hEGF、TGFβ_2对兔角膜内皮细胞损伤的作用及相关蛋白（p27～（kip1）和CDK4）的表达
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摘要
目的：观察hEGF(Human Epidermal Growth Factor)、TGFβ_2(Transforming Growth Factor β_2)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TGFβ_2。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。
Objective: To investgate the different activity of EGF and TGFβ2 on wound healing rabbit corneal endothelial cells cultured
and the expression of p27Kip1 and Cdk4. To Explore the mechanisms
of 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 EGF
and or but 10ng/ml TGFβ2 were added. The control contained no
EGF 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 by
immunofluorescent 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 of
Cdk4 and p27 retain constantly. Reached the confluence of CEC, p27 was upstreamed and Cdk was downstreamed. (4)TGFβ2 inhibit the
wound healing in cultured rabbit corneal endothecial cells (p<0.01), (5)24h after added 40 ng/ml EGF, cell cycle was progressed ; 10 ng/ml
TGFβ2 has no influncess on this process (p<0.01) ; (6)Incubated by
10ng/ml TGFβ2 and normal proportion of EGF, no outcome of rabbit
corneal wounding healing was found. (p>0.05) ; (7)24h after added
10ng/ml TGFβ2, EGF has no effect on this. Conclusion: EGF of
certain concentration enhances the wound healing in cultured rabbit
cornea endothelial cells. Exogenous TGFβ2 inhibits the progress of the
wound healing in cultured rabbit cornea endothelial cells. During this
course, p27 and Cdk4 can act a marker of different period of
wound healing.
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。
Objective: To investgate the different activity of EGF and TGFβ2 on wound healing rabbit corneal endothelial cells cultured
and the expression of p27Kip1 and Cdk4. To Explore the mechanisms
of 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 EGF
and or but 10ng/ml TGFβ2 were added. The control contained no
EGF 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 by
immunofluorescent 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 of
Cdk4 and p27 retain constantly. Reached the confluence of CEC, p27 was upstreamed and Cdk was downstreamed. (4)TGFβ2 inhibit the
wound healing in cultured rabbit corneal endothecial cells (p<0.01), (5)24h after added 40 ng/ml EGF, cell cycle was progressed ; 10 ng/ml
TGFβ2 has no influncess on this process (p<0.01) ; (6)Incubated by
10ng/ml TGFβ2 and normal proportion of EGF, no outcome of rabbit
corneal wounding healing was found. (p>0.05) ; (7)24h after added
10ng/ml TGFβ2, EGF has no effect on this. Conclusion: EGF of
certain concentration enhances the wound healing in cultured rabbit
cornea endothelial cells. Exogenous TGFβ2 inhibits the progress of the
wound healing in cultured rabbit cornea endothelial cells. During this
course, p27 and Cdk4 can act a marker of different period of
wound healing.
引文
1．谢立信主编 《角膜移植学》第一版 人民卫生出版社 2002年53～56页
2. Laing R.A, Neubauer L, Oak SS, Kayne HL, Leibowitz HM. Evidence for mitosis in the adult corneal endothelium. Ophthalmology 1984;91:1129—34.
3. Landshman N. Relationship between morphology and function of healing cat corneal endothelium. Invest Ophthalmol Vis Sci, 1988,29:1100.
4．谢立信，李绍伟，董晓光，袁风波．人各种角膜病变内皮细胞核变化的初步观察．中华眼科杂志，1998，34：19—21．
5. Joyce NC, Navon SE, Roy S, Zieske JD. Expression of cell cycle—associated proteins in human and rabbit corneal endothelium in situ. Invest Ophthalmology Vis Sci 1996; 37; 1566—75.
6．吴静，徐锦堂．EGF对不同区域角膜上皮细胞体外传代培养的影响，眼科研究，1999：17；329—331．
7. Tae Yon Kim ,Won—Il Kim, Ronald E. Smith, etc Different activity of TGF—β2 on the expressions of p27KIP1 and Cdk4 in actively cycling and contact inhibited rabbit corneal endothecial cells, www. Molvis.org/molvis/v7/a37/
8. Reed SI, Bailly E, Dulic V, Hengst L, Resnitzky D, Slingerland J. G1 control in mammalian cells. J Cell Sci Suppl 1994;18:69—73.
9. Pines J. Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Gengs Dev 1999;13:1501—12.
10. Polyak K, Kato JK, Solomon MJ, Sherr CJ, Massague J, Roberts JM,Koff A. p27Kipl, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contract inhibiton to cell cycle arrest. Genes Dev 1994;8:9—22.
11. Pavletich NP. Mechanisms of cyclin-dependent kinase regulation structure of Cdks, their cyclin activators, and Cip and Ink4 inhibitors. J Mol Biol.1999;287:821—828.
12. Massague J, Blain SW, Lo RS .TGFβ signaling in growth control, cancer, and heritable disorders. Cell.2000;103:295—309.
