Effect Of CXCR4 Gene On The Physiological Function Of Rat Bone Marrow Endothelial Progenitor Cells By PI3K Pathway

Objective:Effect of CXCR4 gene on the physiological function of Rat bone Marrow endothelial progenitor cells by regulating the PI3K pathway activityMethods:1.Build the rAAV6 with CXCR4 over-expression;Infection of BM-EPCs by rAAV6 with CXCR4 over-expression;Using the q-polymerase chain reaction?Western Blot method to detect the expressive level of CXCR4 in BM-EPCs;2.By use of the experiment of CCK8 evaluated whether hypoxia preconditioning had influence on prtoliferation of BM-EPCs;Using flow cytometry evaluated whether hypoxia preconditioning have influence on apoptosis of BM-EPCs;using Transwell migration experiment evaluated whether hypoxia preconditioning had influence on migration of BM-EPCs;By use of the experiment of CCK8 evaluated whether CXCR4 over-expression had influence on proliferation of BM-EPCs with hypoxia preconditioning;Using flow cytometry evaluated whether CXCR4 over-expression had influence on apoptosis of BM-EPCs with hypoxia preconditioning;using Trans well migration experiment evaluated whether CXCR4 over-expression had influence on migration of BM-EPCs with hypoxia preconditioning;3.Using Western Blot method to detected whether hypoxia preconditioning and/or cxcr4 over-expression had influence on the expression of PI3K/AKT signal pathway;4.Use of CCK8 experiment evaluated whether LY294002(PI3K inhibitors)had influence on prtoliferation that CXCR4 over-expression of BM-EPCs with hypoxia preconditioning;Using flow cytometry evaluated whether LY294002(PI3K inhibitors)had influence on apoptosis that CXCR4 over-expression of BM-EPCs;By using Transwell migration experiment evaluated whether LY294002(PI3K inhibitors)had influence on migration that CXCR4 over-expression of BM-EPCs with hypoxia preconditioning;Results:1.CXCR4 high-expression group of endothelial progenitor cells in CXCR4 mRNA and protein were higher than empty expression vector control group(p<0.01);2.CCK8 experiment,Transwell migration and flow cytometry respectively confirmed hypoxia preconditioning could inhibit proliferation of BM-EPCs(to determine single variable,individual OD values between the two groups had statistically significant,p<0.05),also inhibit migration of BM-EPCs(to determine single variable,individual migration cells was statistically difference between the two groups,p<0.01),and increased apoptosis of BM-EPCs(to determine single variable,individual apoptosis percentage between the two groups have statistical significance,p<0.01);Cck8 experiment,Transwell migration and flow cytometry respectively had confirmed the CXCR4 over-expression can promote the proliferation of BM-EPCs with hypoxia preconditioning(to determine single variable,individual OD values between the two groups have statistically significant,p<0.05),also could promote the migration of BM-EPCs with hypoxia preconditioning(to determine a single variable,their migration cells was statistically difference between the two groups,p<0.01),and inhibit apoptosis of BM-EPCs with hypoxia preconditioning(to determine single variable,individual apoptosis percentage between the two groups have statistical significance,p<0.01);3.Western Blot results showed that hypoxia preconditioning can decrease the expression of CXCR4/PI3K/Akt in BM-EPCs;CXCR4 over-expression can increase the expression of CXCR4/PI3K/Akt in BM-EPCs with hypoxia preconditioning;4.CCK8 experiment,Transwell migration and flow cytometry respectively had confirmed LY294002 could eliminate that CXCR4 over-expression had positive influence on proliferation of BM-EPCs with hypoxia preconditioning(individual OD values between the two groups had statistically significant,p<0.05),also could eliminate that CXCR4 over-expression had positive influence to migration of BM-EPCs with hypoxia preconditioning(individual migration cells was statistically difference between the two groups,p<0.01);could eliminate CXCR4 over-expression had negative influence on apoptosis of BM-EPCs with hypoxia preconditioning(individual apoptosis percentage between the two groups had statistical significance,p<0.01).Conclusion:Under the induction of SDF-1,CXCR4 over-expression regulated PI3K pathway activity to enhance proliferation and migration of BM-EPCs function with hypoxia preconditioning,and inhibited apoptosis.