| Part oneThe effect of sonic hedgehog to Endothelial Progenitor CellFunctions in Type1Diabetes.Objective1. To investigate the effect of sonic hedgehog on function ofdiabetic endothelial progenitor cells.Methods1.C57/6male rats were used to induce type1diabetic mode withStreptozotocin (STZ)，after10weeks，cultured endothelial progenitor cells were giventhe signal pathway of sonic hedgehog agonist (SAG) at7thday for24h, Western blotmethod were used to detect expression of Shh and the downstream factor GLI-1，atthe same time，vitro angiogenesis experiments and cell migration experiments wereused to observe the function of endothelial progenitor cells to tubular formation andmigrate in type1Diabetes. 2.C57/6male rats were used to induce type1diabetic mode withStreptozotocin (STZ)，after10weeks，cultured endothelial progenitor cells weretransfected by lentiviral vectors highly expressing sonic hedgehog. Western blotmethod were used to detect expression of Shh and the downstream factor GLI-1，at the same time，the transfected endothelial progenitor cells were observed byfluorescence microscopy.vitro angiogenesis experiments and cell migrationexperiments were used to observe the function of endothelial progenitor cells totubular formation and migrate in type1Diabetes.RESULT1. GLI-1were highly expressed in type1diabetic endothelial progenitor cells,given Shh signaling pathway agonist (SAG) in western blot method(P<0.05).theability of tube formation and migration were improved(P<0.05).2. GLI-1were highly expressed in type1diabetic endothelial progenitor cells,transfected with lentiviral highly expressing Shh in western blot method(P<0.05).the ability of tube formation and migration were improved(P<0.05).Summary1. Shh signal pathway agonist (SAG) could improve the expression of GLI-1ofendothelial progenitor cell in type1diabetic.2. Shh signal pathway agonist (SAG) could improve the tubular formation andmigration function of impaired endothelial progenitor cell in type1diabetic.3.Diabetic EPCs were infected with lentivirus highly expressing Shhsuccessfullyby MOI10.4. lentivirus highly expressing Shh could improve the expression of GLI-1ofendothelial progenitor cell in type1diabetic.5. lentivirus highly expressing Shh could improve the tubular formation andmigration function of impaired endothelial progenitor cell in type1diabetic. Part twoSonic hedgehog improves wound healing via restoration ofEndothelial Progenitor Cell Functions in Type1Diabetes.Objective1. To investigate sonic hedgehog improve wound healing via restoration ofendothelial progenitor cell functions in Diabetes.Methods1. full skin defect model1,established with type1Diabetic model feeding10weeks, were randomly divided into Control group, DM group, DM+EPCS group byinjected of saline, endothelial progenitor cells (derived from GFP) around thewound. Wound healing of the experimental group and control group were regularlyobserved.full thickness skin tissue1cm in diameter area around the wound weretaken to make frozen sections in the eighteenth day. the frozen sections were directlyobserved to investigate the effect of endothelial progenitor cells on wound healingunder the fluorescence microscope. Immunohistochemistry, HE staining, were used toobserved angiogenesis and collagen tissue repair in skin tissue.2. full skin defect model2,established with Diabetic model feeding10weeks,were randomly divided into Control group, DM group, DM+EPCSgroup,DM+EPCS(SHH) group by injected of saline, endothelial progenitor cells(derived from diabetic rats) around the wound. full thickness skin tissue1cm indiameter area around the wound were taken to make frozen sections in the eighteenthday. the frozen sections were directly observed to investigate the effect of endothelialprogenitor cells on wound healing under the fluorescence microscope. Immunohistochemistry, HE staining, Mason staining were used to observedangiogenesis and collagen tissue repair in skin tissue.RESULT1. Full skin damage model (1) shows: compared with the control group, therapid of diabetic wound healing is slow (P<0.05), endothelial progenitor cellscould promote wound healing in diabetes (P<0.05). Under the fluorescencemicroscope, subcutaneous tissue showed scattered punctate fluorescence inexperimental group. the number of small blood vessels in experimental groupwere more than control group by immunohistochemistry. the skin structure ofthe experimental group are similar to those of normal skin structure, the skin ofthe control group showed epithelization, but thin epithelium by HE staining andMason staining.2. Full skin damage model (2) showed: compared with the control group, therapid of diabetic wound healing is slow (P<0.05).diabetic wound healing is slow(P<0.05), the endothelial progenitor cells transfected with lentivirus highlyexpressing SHH promoted wound healing in diabetes (P<0.05). the number ofsmall blood vessels in experimental group were more than control group byimmunohistochemistry. the skin structure of the experimental group are similarto those of normal skin structure, the skin of the control group showedepithelization, but thin epithelium by HE staining and Mason staining method.Summary1. Endothelial progenitor cells could promote wound healing by improving theability of form new vessels in diabetic rats.2. Shh improved endothelial progenitor cell，promoted wound healing byrepairing the ability of form new blood vessels in diabetic rats. Conclusion1. Shh signaling pathway can improve the function of EPCs by improve the epressingof Shh in type1diabetic.2. lentivirus highly expressing Shh improve the ability of angiogenesis promotingwound healing in type1diabetic.. |
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