Dual Lineage Tracing Pdx1 And Ptf1a In The Pancreatic Development Process Of Mice

Pancreatic is one of the body's main organs and endocrine dyfunction of beta cells can cause diabetes.To study pancreatic stem cells and pancreatic development highlights the growing importance,becoming one of the most important and one of the most active areas of life science research.The core problem of pancreatic development is cell fate decisions and the molecular mechanisms of cell proliferation and differentiation.Pancreatic and duodenal homeobox 1(Pdx1)and pancreas specific transcription factor 1a(Ptf1a)differentiation play a key role in the process of progenitor cells changing into pancreatic endocrine progenitor cells.The fate of pancreatic development depends on these two factors.How to trace transcription factor Pdx1 and Ptf1 a to observe pancreatic cell differentiation and fate? It is a technical difficulty.Lineage tracing system can trace all descendant cells derived from the same cells,which is an important tool in the mammalian to research characteristics of stem cell.Therefore,using the gene lineage tracing technology to establish lineage tracing model of hybrid double gene in mouse(Pdx1Dre/w;Ptf1aCre/w;Rosa26CAG-RSR-AcGFP1/CAG-LSL-Tomato),to solve the tracer token in the d evelop ment of pancreatic P dx1 +P tf1 a+ cell fate is a feas ib le optio n.Objective:This study aims to use selective gene targeting techonology,combined withthe Cre\Dre recombinant enzyme technology,to biuld Pdx1 & Ptf1 a dual-gene lineage tracing mouse model(Pdx1Dre/w;Ptf1aCre/w;Rosa26CAG-RSR-AcGFP1/CAG-LSL-Tomato).With advanced optical equipment and biotechnology,we can realize living cell lineage tracing,observe the mechanism for pancrease developing and repairing in the disease condition.Method:To research mainly by three parts including: to biuld up dual gene lineage tracing in mice models,to observe Pdx1+ & Ptf1a+ cell fate in pancreatic cell differentiation and to observe Pdx1+ & Ptf1a+ cell expression after partial pancreatectomy.1.To biuld up Ptf1 a & Pdx1 double gene lineage tracing in mice model and gene identification.(1)Propagating and screening Pdx1-Dre\Ptf-1a-Cre Knock in mice as well as Dre & Cre reporter mice of Rosa26CAG-RSR-AcGFP1 or CAG-LSL-Tomato.(2)Time mating to get the lineage tracing model of Pdx1Dre/w;Ptf1aCre/w;Rosa26CAG-RSR-AcGFP1 /CAG-LSL-Tomato.(3)Genotping to identity the character of mice.(4)Wester-Blot to observe Pdx1 & Ptf1 a protein expression in pancrease.(5)Observation of transgenic mice bodyweigh,fasting blood glucose andglucose tolerance test(IPGTT).2.To observe expression of Ptf1a+ & Pdx1+ pancreatic cell differentiation during the development of the tracer.(1)Obtaining transgenic mice pancreas samples.(2)To observe GFP and Tomato expression position with Confocal laser scanning microscopy.(3)Real-time and dynamic monitoring of Pdx1+ & Ptf1a+ cells in pancreatic development.(4)Islet Isolation and purification.(5)To observe Pdx1+ & Ptf1a+ cell expression in islet and pancreatic duct with flow cytometry.(6)Immunofluorescence staining of Ki67 and Caspase3 expression.3.To observe expression of Ptf1a+ cells and Pdx1+ cells in pancreas after partial pancreatectomy.(1)Partial pancreatectomy.(2)Observation of transgenic mice glucose tolerance test(IPGTT)after ppx.(3)To observe Ptf1a+ & Pdx1+ pancreas cells expression position after ppx.(4)Immunofluorescence staining of Insulin expression.Results:1.Successfully build mouse dual Ptf1a+ & Pdx1+ cell lineage tracing model.(1)In fluorescence microscopy,we found that genotype Ptf1a-Cre/w;Pdx1-Dre/w;CAG-AcGFP/Tom of pancreatic tissue can be represented as GFP+Tomato+ double fluorescence staining,Ptf1a-Cre/w;CAG-AcGFP/Tom genotype of pancreatic tissues in mice was only Tomato+,Pdx1-Dre/w;CAG-AcGFP/Tom genotype of pancreatic tissues of mice showed GFP+.(2)Neither weight nor glucose tolerance has significant difference between the control group and transgenic mouse model.2.Pdx1+ cells are embryonic pancreatic progenitor cells and Pdx1+Ptf1a-cells lie in the islet at the end.(1)The expression area of Pdx1+ and Ptf1a+ cells changes in pancreatic development.With development,the number of Pdx1+Ptf1a+ cells increase and almost all of the acinar cells are replaced by the DX1+Ptf1a+ cells.(2)Pdx1+Ptf1a-in embryonic cells go up as embryonic development.But Pdx1+Ptf1a-cell number gradually reduces and are eventually expressed in the pancreas and pancreatic duct cells.Pdx1-Ptf1a-are little discovered in pancreatic development as a whole.(3)With continuous observation E15.5 cultured in media,Pdx1+Ptf1a-cells clearly exist and some were replaced by GFP+/Tomato+ cell.(4)Flow cytometry found that,in pancreatic islet cells most were the Pdx+Ptf1a+ double positive cells,followed by Pdx+Ptf1a-cells,virtually no Ptf1a+ Pdx-cells and in the pancreatic duct cells Pdx+Ptf1a+ double positive cells are still in the majority,followed by Ptf1a+ Pdx-cells,Pdx+Ptf1a-cells at least.3.Ki67 expression decreases in pancreatic development.In the Pdx+Ptf1a+ lineage cells,we found that during embryonic development Ki67 expression in Pdx+Ptf1a+ cells of up to,Ki67-positive rate is gradient descent after birth.Ki67 expression in Pdx+Ptf1a-cells of E15.5 was highest expression in the embryonic period,gradually decrease with development.4.Hyperglycemia with stress after ppx probably stimulats Pdx1+Ptf1a-cell hyperplasia.(1)IPGTT data show that preoperative intravenous glucose-glucose was significantly higher in mice,but mice in each group were able in a short period of relative stability returned to near normal levels.However,postoperative glucose tolerance in mice have changed.(2)?cells mass in the pancreatic islet become larger after ppx.(3)We also found that Pdx1+Ptf1a-cells ratio increased in the islet after surgery.Conclusion:1.The experiment has been successfully constructed mice Pdx1 & Ptf1 a dual-gene lineage tracing model,which will help to explore the molecular mechanisms of development,repairment of pancreas and provide more intuitive?scientific and powerful research methods.Also this model is expected to open study on gene therapy of diabetes mellitus and pancreatic developmental biology chapter.2.Pdx1+ cells maybe the embryo pancreatic progenitor cells of mouse and Ptf1a+ cells differiate based on the Pdx1+ cells.With the development of the pancreas,almost all of the pancreatic cells including islet and pancreatic duct,acinar cells come from Pdx1+ cells transformation.3.Pdx1+Ptf1a-cells decrease after birth and focus on the islet and pancreatic duct cells,which probably become the source of beta-cell regeneration.After Partial ectomy of the pancreas,? cell regeneration occured,which may be linked to Pdx1+Ptf1a-cells proliferation.