用。(4)明确内质网应激基因PERK的敲除是否对心衰大鼠心功能有影响。方法：(1)将原代培养的大鼠心肌细胞随机分为5组,1μM衣霉素分别培养1h,3h,6h,12h和24h,另设不加衣霉素为对照,提取细胞内的总RNA用于转录组测序。(2)ATF6是证明内质网应激是否诱发的核转位因子。将稳定表达ATF6-GFP融合蛋白的U20S细胞模型随机分为对照组、1μM衣霉素诱导组及衣霉素诱导前给药组(分别给予待筛选的80个化合物),采用高内涵技术监测ATF6的核转位。(3)将大鼠心肌细胞随机分为对照组、缺氧组(缺氧24h)、缺氧并给予PP1-14(提前30min给予)共培养组,通过CCK8,ATP等试剂盒检测细胞活力,高内涵及透射电镜观察细胞骨架及细胞形态;将急性心梗大鼠随机分为溶剂对照组、salubrinal组、PP1-14组和metoprolol组,在构建急性心梗模型前3天每天给药1次,另设正常对照大鼠组,通过TTC染色测量心梗面积,检测血清中肌钙蛋白T(c-TNT),肌酸激酶同工酶(CK-MB),C反应蛋白(CRP)的活力,TUNEL及western-blot测量大鼠心室组织的凋亡量及内质网应激分子的表达水平;将心衰大鼠随机分为溶剂对照组、salubrinal组、PP1-14组和captopril组,持续饲养动物8周每隔一天给药1次,另设正常对照大鼠组,应用超声心动图和血流动力学评价心脏的结构及功能,计算心室指数,压力负荷下的压力容积斜率,检测血液中脑钠肽(BNP)和房钠肽(ANP)的表达水平,应用电镜及TUNEL分别观察心肌结构、检测细胞凋亡。提取细胞或心肌组织的总RNA用于转录组测序。(4)构建Si-PERK的心肌细胞并构建PERK-KO的大鼠心衰模型,分别通过ATP检测细胞活力并血流动力学评价心功能、计算心室指数。结果：(1)TM在3h,6h,12h和24h分别诱导7,10,11,13内质网应激相关的基因上调,除去已知Hspa5,Hsp90b1,Calr,Ddit3,Atf4基因,新发现6个(Hyou1,Herpud1,Manf,Creld2,Sdf211,Slc3a2)表达显著变化的基因。(2)在初筛的80个化合物中共发现17个化合物能够抑制TM引起的ATF6核转位,复筛发现PP1-13、PP1-14、PP1-19浓度依赖的抑制ATF6核转位。(3)PP1-14能够增加由于缺氧诱导的心肌细胞存活率下降,减轻缺氧诱导的染色体皱缩及边缘化,PP1-14引起的表达差异基因主要聚类于对内质网应激的反应和细胞周期两方面;急性心梗模型大鼠中出现梗死面积增加,部分心肌细胞伴少量炎细胞浸润,血清中CK-MB、c-TNT、CRP水平上升,细胞凋亡增加,内质网应激相关蛋白表达增加,预防性给予PP1-14,以上各指标均有明显改善,内质网应激相关蛋白表达量下调,被PP1-14下调的基因大部分聚类于代谢过程,氧化还原过程及免疫反应;大鼠慢性心衰模型上出现心室腔扩大、心肌变薄、心体指数变大,血清中BNP、ANP升高,凋亡蛋白表达量升高,心室做功(SW)、每搏输出量(CO)、心输出量(SV)、射血分数(EF)、压力变化最大速率(dp/dtmax)、容积变化最大速率(dv/dtmax)均有所下降,结扎下腔静脉后显示代表心肌收缩功能最大斜率(Dmax)下降,PP1-14治疗组的各项指标均有所改善。PP1-14下调的表达差异基因最多聚类于吞噬体。(4)Si-PERK的心肌细胞上药物的保护作用消失,PERK-KO纯合大鼠出现胚胎期致死,PERK-KO杂合组大鼠心功能各指标不及野生组,PERK-KO杂合心衰大鼠中药物的保护作用不及野生心衰大鼠。结论：(1)TM从3h开始引起内质网应激反应,并新发现Hyoul,Herpud1,Manf,Creld2,Sdf211,Slc3a2这六个基因与内质网应激相关。(2)筛选到3个(PP1-13,PP1-14,PP1-19)内质网应激抑制因子。(3)PP1-14可以减少缺氧诱导的心肌细胞死亡,减少急性心梗引起的心肌损伤,抑制心衰引起的心室重塑,改善心功能。(4)PERK基因在维持心肌正常生理功能及药物保护作用中扮演重要角色。
[Abstract]:Objective: endoplasmic reticulum homeostasis is of great significance in maintaining a stable and orderly cell physiological activity. Many diseases such as myocardial infarction and heart failure are associated with endoplasmic reticulum stress. The purpose of this study is to identify the time dependent effect of endoplasmic reticulum stress induced by TM (TM). (2) screening for inhibition of endoplasmic reticulum stress Small molecular compound. (3) whether small molecule compound PP1-14 has protective effect in 3 models of rat cardiac myocyte anoxia, acute myocardial infarction model and chronic heart failure model of rats. (4) whether the knockout of endoplasmic reticulum stress gene PERK affects heart function of rats with heart failure. Method: (1) the primary rat heart was cultured. The muscle cells were randomly divided into 5 groups. 1 M ycomycin, respectively, cultured 1H, 3h, 6h, 12h and 24h, and the total RNA in the cell was used as control. (2) ATF6 was a nuclear transposition factor that proved whether the endoplasmic reticulum stress was induced. The U20S cell model of the stable expression of ATF6-GFP fusion protein was randomly divided into the control group and 1 mu M ycomycin. The induction group and ycomycin induction group (80 compounds to be screened respectively) were used to monitor the nuclear transposition of ATF6. (3) the rat cardiac myocytes were randomly divided into control group, hypoxia group (anoxic 24h), hypoxia and PP1-14 (early 30min) co culture group, and the cell viability was detected by CCK8, ATP and other kits. The acute myocardial infarction rats were randomly divided into the solvent control group, the salubrinal group, the PP1-14 group and the metoprolol group. The rats were given 1 times a day 3 days before the construction of the acute myocardial infarction model, and the normal control rats were set up. The myocardial infarction area was measured by TTC staining, and the serum troponin T (c-TNT) and creatine kinase were detected in the serum. The activity of isozyme (CK-MB), C reactive protein (CRP), TUNEL and Western-blot were used to measure the apoptosis of ventricular tissue and the expression level of endoplasmic reticulum stress molecules. The rats were randomly divided into the solvent control group, the salubrinal group, the PP1-14 group and the captopril