| Background:Myocardial fibrosis (MF)isthe common pathology various cardiovascular diseasesdeveloped to a certain stage,whichis also one of the main performance of myocardialremodeling.Cardiac fibroblasts (CFs) account for90%of the cardiac mesenchymal cells.Inthe structure of the compensatory after myocardial injury,CFs tissue repair throughmigration, proliferation and collagen secretion.However, the development of myocardialfibrosis will cause ventricular remodeling, lead to heart dysfunction. CFs transform intomyofibroblasts (MFs) is one of the important mechanisms in myocardial fibrosis4.Notch signaling pathway is a recently discovered molecular closely related to cellulardifferentiation. There are4subtypes receptor for Notch (Notchl-4),They precisely regulatedifferentiation of various cells and the pathophysiological process of each organ throughtheir interaction between neighboring cells5,butthe mechanism is unclear.Recently, severalstudies have reported, Notch signaling pathway involved in many fibrotic diseases,however in the study of myocardial fibrosis has not been carried out. Research aims:1.Whether the expression of Notch3changed when CFs transformed to MFs?2.Whether the transformation of CFs to MFs increased after Notch3expressioninhibition?3.Whether inhibited the transformation of CFs to MFs when Notch3overexpressed?Research methods:1.CFs were isolated by collagenase and trypsin digestion methodand incubated for24h， then CFs were induced24h by different concentrations of with differentconcentrations of TGF-β1. Western blot analysis was used to measure α-smooth muscleactin (α-SMA) expression,immunofluorescencewas used to detectα-SMA expression inCFs andmeasured hydroxyproline content in the supernatants. Real-Time PCR was used tomeasure Notch3mRNA expression,western blot analysis was used to measure Notch3protein expression.2.CFs were incubated for24h, RNAi method was employed to interfere with theexpression of Notch3.CFs were divided into3groups: control, negative control andsiRNA interference.Real Time PCR and Western blot analysis were used to detectinterference effects,western blot analysis was used to measure α-SMAexpression,immunofluorescencewas used to detectα-SMA expression in CFs andmeasuredhydroxyproline content in the supernatants.3.CFs were incubated for24h,lentivirus were used to transfect intoCFs,Notch3overexpression.CFs were divided into5groups:control, negativecontrol,Notch3overexpression, TGF-β1-induced, TGF-β1-induced+Notch3overexpression group.After72h, Real Time PCR and Western blot analysis were used todetect transfection effects,western blot analysis was used to measure α-SMAexpression,immunofluorescencewas used to detectα-SMA expression in CFs andmeasuredhydroxyproline content in the supernatants.Research results:1.CFs weresuccessfullyisolated by collagenase and trypsin digestion method.Weincubated for24h using DMEM low sugar containing10%FBS.Cells were observed with an inverted microscope, we saw diverse morphology, multi-protrusion spindle-shaped orstar-shaped flat cells, no spontaneous pulse, nuclear were ovoid and centered.2.We cultured CFs24h,used serum-free DMEM synchronizing24h, then CFs wereinduced24h by0,1,2,5,10ng/ml5different concentrations TGF-β124h.TGF-β1inducedincreases of hydroxyproline content in the supernatants and α-SMA expression dosedependently in CFs and induced decreases of Notch3mRNAand protein expression.Compared with those in control group, significant differences were observed in thechanges at the concentrations of2ng/ml,5ng/ml and10ng/ml (P<0.05).These resultssuggested that TGF-β1can dose dependently induce transformation CFs to MFs,whileinhibit Notch3expression.3. We cultured CFs24h,used serum-free DMEM synchronizing24h,then used siRNAto interfered Notch3expression. Compared with those in control group, significantdifferences were observed in the decreases of Notch3mRNA and protein expression atinterferinggroup(P<0.05); increases of hydroxyproline content in the supernatants andα-SMA expression(P<0.05). These results suggested that transformation of CFs to MFsincreased if Notch3downregulated.4.We used negative control lentivirals to transfect into CFs with different titers.Whenthe MOI was20, transfected into72h,cells were observed with a fluorescencemicroscopy,we saw transfection effects was about80%.Thus, we choosed MOI=20lentiviral transfection in present study.5. We cultured CFs24h,used serum-free DMEM synchronizing24h,then lentiviruswere used to transfect into CFs,Notch3overexpression,while adding10ng/mlTGF-β1-induced24h group and transfected into Notch3after10ng/ml TGF-β1-induced12h group. Compared with those in control group, significant differences were observed inthe increases of Notch3mRNA and protein expression(P<0.05) anddecreases ofhydroxyproline content in the supernatants and α-SMA expression(P<0.05) atNotch3overexpression group; meanwhilesignificant differences were observed in thedecreases of Notch3mRNA and protein expression(P<0.05) andincreases ofhydroxyproline content in the supernatants and α-SMA expression(P<0.05) at TGF-β1-inducedgroup;but there were no significant differencesat TGF-β1+Notch3group.These results suggested thatwhenNotch3overexpressed, the transformation of CFsto MFs was inhibited.Conclusion:Notch3downregulates, the transformation of CFs to MFs will increase.Notch3upregulates, the transformation of CFs to MFs will decrease. |
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