摘要
【目的】研究肝细胞生长因子(hepatocyte growth factor ,HGF)对体外培养的人晶状体上皮细胞(human lens epithelial cells ,hLECs)促增生及促移行的作用,初步探讨HGF在后发性白内障(posterior capsule opacification, PCO)中可能存在的机制,为PCO的临床治疗及预防提供新的依据。
【方法】1.取人晶状体上皮细胞株(hLECs—B3)进行传代培养,通过细胞形态学及免疫组化染色法进行鉴定后,取3-5代细胞用于实验。2.噻唑盐比色实验(MTT法)测定细胞的增生情况:(1)取第3代传代培养的hLECs,分别加入不同浓度的HGF(2.5,5,10,20,40,50,100μg/L),24h后MTT法测定细胞的增生情况;(2)选择能极显著促进hLECs增生的HGF浓度(20μg/L),MTT法测定该浓度的HGF作用不同时间(6,12,24,48h)后hLECs的增生情况。3.流式细胞术(flow cytometry,FCM)检测细胞周期:选择能极显著促进hLECs增生的HGF浓度(20μg/L),FCM检测该浓度的HGF作用24 h后hLECs细胞周期的改变。4.细胞损伤愈合模型测定细胞的移行情况:对已融合的6孔培养板中的细胞进行划线,用不同浓度的HGF(10,20,50μg/L)进行干预,24小时后细胞计数法计数进入裸露区的细胞,判定细胞的移行效应。5.采用HMIAS-2000医学图文分析系统采集、处理图像和应用SPSS13.0软件进行统计分析,探讨HGF对hLECs增殖及移行的影响。
【结果】1.根据细胞的生长特性,形态学特征及波形蛋白、角蛋白免疫组化染色阳性等确定细胞为hLECs。2.MTT法:(1)HGF浓度为10,20,40,50μg/L时,24h后细胞吸光度分别为0.179±0.011,0.211±0.010,0.208±0.007,0.180±0.009与阴性对照组0.162±0.028相比,差异有显著性(P<0.05或P<0.01),增生率分别为10.5﹪,30.2﹪,28.3﹪,11.1﹪,其中浓度为20μg/L时具有最大促增生效应;(2)20μg/L的HGF作用时间为12h,24h时与阴性对照组相比,差异有显著性(P<0.05或P<0.01)。HGF对hLECs促增生效应在一定范围内呈剂量-时间依赖关系。3.流式细胞仪分析:20μg/L的HGF作用24小时后,与阴性对照组相比,实验组hLECs细胞周期发生明显改变,差异有显著性(P<0.05),表现为G0/G1期细胞显著减少,S期和G2/M期细胞增多。4.细胞损伤愈合模型:HGF浓度为10,20,50μg/L时,24h后移行的细胞数分别为10.583±1.594,13.667±1.080,26.750±2.092与阴性对照组8.667±1.080相比,差异有显著性(P<0.05或P<0.01),其移行能力分别为22.1%,57.7%,208.6%。HGF对hLECs促移行效应呈剂量依赖关系。
【结论】HGF可促进hLECs的增生和移行,是hLECs的有丝分裂原和强有力的促移行因子,参与PCO的发生与发展。
【Objective】To investigate the effect of hepatocyte growth factor(HGF)on proliferation and migration of cultured human lens epithelial cells(hLECs) and study the possible mechanism of HGF in posterior capsule opacification(PCO) while to explore a new possibility for anti-PCO therapy.
【Methods】1.hLECs were cultured and sub-cultured. After identified with the shape and immunohistochemical technique, the cells from the third to fifth generation were used in this experiment. 2.MTT assay was used to detect the proliferation of hLECs:(1)The third generation of hLECs were treated with different concentrations of HGF(2.5,5,10,20,40,50,100μg/L),and the growth status of hLECs was analyzed after 24h using MTT;(2) hLECs were treated with 20μg/L of HGF which induced the maximal proliferation of the cells, and the growth status of hLECs was analyzed after different time(6,12,24,48h) using MTT. 3.FCM was used to detect the cell growth cycle: hLECs were treated with 20μg/L of HGF which induced the maximal proliferation of the cells, and the changes of the growth cycle of the cells were detected after 24h using FCM. 4. an in vitro wound-healing model was used to detect the migration of hLECs: A denuded area was made when hLECs cultured in 6-well-plates were confluent into a monolayer. hLECs were then treated with different concentrations of HGF(10,20,50μg/L),and after 24 hours the cells having migrated into the denuded area were enumerated under light microscope. 5. All figures were transformed to data by HMIAS-2000 system and analyzed by SPSS13.0 to study the effect of HGF on proliferation and migration in cultured hLECs.
