| Epicardial progenitor cells have been proven to play important roles in cardiogenesis.They can undergo epithelial-to-mesenchymal transition(EMT)to form a pool of epicardial-derived cells(EPDCs),and then differentiate into cardiac interstitial cells and coronary smooth muscle cells which help to build myocardial fiber framework and coronary blood vessels during heart development.Characterized with multipotential differentiation,epicardial progenitor cells have been identified as the third heartforming field and a pool of progenitor cells specifically expressing Tbx18,Wt1 and Tcf21.Epicardial cells of adult heart loss the characteristics of the embryonic ones,but studies have found that adult quiescent epicardium of the border zone would reactivate and begin to re-express epicardial progenitor cell markers,like WT1 and Tbx18 after myocardial infarction,and then participate in heart repair and neovascularization.Hence,delving into the proliferation and EMT of epicardial progenitor cell not only is important to explore the development-related mechanisms of embryonic heart,but also can help to promote the development of epicardial-dependent treatment for heart injury.Insulin-like growth factor 1 receptor(IGF1R)signaling has been proven to take part in the proliferation and EMT processes of various kinds of cells and it's confirmed that IGF1 R and its ligands IGF1 and IGF2 can be detected at epicardial at E11.5d to E14.5d through hybridization in situ.Since,we hypothesis that IGF1 R may participate in regulating epicardial progenitor cell proliferation and EMT.The most classical downstream signaling pathways of IGF1 R are PI3K/AKT and MAPK/ERK,but focal adhesion kinase(FAK)has also been found to mediate IGF1 R signaling to produce similar physiological or pathological effects in recent years.So,we designed this trial to evaluate the effects of IGF1 R singling on epicardial progenitor cell proliferation and EMT,and testify whether FAK mediate these effects.Part ? The spatio-temporal expression of IGF1 R system in mouse embryonic heartObjective: To determine the expression of IGF1 R and its ligands IGF1 and IGF2 in mouse embryonic heart.Methods: The female and male C57BL/6 mice were copulated,then the embryonic tissues of E11.5d-E17.5d were collected and made into frozen sections.Immunofluorescent staining was used to detect the expression of IGF1R,IGF1 and IGF2 in the embryonic heart.Results: IGF1 R was visually spotted at the epicardium layer through out E11.5d to E17.5d period.IGF1 was weakly expressed originally from E11.5d to E13.5d,but then could be clearly detected from E14.5d to E17.5d.As the embryonic growth factor,IGF2 was shown to be expressed more apparently than IGF1 during the embryonic epicardium development period from E11.5d to E17.5.Conclusions: IGF1 R,IGF1 and IGF2 all could be visually spotted at the epicardium layer throughout E11.5d to E17.5d period suggesting that IGF1 R system might participate in the development of mouse embryonic epicardium.Part ? Effects of IGF1 R signaling on the proliferation of epicardial progenitor cellsObjective: To establish the in vitro cell culture system of mouse epicardial progenitor cells and investigate the effects of IGF1 R signaling on the proliferation of epicardial progenitor cells in vitro.Also,we aimed to find out whether FAK mediate the IGF1R-related proliferation.Methods: E12.5d mouse ventricular heart tissues were harvested to culture the epicardial progenitor cells.Immunofluorescence staining was used to detect the expression of Tbx18,Wt1 and IGF1 R.And then epicardial progenitor cells were cultured with Picropodophyllin(PPP)at different concentrations(0,0.2?M,0.4?M,0.8?m and 1.6?M)for 72 hours and CCK-8 was used to detect the cell proliferation situation.Then,cultured cells were randomized into two groups: Control group and PPP group(treated with 0.4?M PPP).After cultured for 72 h,immunofluorescent staining was used to determine the expression of Ki67 and phosphorylation of FAK(p Y397).q RT-PCR was used to determine the expression of Ki67,Cyclin D1 encode gene(CCND1)and FAK encode genes(Ptk2 and Ptk2b)at m RNA level and flow cytometry was used to test