【作者】 刘澍；

【导师】 陈美玲；

【作者基本信息】 青岛大学 ， 眼科学， 2004， 硕士

【摘要】 目的：观察hEGF（Human Epidermal Growth Factor）、TGFβ₂（Transforming Growth Factor β₂）对家兔角膜内皮细胞损伤后的作用及相关蛋白(p27^Kip1[以下简称p27]和CDK4)的表达，探讨角膜内皮细胞损伤修复的相关机制。方法：利用消化法和贴壁培养法体外培养家兔角膜内皮细胞，传一代融合后，定量损伤直径3.5mm范围内的细胞，培养液中分别加入不同浓度的hEGF和或浓度为10ng／ml的TGFβ₂。对照组不加任何药物。损伤后第1、3、5，7天倒置显微镜下照相并转入计算机测量家兔角膜内皮细胞损伤模型的未愈合面积，作为判断不同浓度、不同药物对角膜内皮细胞损伤愈合速度的指标。培养液中同时加入p27和Cdk4（cyclin dependent kinase 4）的一抗，应用免疫荧光染色方法（immunofluorescent staining），通过荧光强度的高低来判断在细胞周期不同阶段p27和Cdk4的含量。结果：①在正常家兔角膜内皮细胞，p27为阳性，Cdk4为阴性或弱阳性。②一定浓度的EGF可以加快体外培养的家兔角膜内皮细胞的损伤愈合速度，EGF对体外培养的家兔角膜内皮细胞的损伤愈合的促进作用具有剂量依存性，以10ng／ml～40ng／ml为最适（p＜0.05，p＜0.01）。③损伤模型制作后，在只加入含胎牛血清的培养液中培养的第一天，Cdk4含量增加，p27的含量下降，甚至消失；在家兔角膜内皮细胞创面愈合的过程中，Cdk4的含量基本保持不变，p27的含量无变化；达到完全愈合后，p27含量增加，接近原代未损伤细胞阶段，中英文摘要Cdk4含量降低，甚至消失。④TGF民对体外培养的家兔角膜内皮细胞损伤模型的修复过程起抑制作用（p<0.01）⑤EGF孵育角膜内皮细胞损伤模型24h后，TGF贬对此阶段的细胞修复过程无抑制作用;⑥TGFpZ可抑制同时加入的EGF对角膜内皮细胞损伤的修复作用;⑦正常浓度（40n留ml）的EGF对被10n留ml的TGFpZ孵育24h后的家兔角膜内皮细胞损伤模型的修复作用较低，甚至无促进作用。（P>0.05）结论:l一定质量浓度的EGF可以促进体外培养的家兔角膜内皮细胞的损伤修复。2.在培养液中加入外源性的TGFpZ可抑制家兔角膜内皮细胞的损伤修复。3.在此过程中，细胞周期蛋白抑制因子p27 KIPI和细胞周期蛋白激酶cdk4可作为定性判断角膜内皮细胞损伤不同修复阶段的参考指标。更多还原

【Abstract】 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.更多还原