Effect Of Apolipoprotein J Overexpression On Proliferation And Migration Of Rat Vascular Smooth Muscle Cells

Objective: In this study,the recombinant plasmid p CMV3-C-Apo J and empty plasmid p CMV3-C--CV was transfected into rat vascular smooth muscle cells by lipofection methods,and detecting its expression in rat vascular smooth muscle cells in vitro.Observe the effect of apolipoprotein J overexpression on the proliferation and migration of rat vascular smooth muscle cells,and research the relationship between Apo J and vascular restenosis and its possible mechanism.Methods:The rat vascular smooth muscle cells were cultured in vitro.The recombinant plasmid p CMV3-C-Apo J and empty plasmid p CMV3-C--CV was transfected into rat vascular smooth muscle cells by lipofection methods.Set up Apo J group,Control group.Observe green fluorescence protein expression under fluorescence microscope after 48 hours of plasmid transfection.The vascular smooth muscle cells were lysed with using RIPA cell lysate to collect cellular proteins.Using Western blot method to detect the expression of apolipoprotein J(Apo J),apolipoprotein E receptor 2(Apo ER2)and very low density lipoprotein receptor(VLDLR).The proliferation ability of Apo J group and control group were evaluated by CCK-8,and the cell migration was determined with transwell chamber and wound healing assay.Results:1、Recombinant plasmid p CMV3-C-Apo J was successfully transfected into rat vascular smooth muscle cells,resulting in overexpression of apolipoprotein J.The results of Western blot method showed that expression level of Aop J in Apo J group(0.203±0.002)was significantly higher than that in Control group(0.129±0.003)(P<0.05);2、The expression level of apolipoprotein E receptor 2(Apo ER2)in Apo J group(0.219±0.002)was significantly higher than that in Control group(0.118±0.002)(P<0.05);3、Very low density lipoprotein receptor(VLDLR)was not detected in Apo J group or control group;4、The results of CCK-8 experiments showed that after adding 10 μl CCK-8 solution to rat vascular smooth muscle cell culture medium for 2 h,the absorbance(OD value)at 450 nm-490 nm of Apo J group(0.961±0.097)was significantly lower than the OD value of Control group(1.420±0.302)(P<0.05),indicating that the overexpression of apolipoprotein J can inhibit the proliferation of vascular smooth muscle cells.5、The results of Transwell experiments showed that Transwell cell chambers were removed from cultured rat vascular smooth muscle cells incubated for 8 hours,and then crystal violet staining was performed.The cells in the subventricular layer were observed under a microscope and photographed.The cells were counted with Image J software.The number of cells migrated to the membrane in Apo J group(220.000±44.550)was significantly less than that in Control group(435.560±100.387)(P<0.05),indicating that the overexpression of apolipoprotein J can inhibit the migration of vascular smooth muscle cells.6、Wound healing assay:Recording scratches after crystal violet staining at 0 hours and 24 hours after scratching the fused monolayer cells.The Image J software was used to calculate the mobility of each group of cells.The cell migration rate in Apo J group(0.058±0.028)was significantly lower than that in Control group(0.203±0.051)(P<0.05),indicating that the overexpression of apolipoprotein J significantly inhibit the migration of rat vascular smooth muscle cells.Conclusion:1、The recombinant plasmid p CMV3-C-Apo J was successfully transfected into rat vascular smooth muscle cells in vitro by lipofection methods,resulting in overexpression of apolipoprotein J.2、The results of CCK-8 showed that overexpression of apolipoprotein J can inhibit the proliferation of rat vascular smooth muscle cells.Transwell and wound healing assay showed that overexpression of apolipoprotein J can inhibit the migration of rat vascular smooth muscle cells.3、Overexpression of apolipoprotein J may up-regulate Apo ER2,inhibit the proliferation and migration of rat vascular smooth muscle cells.Apo J may have inhibitory effect on vascular restenosis.4、Very low density lipoprotein receptor(VLDLR)was not detected in Apo J group or control group,suggesting that the inhibitory effect of apolipoprotein J overexpression on the proliferation and migration of rat vascular smooth muscle cells may not be related to VLDLR.