the cell cycle changes.Considering that fetal bovine serum(FBS)which was used in cell culture medium contained insulin-like growth factors(IGFs),we reduced the FBS concentration from 10% to 1% before the intervention with IGFs.Then,different concentrations of IGF1(0,5ng/ml,10ng/ml and 50ng/ml)and IGF2(0,10ng/ml,50ng/ml and 100ng/ml)was used to treat cells for 72 h,CCK-8 was applied to check the cell proliferation.Cells were also randomly divided into five grous: Control group,IGF1 group(50ng/ml),IGF2 group(100ng/ml),IGF1+Y15 group(50ng/ml IGF1+1n M Y15)and IGF2+Y15 group(100ng/ml IGF2+1n M Y15).The proliferation condition of the five groups were checked by CCK-8.Immunofluorescent staining was used to determine the expression of Ki67 and phosphorylation of FAK(p Y397).q RT-PCR was applied to test the expression of Ki67,CCND1,Ptk2 and Ptk2 b at m RNA level.flow cytometry was used to test the differences of cell cycle among these five groups.Results: The cultured cells was in a cobblestone-like performance and highly expressed the epicaridal progenitor cell markers Tbx18 and Wt1.IGF1 R was also found to be dotted on the cytomembrane.CCK-8 test showed that PPP could markedly decress the proliferation of epicardial progenitor cells in a concentration-dependent manner.And immunofluorescent staining demonstrated that the expression of Ki67 and phosphorylation of FAK(p Y397)were significantly decrease in the PPP group.q RT-PCR also showed significant decrease of Ki67,CCND1,Ptk2 and Ptk2 b at m RNA level in the epicardial progenitor cells in the PPP group.Flow cytometry revealed that PPP significantly increased the proportion of G1-phased cells and reduced the proportion of S and G2-pahsed cells.On the contrary,CCK-8 assay showed that IGF1 and IGF2 could enhanced epicardial progenitor cell proliferation in a concentration-dependent manners.Compared to the control group,IGF1 and IGF2 group demonstrated a higher expression of Ki67 and phosphorylation of FAK at protein level,a higher expression of Ki67,CCND1,Ptk2 and Ptk2 b at m RNA level,a higher proportion of S and G2-phased cells and a lower proportion of G1-phased cells.Usage of Y15,a specific inhibitor for the phospherylation of FAK(p Y397),would result in expression reduction on Ki67 and CCND1 without affecting Ptk2 and Ptk2 b at m RNA level.Also,Y15 were demonstrated significantly inhibit the effects of IGFs on cell proliferation and cell cycle changes from G1 to S/G2 phase.Conclusions: Using the epicardial progenitor cells successfully cultured in vitro,we found that IGF1 R signaling played a role in regulating the proliferation of epicardial progenitor cells and also FAK was involed in mediating this effect.Part ? Effects of IGF1 R on EMT of epicardial progenitor cellsObjective: To explore whether IGF1 R have an effects on the EMT process of epicardial progenitor cells.Method: Similar to the above case,cultured cells were randomly divided into two groups: Control group and PPP group(treated with 0.4?M Picropodophyllin).After cultured for 72 h,immunofluorescent staining was used to determine the expression of ZO-1 and Vimentin.q RT-PCR was used to determine the expression of Chd1,Tjp1,Chd2 and Vim at m RNA level.After reducing the FBS concentration from 10% to 1%,cells were randomly divided into three grous: Control group,IGF1 group(50ng/ml)and IGF2 group(100ng/ml).Immunofluorescent staining and q RT-PCR were used to determine the expression differences of EMT-related markers which we determined to detected among the three groups.Transwell assay was further used to test the cell migration capability changes induced by IGFs.Results: Immunofluorescent staining and q RT-PCR resulted in no evidences of differences in ZO-1 and Vimentin expression and Chd1,Tjp1,Chd2 and Vim expression at m RNA level.Intervention with IGF1 and IGF2 also did not cause significant changes in the expression of the EMT-related markers we determined to detected.Results of Transwell assay showed on effects of IGFs on cell migration.Conclusion: We detected no effects of IGF1 R signaling on regualating EMT process of epicardial progenitor cells. |
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