【Result】1. According to growth characteristics, morphology features and staining features (vimentin and keratin) determine cells were hLECs. 2.MTT assay:The proliferation number of hLECs was 0.179±0.011,0.211±0.010,0.208±0.007 and 0.180±0.009 respectively at the concentration of 10,20,40 and 50μg/L of HGF, showing a significantly increased proliferation in comparison with the negative control group (0.162±0.028)(P<0.05或P<0.01),and the proliferation rate was respectively 10.5﹪,30.2﹪,28.3﹪,11.1﹪.At the concentration of 20μg/L, HGF induced the maximal increase of proliferation rate(P<0.01)after 24 hours. HGF promoted the growth of hLECs in a dose-and–time dependent manner. 3.FCM analysis: There was significant difference of the cell cycle between the negative control group and the HGF group after 24 hours (P<0.05). Showing that in the HGF group the G0/G1-phase cells decreased while S + G2/M-phase cells increased. 4.The wound-healing model: The number of migrated hLECs was10.583±1.594,13.667±1.080 and 26.750±2.092 respectively at the concentration of 10,20 and 50μg/L of HGF after 24 hours, showing a significantly migration in comparison with the negative control group (8.667±1.080)(P<0.05或P<0.01),and the migration ability was increased by 22.1%,57.7%,208.6% respectively. HGF promoted migration of hLECs in a dose dependent manner.
【Conclusion】HGF can induce the proliferation and obvious migration of hLECs, HGF was a mitogen and potent migratory factor of hLECs.
引文
1. Arita T, Murata Y, Lin LR, Tsuji T, Reddy VN. Synthesis of lens capsule in long-term culture of human lens epithelial cells[J]. Loves, 1993;34(2):355-362.
2. Nishi O, Nishi K, Fujiwara T, Shirasawa E. Types of collagen synthesized by lens epithelial cells of human cataracts[J]. Br J Ophthalmol, 1995;79(10):939-943.
3. Zarnegar R, Michalopoulos GK. The many faces of hepatocyte growth factor: from hematopoiesis to hematopoiesis[J]. Cell Biol, 1995;129(5):1177–1180.
4.黄玲,惠延年,韩泉洪,等.肝细胞生长因子对培养的视网膜色素上皮细胞移行及增生的作用[J].中华眼底病杂志,2001;17(4):307-310.
5. Wilson SE, He YG, Weng J, et al. Effect of epithelial growth factor, hepatocyte growth factor, keratinocyte growth factor on the motility, differentiation of human corneal epithelial cells[J]. Exp Eye Res,1994;59:665-678.
6. Wordinger RJ, Clark AF, Agarwal R, et al. Cultured human trabecular meshwork cells express functional growth factor receptors[J]. Invest Ophthalmol Vis Sci, 1998;39(9): 1575 -1589.
7. Umeda N, Ozaki H, Hayashi H, et al. Non-paralleled increase of hepatocyte growth factor and vascular endothelial growth factor in the eye with angiogenic and nonangiogenic fibroproliferation[J]. Ophthalmic Res,2002;34(1):43-47.
8.鄂征,主编.组织培养与分子学生物技术(第1版).北京:北京出版社.1995,50.
9.司徒镇强,主编.细胞培养.修订版,西安:世界图书出版公司.2004,44.
10.刘小蓉,李进.晶状体上皮细胞的体外培养方法的改良.解剖科学进展,1997;3(3):228.
11.司徒镇强,主编.细胞培养.修订版,西安:世界图书出版公司.2004,189.
12.梁皓,赵军,谭少健,等.去整合素对人眼晶状体上皮细胞的粘附移行作用[J].眼科新进展,2006;26(4):262-264.
13. Wormstone IM, Tamiya S, Marcantonio JM, et al. Hepatocyte growth factor function and c-Met expression in human lens epithelial cells[J]. Invest Ophthalmol Vis Sci,2000;41 (13): 4216–4222.
14.黄瑶,李立,汤永强.肝细胞生长因子对牛视网膜血管内皮细胞的促增生移行效应[J].眼科研究,2006;24(1):50-53.
15. Sunderlin K, Sjostrand J. Posterior capsule opacification 5 years after extracapsular cataract extraction[J]. Cataract Refract Surg,1999;25(2):246-250.
16. Zeng Q, Chen S, You Z, et al. Hepatocyte growth factor inhibits anoikis in head and neck squamous cell carcinoma cells by activation of ERK and Akt signaling independent of NF-kappa B[J]. Biol Chem, 2002;277(28):25203-25208.
17. Maggiora P, Gambarrotta G, Olivero M, et al. Control of invasive growth by the HGF receptor family[J]. Cell Physiol, 1997;173(2):183-186.
