Analysis Of Increased Fibrosis After Anti-VEGF Treatment And Construction Of Lentiviral Particle That Inhibit Endogenous CTGF Expression

Objective:To verify the factors that increase fibrosis after anti-VEGF treatment,construct the connective tissue growth factor(CTGF)recombinant interference vector(shRNA)and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells.Methods:Classify cells into three groups:blank control,high glucose and high glucose with anti-VEGF groups,to simulate cellular state in patients with anti-VEGF drugs treatment PDR patients or not.Apply real-time PCR to verify the expression of CTGF,FN,a-SMA and ColI.Collect fibroproliferative membranes in patients with PDR injected with anti-VEGF drugs or not to perform semi-quantitative fluorescence analysis.Construct human CTGF recombinant interference vector(CTGFshRNA)marked with the red florescent mcherry protein and acquire high-titer CTGF shRNA lentivirus particles via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells.Identify the optimal multiplicity and onset time of lentivirus infection by tracing down the red florescent protein in interference vector and classify cells into three groups:blank control,infection control and CTGF knockdown groups.Observe the differences in cells migrating ability through Transwell allay and quantify CTGF knockdown efficiency and expression of FN,a-SMA and ColI protein through real-time PCR testing and Western blot system.Results:The expression of each factor in the high glucose with anti-VEGF group(CTGF:197.3 5± 14.95,FN:3.08±0.47,α-SMA:4.50± 1.07,ColI:2.14±0.06)was significantly higher than that in the high glucose group(CTGF:2.00±0.14,FN:1.54±0.10,α-SMA:2.24±0.35,ColI:1.97±0.08),the difference was statistically significant(F=7.86、4.24、6.03、0.16,P=0.000、0.005、0.03、0.041).Proliferation membrane fluorescence results are consistent with it.Our experiment finds that an MOI of 20 and onset time of 72 hours are the requirements to optimize lentivirus infection and effectively bring down the expression level of endogenous CTGF.Transwell allay result suggests a contrast in the number of migrated cells in knockdown group(117.33± 19.76)and that in blank control group(268.00±30.05)and infection control group(241.67±39.12),which is of statistical significance.Real-time PCR testing finds a contrast in related gene expression in knocked-down group(CTGF:0.11 ±0.11,FN:0.14±0.13,α-SMA:0.10±0.09,ColI:0.09±0.04)and that in blank control,which is of statistical significance.(F=128.83、124.44、144.76、1374.44,P=0.000、0.000、0.000、0.000).Western blot system finds the statistical significance of the contrasted number of related protein expression in knockdown group(CTGF:0.96±0.07,FN:0.99±0.06,α-SMA:0.61 ±0.06,ColI:0.60±0.01)and that in blank control(CTGF:1.76±0.19,FN:1.72±0.15,α-SMA:1.30±0.16,ColI:1.19±0.08).(F=22.55、41.60、25.73、161.68,P=0.002、0.000、0.001、0.000).Conclusion:Anti-VEGF drugs increase the expression of CTGF and other related factors in retinal vascular endothelial cells,which has been verified in clinical tissues.The success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression and inhibit cell migration and down regulates expression of FN,α-SMA and ColI,hence proved to be a potential molecular tool to inhibit endogenous CTGF expression.