| Objective: Heart failure is the end stage of every heart disease and the incidence of heart failure has been increased.Cardiac fibrosis is strongly related to heart failure.Cardiac fibrosis is pivotal in the pathologic progress of heart disease and is also cause factor of plenty of heart diseases.Most of the studies about cardiac fibrosis have focused on cardiac fibroblasts,however,in this study,we focused on the influence of cardiomyocytes on fibroblasts and the role of cardiomyocytes in cardiac fibrosis.By studying the influence of cardiomyocytes on fibroblasts through exosomes,this article will shed some lights on cardiac fibrosis.Methods Chapter one:(1)The instruction of two heart failure model of rats,one of which is induced by ligation of left anterior descending coronary artery,and another one is induced by transfusion of doxorubicin.(2)Examine miR-1,miR-133,miR-208 a and miR-499 in serum and heart tissue of two heart failure rats model.(3)Isolation of cardiomyocytes and fibroblasts from adult rats(4)Cardiomyocytes were stimulated by hypoxia and angiotensin ? to simualte the two different heart failure models.Examine the expression of miR-208 a in stimulated cardiomyocytes and in fibroblasts when fibroblasts were co-cultured with the supernatant of cardiomyocytes.(5)Isolation and identification of exosomes from stimulated cardiomyocytes and identification their up-taken by fibroblasts.(6)Examine the expression of miR-208 a in fibroblasts when exosomes were added in the supernatant of fibroblasts.Chapter two:(1)Examine the influence of stimulated cardiomyocytes on the proliferation and differentiation of fibroblasts.(2)Examine the changes of cardiac function of rats when microRNA was inhibited by microRNA inhibitor.(3)Examine the changes of cardiac function of rats when microRNA was inhibited by microRNA sponge AAV9.(4)Examine the changes of cardiac function of rats when microRNA were transfused into normal rats Chapter three:(1)Search the target of microRNA and confirm it by dual luciferase report gene assay.(2)Compare the influence of depressing the target gene and using microRNA agomir on fibroblasts(3)Examine whether over-expressing the target gene could counter the using of microRNA on influencing fibroblasts(4)Identify the target gene of microRNA and its function on downstream signal.Results Chapter one:(1)We have successfully instructed two heart failure model of rats,one of which is induced by ligation of left anterior descending coronary artery,and another one is induced by transfusion of doxorubicin.(2)By examining miR-1,miR-133,miR-208 a and miR-499 in serum and heart tissue of two heart failure rats model,we found that miR-208 a were the only one that increase in serum and heart tissue of both two models.(3)We have successfully isolated cardiomyocytes and fibroblasts from adult rats.(4)In vitro,Cardiomyocytes were stimulated by hypoxia and angiotensin ? to simulate the two different heart failure models.The expression of miR-208 a in stimulated cardiomyocytes and in fibroblasts when fibroblasts were co-cultured with the supernatant of cardiomyocytes were both increased.(5)We have successfully isolated and identified exosomes from stimulated cardiomyocytes and identified their up-taken by fibroblasts as well.(6)By examining the expression of miR-208 a in fibroblasts when exosomes were added in the supernatant of fibroblasts,we found that miR-208 a were increased in a time dependent manner.Chapter two:(1)Exosomes from stimulated cardiomyocytes could stimulate the proliferation and differentiation of fibroblasts.(2)The cardiac function of heart failure rats were improved and cardiac fibrosis were decreased when miR-208 a was inhibited by miR-208 a inhibitor.(3)The cardiac function of heart failure rats were improved and cardiac fibrosis were decreased when miR-208 a was inhibited by miR-208 a sponge AAV9.(4)The cardiac function of normal rats were injured and cardiac fibrosis were increased in a dose-dependent manner when miR-208 a were transfused into normal rats.Chapter three:(1)DYRK2 was the target gene of miR-208 a and it has been confirmed by dual luciferase report gene assay.(2)The influence of depressing the target gene and using microRNA agomir on fibroblasts were the same.(3)Over-expressing of the target gene could counter the using of microRNA on influencing fibroblasts.(4)DYRK2 could phosphorylate NFAT to keep it in cytoplasm,avoiding its entry of nuclear to starts the transcription of pro-fibrosis genes.Conclusion Cardiomyocytes stimulated by hypoxia or angiotensin ? has increased miR-208 a and exosomes,as well as miR-208 a in exosomes.These exosomes can stimulate the proliferation and differentiation of fibroblasts.Fibroblasts takes up the exosomes and miR-208 a in these exosomes will enter into fibroblasts.Then miR-208 a will inhibit the expression of dyrk2 in fibroblasts,which will dephosphorylate NFAT.Then the dephosphorylated NFAT will enter into the nuclear of fibroblasts,which starts the transcription of pro-fibrosis genes. |
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