18. Parr C, Hiscox S, Nakamura T, et al. Nk4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invaision of colon cancer cell[J]. Int J Cancer, 2000; 85(4):563-570.
19. Bardelli A, Longati P, Willams TA, et al. A peptide representing the carboxy-terminal tail of the met receptor inhibits kinase activity and invasive growth[J]. Biol Chem, 1999; 274(41):29274-29281.
20. Nakamura Y, Morishita R, Nakamura S, et al. A vascular modulator, hepatocyte growth factor is associated with systolic pressure[J]. Hypertension,1996:28:409-413.
21. Rosen EM, Lamszus K, Laterra J, et al. HGF/SF in angiogenesis[J]. Ciba Found Symp, 1997;212:215-226.
22. Wilson SE, Weng J, Chwang EL, et al. Hepatocyte growth factor, keratinocyte growth factor, their receptors, fibroblast growth factor receptor-2, and the cells of the cornea[J]. Inwest Ophthalmol Vis Sci, 1993;34(8): 2544-2561.
23.黄玲,惠延年.肝细胞因子的眼组织表达及作用.国外医学眼科学分册,2000;24(4):236-240.
24. Wormstone IM, Liu CS, Rakic JM, et al. Human lens epithelial cell proliferation in a protein-free medium[J]. Invest Ophthalmol Vis Sci,1997;38(2):396–404.
25. Choi J, Park SY, Joo CK, et al. Hepatocyte growth factor induces proliferation of lens epithelial cells through activation of ERK1/2 and JNK/SAPK[J]. Invest Ophthalmol Vis Sci,2004;45(8):2696-2704.
26. Weng J, Liang Q, Mohan RR, Li Q, Wilson SE. Hepatocyte growth factor, keratinocyte growth factor, and other growth factor-receptor systems in the lens[J]. Invest Ophthalmol Vis Sci, 1997;38(8):1543-1554.
27. Wormstone IM, Tamiya S, Marcantonio JM, Reddan JR. Hepatocyte growth factor function and c-met expression in human lens epithelial cells[J]. Invest Ophthalmol Vis Sci,2000;41(13):4216-4222.
1. Lois N, Dawson R, McKinnon AD, et al. A new model of PCO in rodents[ J ]. Invest Ophthalmol Vis Sci, 2003;44 (8) :3450～3457.
2. Meacock WR, Spalton DJ , Boyce J, et al. The effect of PCO on visual function[ J ]. Invest Ophthalmol Vis Sci, 2003;44 (11) :4665～4669.
3. Sunderlin K, Sjostrand J. Posterior capsule opacification 5 years after extracapsular cataract extraction[J] .Cataract Refract Surg,1999;25(2):246-250.
4. McDonnell PJ, Green WR, Manmenee AE, et al. Pathology of intraocular lenses in 33 eyes examined postmortem[J].Ophthalmology,1983,90:386-403.
5. Kurosaka D, Kato K, Nagamoto T. Presence of alpha smooth muscle actin in lens epithelial cells of aphakic rabbit eyes[J].Br J Ophthalmol, 1996;80(10):906-910.
6. Sanders DR, Kraff MC, Lieberman HL, et al. Breakdown and reestablishment of blood-aqueous barrier with implant surgery [J]. Arch Ophthalmol, 1982; 100:588-593.
7. Ayaki M, Ishida Y, Nishimura E, et al. Lens epithelial migration between posterior capsule and intraocular lens with various finished posterior optic edge and hepatic angulations [J] .Ophthalmic Res, 2003; 35 (5) : 261～267.
8. Vargas LG, Izak AM, Apple DJ, et al. Implantation of a single piece, hydrophilic, acrylic, minus power foldable posterior chamber IOL in a rabbit model: clinic pathologic study of PCO [J] .Cataract Refract Surg, 2003;29 (8) :1613～1620.
9. Kato K ,Kurosaka D, Nugamoto T. Apoptotic cell death in rabbit lens after lens extraction[J]. Invest Ophthalmol Vis Sci,1997;38(11):2322-2330.
10. Saika S, Kawashima Y. Immunolocalization of prolyl-4-hydroxylase in rabbit lens epithelial cells[J].Cataract Refract Surg, 1998;24:1261-1265.
11. Saika S, Kawashima Y. Immunolocalization of prolyl-4-hydroxylase subunits, a-smooth muscle action, and extracelluar matrix components in human lens capsules with lens implants[J]. Exp Eye Res, 1998;66:283-284.
12.刘玉福,袁佳琴,王德文,等.碱性成纤维细胞生长因子受体mRNA在人晶体上皮细胞内的检测研究.中国实用眼科杂志,1999;17(5):265-267.
