| Background and aims:In the last three decades,the incidence of diabetes,including type 1 diabetes and type 2 diabetes,has increased year by year.For diabetes,especially the type 1 diabetes,insulin replacement therapy is the preferred treatment,but the diabetes is not managed very well due to many kinds of reasons,which results in all kinds of chronic complications and comorbidities,such as diabetic nephropathy,diabetic neuropathy and diabetic angiopathy.Furthermore,the diabetes and its complications not only cause severely health problem of children and adolescents,but also make diabetes and its complications one of the leading causes of human death worldwide.Currently,it has been suggested that notch signaling plays a pivotal role in pancreatic development and differentiation,and autophagy is involved in the progression of diabetes by regulating beta cells function.In addition,using beta cells differentiated from pancreatic epithelial-derived cells after in vitro expansion for beta cell replacement treatment can be a new way for the diabetes treatment.So we conduct the study is to explore how notch signaling and autophagy involve in the pancreatic epithelial-cells differentiation toward beta cells,which may help find a new way for the diabetes treatment.Methods:1.This study was aimed to use the PDEC cells isolated from SD rats as the model.When notch signaling was inhibited or induced,the expressions of Hesl,Pdxl,Rfx6,p21 and p27 were checked by RT-PCR and western blot.Meanwhile,the expressions and levels of insulin and ghrelin were measured by RT-PCR,immunofluorescence and flow cytometry,which can reveal how the notch signaling involved in the proliferation of PDEC.2.When autophagy was inhibited or induced,the expression of Hes1,Beclinl,LC3,p21 and p27 were checked by immunofluorescence and western blot.Meanwhile,the expressions and levels of insulin and ghrelin were measured by immunofluorescence and flow cytometry,which can reveal how the autophagy-mediated notch signaling involved in the proliferation of PDEC.Results:1.Compared with the control group,the insulin-positive and ghrelin-positive cell rates in Hesl overexpression cells were both significantly decreased by flow cytometry(p<0.01),while the insulin-positive and ghrelin-positive cell rates in DAPT-treated cells were significantly increased(p<0.01).The expressions of Pdx1,Rfx6,p21 and p27were down-regulated in Hesl overexpression cells by RT-PCR and western blot,and the expressions were reversed in DAPT-treated cells.Furthermore,the expressions and levels of ghrelin and insulin were decreased in Hes1 overexpression cells,while the expressions and levels of ghrelin and insulin were increased in DAPT-treated cells by RT-PCR,immunofluorescence and Elisa assay.2.Compared with the control group,the expressions of Beclinl and LC3-? were increased in starved cells,while the expressions of Beclinl and LC3-?were decreased in 3-MA-treated cells by immunofluorescence and western blot.And the western blot results showed that expression of Hesl was down-regulated while the expressions of p21 and p27 were up-regulated in the starved cells(p<0.01),and in 3-MA-treated cells all the results were reversed(p<0.01).Furthermore,the expressions and levels of ghrelin and insulin were increased in starved cells(p<0.01),while the expressions and levels of ghrelin and insulin were decreased in 3-MA-treated cells by immunofluorescence and Elisa assay.Conclusions:Autophagy can negatively regulate the notch signaling pathway,while the inhibition of notch signaling may up-regulate the pancreatic transcript factors and then enhance the secretion of insulin.And then considering the notch signaling and autophagy involved in the process of PDEC proliferation,notch signaling and autophagy may be used to promote PDEC differentiation toward beta cells,which may be a basis for the beta cell replacement treatment of diabetes. |
PDF Full Text Request