| Necrosis after skin flap transplantation is always a difficult problem in clinic,and I/R(Ischemia/Reperfusion)injury is one of the main causes of necrosis after transplantation.Stromal cell-derived factor-1?(SDF-1?)plays an important role in the neovascularization of ischemic flaps through the SDF-1?/CXCR4 biological axis.Activation of calcium sensing receptor(Ca SR)is associated with myocardial apoptosis induced by ischemia reperfusion injury(I/R).The present study evaluated the feasibility of applying SDF-1? to alleviate Ca SR activation-mediated I/R injury in ischemic axial skin flaps.The research content is divided into the following three aspects:Section One Establish the SD rat model of ischemia reperfusion and to select the right ischemic timeObjective: To establish a SD rat model of ischemia reperfusion,and to select a suitable ischemic time.Methods: Epigastric axial skin flaps based on medial and lateral branches of superficial inferior epigastric vessels were elevated in Sprague-Dawley rats.12 rats were randomly divided into 4 groups and ischemia time was 1 h,2 h,3 h and 4 h,respectively.After reperfusion 2 h to collect the flap tissue,Ca SR was detected with WB in each group.Results: The expression of Ca SR was the highest at 3 h group.When it was lower than 3 h or higher than 3 h,the expression of Ca SR was decreased.Conclusion: 3 h was chosen as the ischemic time of the next experiment.Section Tow Effects of Ca SR and SDF-1? on necrosis,apoptosis and angiogenesis of skin flapsObjective: To study the effect of Ca SR expression on flap necrosis,apoptosis and angiogenesis,and the antagonism of SDF-1? to Ca SR.Methods: 30 rats were randomly divided into 5 groups administered Ca Cl2(i.e.,a specific Ca SR agonist),NPS2143(i.e.,a Ca SR inhibitor)+Ca Cl2,SDF-1?(i.e.,a CXCR4agonist)+Ca Cl2,AMD3100(i.e.,a CXCR4 antagonist)+SDF-1?+Ca Cl2,and normal saline through the femoral vein.After 2 h or 7 days of reperfusion,the skin flaps were harvested to evaluate flap necrosis and neovascularization.Results: Ca Cl2 group had higher necrosis rate,higher apoptosis rate and lower angiogenesis.The NPS2143+ Ca Cl2 group had obvious inhibitory effect on the above results.The necrosis rate and apoptosis rate of SDF-1 + Ca Cl2 group were both decreased,and the rate of angiogenesis was increased.Conclusion: Ca SR is closely related to the ischemia reperfusion injury of the skin flap,while SDF-1 has an antagonistic action of Ca SR.Section Three Flap necrosis and neovascularization were detected through quantitative real-time PCR,western blotting,and immunohistochemistry analysis.Objective:To detect the expression of Ca SR,CXCR4 and the expression of apoptosis related proteins in the skin flap by PCR,WB and IHC.Methods: The tissues harvested of the second part flaps were examined by immunohistochemistry,Western blot and real-time quantitative PCR.Results: When Ca SR has a higher expression,there was a higher expression of Pro apoptotic protein(FSA,Cyt-c,Cleaved Casepase-3,Casepase-9,Bax)and lower expression of anti apoptotic protein(Bcl-2).The results of NPS2143+Ca Cl2 group were completely opposite.That is,when the expression of Ca SR was lower,the expression of Pro apoptotic protein was decreased and the expression of anti apoptotic protein was increased.The activation of the SDF-1?/CXCR4 axis could reduce Caspase-3/9,FAS,Cyt-c and Bax,and increase Bcl-2.The results initially demonstrated that the activation of the SDF-1?/CXCR4 axis could function as an NPS2143 to significantly neutralize the extensive expression of Ca SR in vascular endothelial cell-induced I/R injury of skin flaps through the reducing Caspase-3/9,FAS,Cyt-c and Bax,and increasing Bcl-2. |
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