13. Senoo T, Joyce NC, et al. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophtholmol Vis Sci.2000;41:660—667.
14. Wilson SE, Schultz GS,Chegini N, et al. Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res. 1994;59:63—71.
15. Moses HL, Yang EY, Pietenpol JA. TGF- beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell 1990;63:245—247.
16. Chen KH, Harris DL, Joyce NC. et al. TGFβ2 in aqueous humor suppresses S-phase enter in cultured corneal endothelial cells. 1999;40:2513—2519.
17. Joyce NC, Joyce SJ, Powell SM, Meklir B. EGF and PGE2: effects on corneal endothelial cell migration and monolayer spreading during wound repair in vitro. Curr Eye Res 1995;14:601—9.
18. Van Setten GB, Fagerholm P, Philipson B, Schultz G, et al. Growth factors and their receptors in the anterior chamber. Absence of epidermal growth factor and transforming growth factor alpha in human aqueous humor. Ophthalmic Res 1996;28:361—364.
19．吴静，徐锦堂．EGF对不同区域角膜上皮细胞体外传代培养的影响．眼科研究，1999：17；329—331．
20．姜琨，秦樾，童坦君．erbB-2表达抑制与表皮生长因子刺激对信号转导与转录激活分子及细胞周期蛋白D1的影响．北京医科大学学报，1998；30：205—206．
21．钟一声，王康孙，石海云．猴、兔角膜细胞成分原代培养的研究．眼科研究，1999；17：191—194．
22．赵靖，谢立信，史伟云，牛晓光．表皮生长因子对猫角膜内皮细胞DNA合成的影响 眼科研究2002；20(5)419—422．
23．卢圣栋主编 《现代分子生物学实验技术》 第二版 中国协和医科大学出版社2001年397—399．
24. Bates AK, Hiorns RW, Cheng H,et al, Modelling of changes in the corneal endothelium after cataract surgery and penetrating keratoplasty. Br J Ophthalmol,1992,76:32—35.
25. Petroll WM,Jester JV ,Bean JJ et al .Myofibroblast transformation of cat corneal endothecial by transforming growth factor- betal,-beta-2,and-beta3 [J].Invest Ophthalmol Vis Sci, 1998,39(11):2018—2032.
26. Carpenter G, Cohen S, Epidermal growth factor. J Biol Chem. 1990,265 ;7709.
27. Zieske JD, Takahashi H, Hutcheon AE. Et al. Activation of epidermal growth factor receptor during corneal epithelial migration. Invest Ophthalmol Vis Sci,20000,41(6):1346.
28. Van Setten GB, Fagerholm P, Philipson B, Schultz G, et al. Growth factors and their receptors in the anterior chamber. Absence of epidermal growth factor and transforming growth factor alpha in human aqueous humor. Ophthalmic Res 1996;28:361—364.
29. Senoo T, Joyce NC, et al. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophthalmol Vis Sci 2000;41:660—667.
30. Chen KH ,Harris DL,Joyce NC, et al. TGF β 2 in aqueous humour suppresses S phase entry in cultured corneal endothecial cells [J]. Invest Ophthalmol Vis Sci, 1999,40(11):2513.
31. Harris DL, Joyce NC, et al. Tansforming growth factor-beta suppresses proliferation of rabbit corneal endothelial cells in vitro.[J]. J interferon Cytokine Res,1999;19:327—334.
32. Chen KH ,Harris DL,Joyce NC, et al. TGFβ 2 in aqueous humour suppresses S phase entry in cultured corneal endothecial cells [J]. Invest Ophthalmol Vis Sci, 1999,40(11):2519.
33. Smith RS. Ultrastructural studies on the blood aqueous barrier, 1.transport of an electron-dense tracer in the iris and ciliary body of the mouse. Am J Ophtholmol 1971;63:143.
34. 闫洪禄主编 眼生理学 北京 人民卫生出版社 2001年第一版 153-159。
2. Laing R.A, Neubauer L, Oak SS, Kayne HL, Leibowitz HM. Evidence for mitosis in the adult corneal endothelium. Ophthalmology 1984;91:1129—34.
3. Landshman N. Relationship between morphology and function of healing cat corneal endothelium. Invest Ophthalmol Vis Sci, 1988,29:1100.
4．谢立信，李绍伟，董晓光，袁风波．人各种角膜病变内皮细胞核变化的初步观察．中华眼科杂志，1998，34：19—21．
5. Joyce NC, Navon SE, Roy S, Zieske JD. Expression of cell cycle—associated proteins in human and rabbit corneal endothelium in situ. Invest Ophthalmology Vis Sci 1996; 37; 1566—75.
