| Reserach Background:The Hedgehog Signaling pathyway plays a very important role in the development of the body and the regulation of cells in the Hedgehog-Gli signal transduction pathway.It is named for its hairy masses,like cowering hedgehogs.Hh signaling pathway has a clear relationship with the development of various diseases.Therefore,the study of the relationship between Hh-Gli signaling pathway and the pathogenesis of myocardial fibrosis plays an important role in the prevention and treatment of clinical diseases and the development of new drugs.Research Objective:To study the impact of blocking Hedgehog-Gli signaling pathway of Transforming growth factor beta1,TGF-beta1 induced myocardial fibroblasts epithelial mesenchymal transdifferentiation occurred in the process.Research Methods:The method choose left coronary artery ligation to be the mouse myocardial infarction model.HE staining and Masson staining were usedto observe the different pathological changes of myocardial tissue in 3 days,7 days and 14 days after myocardial infarction.On the basis of modeling success mice,using Real-Time PCR assay to detect different times mRNA expression in Gli1,using The expression of Gli1 protein was detected by Western blot method,and the time and space expression of Gli1 fluorescent protein was detected by immunofluorescence method.The application of differential sticking wall of primitive culture of SD rat cardiac fibroblasts,and pancreatic enzyme digestion batches,combination of immunofluorescence identifying myocardial fibroblasts specificity protein: Vimentin positive confirmed for that the cultured cells for cardiac fibroblasts.The myocardial fibroblasts from 2-3 generations were selected for the TGF-beta1 intervention.Myocardial fibroblasts were transformed into myofibroblasts after TGF-beta1 intervention.a-SMA fluorescence immunoassay was identified as myofibroblast.The CFs was stimulated by TGF-beta1,1,5,10ng/ml of different concentrations.Gli1 mRNA expression was detected by Real-Time PCR.The expression of Gli1 protein was detected by Western blotting.The space and time expression of Gli1 was detected by fluorescence immunoassay after 10ng/ml TGF-beta 1induced CFs 24 hours.After using GANT61 and Cyclopamine specifically to block Gli1 and Smo for 24 hours in Hedgehog signaling pathway,Thelevel of Gli1 mRNA was detected by Real-Time PCR with 10ng/ml TGF-beta1.The protein expression of Gli1 was detected by Western blot.Research Results:In this study,we chose the method that was most consistent with the pathological process of clinical myocardial infarction: the mouse model of myocardial infarction in the anterior descending branch of the left coronary artery was used,and the pathological process of fibrosis after myocardial infarction was studied.We can see the infiltration of inflammatory cells in the infarction area,abnormal hyperplasia of collagen fibers,rupture of myocardial fibers,and disorder of structure.Choose the success modeled mice,on the basis of application of Real-Time PCR assay to detect different times mRNA expression in Gli1,using Western blot method to detect Gli1 protein expression of the quantity change,the application of immunofluorescence test Gli1 fluorescent protein of time and space expression.Based on the experiment of successful model mice,Real-Time PCR method and Western blot method to detect different times after Gli1 mRNA and protein expression of the situation,found that after the occurrence of myocardial infarction level of mRNA and protein express increased,and be the time dependence,with extended time,the expression increasing.Immunofluorescence tests found that Gli1 fluorescent protein expression of space and time,found that two weeks after myocardial infarction Gli1 express the most obvious,further suggest two weeks after myocardial infarction myocardial fibrosis,the most obvious and Gli1 involved in the occurrence of myocardial infarction and development process.It was confirmed that Gli1 was involved in myocardial fibrosis in vivo test of myocardial fibrosis,and provided a theoretical basis for choosing suitable time window for clinical treatment.1-3 days netel borned SD rats was choosed,differential is chosen to stick wall of primary cell culture and pancreatic enzyme and collagenase digestion and extend the experimental methods,by inverted phase contrast microscope observation: cells is larger,irregular triangle or fusiform,contains about 2 to 3 nucleus,nucleus big center,ovoid,transparent cytoplasm.Immunofluorescence identification of myocardial fibroblast specific protein: Vimentin was positive,confirming that cultured cells were myocardial fibroblasts.The myocardial fibroblasts from 2 to 3 generations were selected to conduct TGF-beta1 intervention,and the fluorescence immunoassay was used to identify the specific markers of myofibroblasts,a-SMA spatiotemporal expression.The spatiotemporal expression of Gli1 was detected by fluorescence immunoassay.The expression of mRNA and protein in Gli1 was increased with the increase of the intervention dose,with different concentrations(0,1,5,10ng/ml)of TGF-beta1 stimulated CFs,fluorescence quantitative PCR and Western blot detection.GANT61 and Cyclopamine specificity were used respectively to block the Hedgehog signaling pathway of Gli1 and Smo after 24 hours,fluorescence quantitative PCR method and fluorescence immunoassay method to detect Gli1 mRNA and protein expression shows are effectively suppresses the mRNA and protein expression.It is confirmed that the expression of Hedgehog downstream factor Gli1 in myocardial fibrotic epithelium transformation has been proved to be involved in the development of myocardial fibrosis.Research Conclusion:It is suggested that Hedgehog signaling pathway participates in the occurrence and development of myocardial fibrosis.To provide effective strategies for early clinical prevention and treatment of myocardial fibrosis. |
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