group, and the animals were continuously fed for 1 times every other day for 8 weeks, and the normal control rats should be set up. The structure and function of the heart were evaluated by echocardiography and hemodynamics, the ventricular index, pressure volume slope under pressure load were calculated, the expression level of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) in the blood was detected. The myocardial structure was observed by electron microscopy and TUNEL, and the apoptosis was detected. The total RNA of the cells or cardiac tissue was used for transcription. Group sequencing. (4) construct Si-PERK cardiomyocytes and construct PERK-KO model of heart failure in rats. The ventricular index was calculated by ATP detection of cell viability and hemodynamic evaluation. Results: (1) TM in 3h, 6h, 12h and 24h induced the up-regulated gene up of the 7,10,11,13 endoplasmic reticulum stress phase, respectively, to remove the known Hspa5, Hsp90b1, Calr, etc. Gene, 6 new genes (Hyou1, Herpud1, Manf, Creld2, Sdf211, Slc3a2) were found to express significant changes. (2) a total of 17 compounds were found to inhibit the ATF6 translocation caused by TM in the initial screening of 80 compounds. The rescreening found PP1-13, PP1-14, PP1-19 concentration dependent inhibition of the nuclear translocation. (3) it could increase the myocardial finer induced by hypoxia. The cell survival rate decreased, reducing the chromosomal contraction induced by hypoxia and marginalization. The PP1-14 induced differentially expressed genes were mainly clustered in two aspects of the response to endoplasmic reticulum stress and cell cycle. The infarct area increased in the acute myocardial infarction model rats, some of the myocardial cells were accompanied by a small number of inflammatory cells, and the levels of CK-MB, c-TNT, and CRP in the serum increased. Apoptosis increased, the expression of endoplasmic reticulum stress related proteins increased, PP1-14 was given prophylactic, the above indexes were obviously improved, the expression of endoplasmic reticulum stress related protein was down, and most of the genes down regulated by PP1-14 were clustered in metabolic process, redox process and immune response, and ventricular cavity enlargement in chronic heart failure model of rats was enlarged. The myocardium became thinner, the heart body index increased, the serum BNP, ANP increased, the expression of apoptotic protein increased, ventricular work (SW), cardiac output (CO), cardiac output (SV), ejection fraction (EF), the maximum rate of pressure change (dp/dtmax), the maximum rate of volume change (dv/dtmax) decreased, and the maximum deviation of the myocardial contractile function after the ligation of the inferior vena cava indicated the maximum oblique systolic function of the myocardium. The rate (Dmax) decreased, and all the indexes of the PP1-14 treatment group improved the expression difference of.PP1-14 in the phagocyte. (4) the protective effect of drug on the Si-PERK myocardial cells disappeared, the PERK-KO homozygous rats died in the embryonic stage, and the cardiac function of the PERK-KO heterozygous group was not as good as the wild group, PERK-KO heterozygous heart failure rats The protective effect of Chinese medicine was less than that of wild heart failure rats. Conclusion: (1) TM began to induce endoplasmic reticulum stress response from 3h, and the six genes of Hyoul, Herpud1, Manf, Creld2, Sdf211, Slc3a2 were associated with endoplasmic reticulum stress. (2) 3 (PP1-13, PP1-14, PP1-19) endoplasmic reticulum stress inhibition factors were screened. (3) PP1-14 could reduce hypoxia induced heart Myocyte death, reducing myocardial injury caused by acute myocardial infarction, inhibiting ventricular remodeling caused by heart failure and improving cardiac function. (4) the PERK gene plays an important role in maintaining the normal physiological function of the myocardium and the protective effect of the drug.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.6

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