6．吴静，徐锦堂．EGF对不同区域角膜上皮细胞体外传代培养的影响，眼科研究，1999：17；329—331．
7. Tae Yon Kim ,Won—Il Kim, Ronald E. Smith, etc Different activity of TGF—β2 on the expressions of p27KIP1 and Cdk4 in actively cycling and contact inhibited rabbit corneal endothecial cells, www. Molvis.org/molvis/v7/a37/
8. Reed SI, Bailly E, Dulic V, Hengst L, Resnitzky D, Slingerland J. G1 control in mammalian cells. J Cell Sci Suppl 1994;18:69—73.
9. Pines J. Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Gengs Dev 1999;13:1501—12.
10. Polyak K, Kato JK, Solomon MJ, Sherr CJ, Massague J, Roberts JM,Koff A. p27Kipl, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contract inhibiton to cell cycle arrest. Genes Dev 1994;8:9—22.
11. Pavletich NP. Mechanisms of cyclin-dependent kinase regulation structure of Cdks, their cyclin activators, and Cip and Ink4 inhibitors. J Mol Biol.1999;287:821—828.
12. Massague J, Blain SW, Lo RS .TGFβ signaling in growth control, cancer, and heritable disorders. Cell.2000;103:295—309.
13. Senoo T, Joyce NC, et al. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophtholmol Vis Sci.2000;41:660—667.
14. Wilson SE, Schultz GS,Chegini N, et al. Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res. 1994;59:63—71.
15. Moses HL, Yang EY, Pietenpol JA. TGF- beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell 1990;63:245—247.
16. Chen KH, Harris DL, Joyce NC. et al. TGFβ2 in aqueous humor suppresses S-phase enter in cultured corneal endothelial cells. 1999;40:2513—2519.
17. Joyce NC, Joyce SJ, Powell SM, Meklir B. EGF and PGE2: effects on corneal endothelial cell migration and monolayer spreading during wound repair in vitro. Curr Eye Res 1995;14:601—9.
18. Van Setten GB, Fagerholm P, Philipson B, Schultz G, et al. Growth factors and their receptors in the anterior chamber. Absence of epidermal growth factor and transforming growth factor alpha in human aqueous humor. Ophthalmic Res 1996;28:361—364.
19．吴静，徐锦堂．EGF对不同区域角膜上皮细胞体外传代培养的影响．眼科研究，1999：17；329—331．
20．姜琨，秦樾，童坦君．erbB-2表达抑制与表皮生长因子刺激对信号转导与转录激活分子及细胞周期蛋白D1的影响．北京医科大学学报，1998；30：205—206．
21．钟一声，王康孙，石海云．猴、兔角膜细胞成分原代培养的研究．眼科研究，1999；17：191—194．
22．赵靖，谢立信，史伟云，牛晓光．表皮生长因子对猫角膜内皮细胞DNA合成的影响 眼科研究2002；20(5)419—422．
23．卢圣栋主编 《现代分子生物学实验技术》 第二版 中国协和医科大学出版社2001年397—399．
24. Bates AK, Hiorns RW, Cheng H,et al, Modelling of changes in the corneal endothelium after cataract surgery and penetrating keratoplasty. Br J Ophthalmol,1992,76:32—35.
25. Petroll WM,Jester JV ,Bean JJ et al .Myofibroblast transformation of cat corneal endothecial by transforming growth factor- betal,-beta-2,and-beta3 [J].Invest Ophthalmol Vis Sci, 1998,39(11):2018—2032.
26. Carpenter G, Cohen S, Epidermal growth factor. J Biol Chem. 1990,265 ;7709.
27. Zieske JD, Takahashi H, Hutcheon AE. Et al. Activation of epidermal growth factor receptor during corneal epithelial migration. Invest Ophthalmol Vis Sci,20000,41(6):1346.
28. Van Setten GB, Fagerholm P, Philipson B, Schultz G, et al. Growth factors and their receptors in the anterior chamber. Absence of epidermal growth factor and transforming growth factor alpha in human aqueous humor. Ophthalmic Res 1996;28:361—364.
29. Senoo T, Joyce NC, et al. Cell cycle kinetics in corneal endothelium from old and young donors. Invest Ophthalmol Vis Sci 2000;41:660—667.
30. Chen KH ,Harris DL,Joyce NC, et al. TGF β 2 in aqueous humour suppresses S phase entry in cultured corneal endothecial cells [J]. Invest Ophthalmol Vis Sci, 1999,40(11):2513.
31. Harris DL, Joyce NC, et al. Tansforming growth factor-beta suppresses proliferation of rabbit corneal endothelial cells in vitro.[J]. J interferon Cytokine Res,1999;19:327—334.
32. Chen KH ,Harris DL,Joyce NC, et al. TGFβ 2 in aqueous humour suppresses S phase entry in cultured corneal endothecial cells [J]. Invest Ophthalmol Vis Sci, 1999,40(11):2519.
33. Smith RS. Ultrastructural studies on the blood aqueous barrier, 1.transport of an electron-dense tracer in the iris and ciliary body of the mouse. Am J Ophtholmol 1971;63:143.
34. 闫洪禄主编 眼生理学 北京 人民卫生出版社 2001年第一版 153-159。