TNF-α Expression In Pterygium Fibroblast Induced By Ultraviolet-exposed And Hydrogen Peroxide And Inhibiting Effect Of Aprotinin On Expression Of TNF-α

PART 1 TNF–αexpression in pterygium fibroblast induced by ultraviolet-exposed and Hydrogen PeroxideObjective: To investigate diferent concentrations of TNF–αexpression in pterygium fibroblast induced by ultraviolet-exposed and Hydrogen Peroxide, and could use Hydrogen Peroxide substitution for ultraviolet-exposed in experiment. This way will be very accurate and quick.Methods : The fibroblasts were isolated from human pterygium and then cultivated in vitro. The cells from the third to fifth generation were used in our experiment. (1) Human pterygium fibroblast cells were identificated by the method of SP and fractionated to threegroups: The first group is negative control group, the second one is ultraviolet-exposed group, the third one is Hydrogen Peroxide(150μmol/L) induction group. (2) Extracting supernate fluid at different effective time(15min,30min,60min,6h,24h) and then centrifuging. Enzyme Linked Immunosorbent Assay(ELISA) was used to evaluate the concentration of TNF–α.Results: TNF-αincreases in pterygium fibroblast both in ultraviolet-exposed and Hydrogen Peroxide induction. The difference is significant compared with control group(P <0.05). There is no significant difference between two handling groups(P >0.05). Conclusions: Hydrogen Peroxide(150μmol/L) could imitate ultraviolet-exposed in pterygium experiment.PART 2 Inhibiting effect of Aprotinin on expression of TNF–αin Pterygium FibroblastObjective : From the first department we can use Hydrogen Peroxide instead of ultraviolet -exposed in experiment. And now we are going to study antiproliferation of aprotinin on fibroblast of pterygium and antiexpression of TNF–αin vitro. This will afford potential agent for prevention and treatment of primary or recurrence pterygium.Methods: In the same manner of the first department, primary culture and subculture of pterygium fibroblasts were established in vitro. Hydrogen Peroxide(150μmol/L) was used to stimulate the TNF-αexpression in pterygium fibroblast. And then different concentrations of aprotinin (1500 U/mL, 3000 U/mL, 6000 U/mL, 12000U/mL) were added to the culture fibroblasts of the third to fifth generation respectively. MTT assay at 492nm was used to evaluate the effect of aprotinin on cells proliferation. Enzyme Linked Immunosorbent Assay(ELISA) at 450nm was used to evaluate the concentration of TNF–α.Results: Aprotinin didn't stimulate pterygium fibroblast proliferation in the experiment. However, it could inhibit the TNF-αexpression of pterygium fibroblast at different concentrations; especially at 1500U/ml and 3000U/ml. The suppression amounts were19.36 pg/ml, 38.46 pg/ml, 56.15 pg/ml, 13.84 pg/ml; 10.2 pg/ml, 31.46 pg/ml, 49.15 pg/ml, 4.62pg/ml(P <0.05). And the effective time of Hydrogen Peroxide were 30min, 60min, 6h and 24h(P <0.05).Conclusions: Aprotinin can inhibit the proliferation of pterygium fibroblast and TNF–αexpression.