Experimental Study On The Effect Of Over Expression Of Periostin On The Function Of Endothelial Progenitor Cells

Objective: To investigate the construction of recombinant lentiviral LV5-PERI Periostin gene expression system of learning technology of exogenous gene by lentivirus transfection,and the transfection efficiency of specific detection;through the comparative analysis of the test results to determine the expression of Periostin gene on vascular endothelial cells in mice in vitro before the effects of cell proliferation and apoptosis,invasion and migration the functions and differences;later envisaged by the method of gene transfer of vascular endothelial precursor cells overexpressing Periostin system,construct endothelial progenitor cells overexpressing Periostin,and from to enhance the biological function of endothelial progenitor cells,the endothelial progenitor cells can migrate into the vessel wall by early and rapidly the site of injury,and can promote better differentiation of precursor cells of vascular endothelial cells to mature.The current control in the treatment of intractable vascular occlusion diseases appear lumen for the treatment of postoperative restenosis,thrombosis treatment ideas,early reendothelialization,repair damaged endothelium,improve the long-term clinical effect.Methods: the target gene(Periostin gene),using the method of lentiviral transfected exogenous gene expression vector LV5 to construct recombinant lentivirus,the formation of LV5-PERI,and then infected target cells to establish a stable to endothelial progenitor cells overexpressing Periostin gene system,ensure cell infection efficiency can reach above 90%;set three group of vascular endothelial precursor cells,namely: plasmid transfection group(over expression group),empty plasmid group and blank control group,by Western blotting,RT-PCR,Elisa and other testing means to clear through building the efficiency of transfection and expression of exogenous.CCK8 was used in 24 h,48h,72 h,96h,120 h time point detection absorbance at 490 nm,and the growth curve,the difference between the three groups in the aspects of cell proliferation activity;the Accuri C6 individuals were detected by flow cytometry cell apoptosis.Detection of cell migration experiments(the scratch method),0h,12 h and 24 h respectively at the time point detection,observed the difference in cell migration;detect the effect of overexpression of Periostin gene on vascular endothelial precursor cells invasion function by using Transwell chamber method.Statistical methods were used to analyze the effects of overexpression of Periostin gene on the proliferation,apoptosis,invasion and migration of endothelial progenitor cells.Results: the recombinant plasmid LV5-PERI was constructed successfully,Periostin gene transfection group of vascular endothelial precursor cells significantly increased the expression of Periostin protein was detected by Elisa method,the light absorption value is about 18 times the corresponding empty vector transfected group and blank control group at 450 nm,the difference was statistically significant(P<0.05).The ability of cell proliferation,detected by CCK8 overexpression group than the control group between the light absorption value of small,low proliferative activity,the difference was statistically significant(P<0.05);found that detection of cell apoptosis in transfected group were the control group the number of apoptotic cells than the control group comparison,performance a significant difference(P<0.05);in cell migration,through the experiments of scratch test expression of transfected cells than the empty plasmid expression group and blank control group(P<0.05)fast transfer percentage the same experiment was repeated;over expression in transfected cells invasion number was more than that in blank control group,the differences between groups were statistically significant(P<0.05).However,there was no significant difference in the detection of cell function(proliferation,apoptosis,migration and invasion)between the plasmid expression group and the blank control group(P>0.05).Conclusions: The recombinant plasmid LV5-PERI transfected by lentivirus can stably and efficiently construct the vascular endothelial progenitor cell overexpressing Periostin gene system,and can continuously express Periostin protein in vitro.Periostin can enhance the activity of cells by the high expression of Periostin protein influences the biological behavior of the cell,the protein plays a certain role in enhancing vascular endothelial migration and invasion of cells before the.However,only up regulation of Periostin had no significant effect on the proliferation of vascular endothelial progenitor cells.