Inhibitory Effect Of Kistrin, A Disintegrin, On Adhesion And Proliferation Of Human Lens Epithelial Cell Line SRA01/04

Objective:Posterior capsule opacification(PCO)is the mainly reason induced to low vision after extracapsular cataract extraction.Even though tremendous advances have been made especially during the last 10-15 yeas in terms of surgical techniques and improvement of implant technology,Posterior capsule opacification(PCO)still remains a serious long-terms complication, which is now the commonest complication of cataract surgery occurring in up to 20～50%of patients after the operation,especially in children,it is up to 100%. Nd:YAG laser posterior capsulotomy is a conventional method for treatment of PCO and brings some complications after operation.And children have to take surgery once more because they can't cooperate with ophthelmologies to receive the Nd:YAG laser posterior capsulotomy.The safe and effective medicine has been looking for treating PCO.The adhesion and proliferation of len epithelial cells retained in the capsular bag following surgery plays an important role in the metastasis of famation PCO,So the block of LECs adhesion and inhibition of proliferation is one important strategy to develop anti-PCO drugs.The RGD-containing peptides espesially disintegrin catch the eyes of reseachers because they can block cell adhesion to ECM and prevent the proliferation of malignant tumors,.So these medicine have been applicated in other area on cell adhesion.To investigate the effects of disintegrin on LECs, we employed cell culture technique in vitro and study the effects of Kistrin,a disintegrin contain RGD peptide,on human lens epithelial cell line,SRA01/04, adhesion and proliferation.Our goal was to provide experimental and theoretical basis for Kistrin in anti-PCO mechanisms.Methods:Our work was composed of two parts:(1)Detecting the effects of Kistrin on the adhesion of SRA01/04 cells.SRA01/04 cells suspended in DMEM/F₁₂medium containing 10%fetal bovine serum.They were pre-incubate for 30 minute in various concentrations of Kistrin(40ng/ml,80ng/ml,160ng/ml,320ng/ml,640ng/ml)and RGD peptide(0.125mg/ml,0.25mg/ml,0.5mg/ ml,1.0mg/ml,2.0mg/ml),then were seeded at 2.5×10⁴ cells well^-1onto 96-well plate coated with rat-tail collagen at 37℃,5%CO₂.After incubation for 9 minutes,non-adherent cells were discarded and adherent cells were washed twice with PBS.MTT assay was employed to determine the effect of Kistrin on SRA01/04 cells that had been adhered.(2)Detecting the effects of Kistrin on the proliferation of SRA01/04 cells.The SRA01/04 cells were plated at a density of 1×10⁴ cells well^-1in 96-well rat-tail collagen coated plates,using DMEM/F₁₂ media containing 10%FBS.After 12 h,the medium was changed to serum-free media and cells were cultured for synchronization.After 24h,the medium was change to various concentrations of Kistrin(40ng/ml,80ng/ml,160ng/ml,320ng/ml,640ng/ml)and RGD peptide(0.125mg/ml,0.25mg/ml,0.5mg/ml,1.0mg/ml,2.0mg/ml)again and SRA01/04 cells were cultured for 24h,48h and 72h at 37℃,5%CO2.The inhibition of proliferation was measured by colorimetric MTT assay.Results:1,Kistrin could inhibit the adhesion of SRA01/04 cells to rat-tail collagen dose-dependently.The inhibitor rates of Kistrin at concentration of 40ng/ml,80ng/ml,160ng/ml,,320ng/ml,640ng/ml were 3.7%,7.8%,15.9%,22.7%,33.4%respectively.And the inhibitor rates of RGD peptide at concentraton of 0.125mg/ml,0.25mg/ml,0.5mg/ml,1.0mg/ml,2.0mg/ml were 5.1%,12.2%,19.0%,25.9%and 35.1%respectively.There was significant difference among the groups except the lowest concentration (P＜0.05).The IC₅₀value of Kistrin and RGD peptide on inhibiting SRA01/04 cells adhesion were 918.1ng/ml and 2.7mg/ml respectively.These results indicate that on rat-tail collagen the anti-adhesive activety of Kistrin was higher 3000 times than that of RGD peptide.2,Kistrin had a strong inhibition on the proliferation of SRA01/04 cells dosage-dependently and time-dependently (P＜0.05).Significant difference of the inhibitory effects was observed after culturing separately for 48 hours,72 hours and 24 hours.After 24h,48h and 72h exposure,the IC₅₀values of Kistrin and RGD peptede were 640.5ng/ml,429.8 ng/ml,303.0ng/ml and 2.03mg/ml,1.27mg/ml,0.87mg/ml respectively.These results indicate that on rat-tail collagen the anti-proliferative activety of Kistrin was higher 2900～3200 times than that of RGD peptide in 24h,48h and 72h. 3,the apoptosis was appeared after exposure of SRA01/04 cells to Kistrin at hign dosage for 72hConclusion:1,These experiments confirmed that Kistrin and RGD peptide could inhibit the adhesive and proliferative effects on SRA01/04 cells,and it would be enhanced greatly as the concentration and the time increased.2,The anti-adhesive effect and anti-proliferative effect of Kistrin to SRA01/04 cells on rat-tail collagen are stronger 2900 times above than that of RGD peptide.3,The apoptosis was appeared after exposure of SRA01/04 cells to Kistrin at hign dosage for 72h.4,The study provided experimental basis for Kistrin in anti-PCO mechanisms.