Different Activity Of HEGF And TGF-β₂ On The Expression Of P27～(kip1) And Cdk4 On Wound Healing In Cultured Rabbit Corneal Endothelial Cells

Objective: To investgate the different activity of EGF and TGFβ2 on wound healing rabbit corneal endothelial cells culturedand the expression of p27Kip1 and Cdk4. To Explore the mechanismsof wound cornea endothelial cell healing. Methods: Rabbit cornea endothelial cells was cultured by Digest method and Joint method in vitro. Wound was created in confluent secondary culture of rabbit corneal endothelial cells. The area of wound is a roundness area and the diameter is about 3.5 mm. After wounding, different dose of EGFand or but 10ng/ml TGFβ2 were added. The control contained noEGF or TGFβ2 . The wound area was evaluated by computer on 1, 3, 5 and 7 days after wounding. Subcellular localization of cyclin dependent kinase 4 (Cdk4 ) and p27 (p27) was determined byimmunofluorescent staining. Judging the content of Cdk4 and p27 by the difference of the intensity of fluorescence. Results:(1)In normal rabbit corneal endothelial cells, p27 is positive and Cdk4 is negrative or faint. (2)EGF enhanced the wound healing in a dose-dependent manner and enhancing wound healing responses peaked at 10-40 ng/ml; (p<0.05,p<0.01) (3)Afetr the wounding, CEC was cultured in MEM only plus bovince serum . In 1st day, Cdk4 increased and p27 decreased even disappeared. During the progress of CEC healing, content ofCdk4 and p27 retain constantly. Reached the confluence of CEC, p27 was upstreamed and Cdk was downstreamed. (4)TGFβ2 inhibit thewound healing in cultured rabbit corneal endothecial cells (p<0.01), (5)24h after added 40 ng/ml EGF, cell cycle was progressed ; 10 ng/mlTGFβ2 has no influncess on this process (p<0.01) ; (6)Incubated by10ng/ml TGFβ2 and normal proportion of EGF, no outcome of rabbitcorneal wounding healing was found. (p>0.05) ; (7)24h after added10ng/ml TGFβ2, EGF has no effect on this. Conclusion: EGF ofcertain concentration enhances the wound healing in cultured rabbitcornea endothelial cells. Exogenous TGFβ2 inhibits the progress of thewound healing in cultured rabbit cornea endothelial cells. During thiscourse, p27 and Cdk4 can act a marker of different period ofwound healing.