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[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.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R541.6

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