13.刘玉福,袁佳琴,王德文,等.碱性成纤维细胞生长因子在人晶体上皮细胞的蛋白检测研究.中国实用眼科杂志,1999;17(7):462-464.
14.孙朝晖,姚克,吴仁毅,等.碱性成纤维细胞生长因子和胰岛素样生长因子-1及其协同作用对牛晶体上皮细胞增殖的影响.中华眼科杂志,1999;35(6):462-464.
15.申屠形超,姚克,孙朝晖,等. bFGF在人眼组织中的表达及其对晶体上皮细胞增殖的影响.中华医学杂志(英文版), 2002;115(2):268-271.
16. Marcantonio JM, Rakic JM, Vrensen GF, et al. Lens cells populations studied in human donor capsular bags with implanted intraocular lenses[J]. Invest Ophthalmol Vis Sci, 2000;41(6):1130-1141.
17. Mcavoy JM. Evidence that fibroblast growth factor promotes lens fiber differentiation [ J ]. Development,1987;10:217-221.
18. Nishi K, Nishi O, Ohmoto Y. The synthesis of cytokines by human lens epithelial cells—interleukin-1(IL-1),tumor necrosis factor (TNF),interleukin-6(IL-6),and epidermal growth factor(EGF).Nippon Ganka Gakkai Zasshi, 1992;96(6):715-720.
19. Namiki M, Tagami Y, Yamamoto M, et al. Presence of human epidermal growth facto r(hEGF), basic fibroblast growth factor(bFGF) in human aqueous[J]. Nippon Ganka Gakkai Zasshi,1992;96(5):652-656.
20. Majima K. Effect of epidermal growth factor upon morphological changes of human lens epithelial cells. Ophthalmologica,1998;212(4):250-256.
21.陶津华,安小玲,孔郡,等. EGF体外对人晶状体上皮细胞迁移的影响.国际眼科杂志,2006;6(1):68-71.
22.刘冬玲.生长因子与后发性白内障.国外医学眼科学分册,1996;20(2):86-91.
23. Nishi O, Nishi K, Wada K, Ohmoto Y. Expression of transforming growth factor(TGF)-alpha,TGF-beta(2) and interleukin 8 messenger RNA in posturgical and cultured lens epithelial cells obtained from patients with senile cataracts[J]. Graefes Arch Clin Exp Ophthalmol, 1999;237(10):806-811.
24. Allen JB, Davidson MG, Nasisse MP, et al. The lens influences aqueous humor level of transforming growth factor-2[J]. Graefes Arch Clin Exp Ophthahnol,1998;236(2): 305- 311.
25. Liu J, Hales AM, Chamberlain CG, et al. Induction of cataract-like changes in rat lens epithelial explants by transforming growth factor beta[J]. Invest Ophthalmol Vis Sci,1994;35(3):388-401.
26. Liu J , Hales AM , Chamberlain CG. Induction of cataract-like changes in rat lens epithelial explants by transforming growth factor beta [J]. Invest Ophthalmol Vis Sci, 1994;34 (2) : 388-401.
27. Ignotz RA , Massague J. Cell adhesion protein receptors as targets for transforming growth factor-βaction [J].Cell ,1987;51:189-197.
28. Aslam TM, Aspinall P, Dhillon B. Posterior capsule morphology determinants of visual function[J]. Graefes Arch Clin Exp Ophthalmol, 2003; 241 (3) : 208～212.
29. Wunderlich K, Knorr M. Effect of platelet-derived growth factor PDGF on replication of cultivated bovine lens epithelial cells[J]. Ophthalmology, 1994;91(1):98-102.
30. Vargas LG, Escobar-Gomez M, Apple DJ, et al. Pharmacologic prevention of PCO: in vitro effects of preservative-free lidocaine on lens epithelial cells [J] . Cataract Refract Surg, 2003;29 (8) :1585～1592.
31. Ayaki M, Ishida Y, Nishimura E, et al. Lens epithelial migration between posterior capsule and intraocular lens with various finished posterior optic edge and hepatic angulation [J] .Ophthalmic Res, 2003;35 (5) : 261～267.
32. Kok A, Lovicu FJ, Chamberlain CG, McAvoy JVV. Influence of platelet-derived growth factor on lens epithelial cell proliferation and differentiation[J]. Growth Factors, 2002;20(1):27-34.
33.孙朝晖,姚克,吴仁毅,等.碱性成纤维细胞生长因子和胰岛素样生长因子-1及其协同作用对牛晶体上皮细胞增殖的影响.中华眼科杂志,1999;35(6):462-464.
34. Civil A, van Genesen ST, Klok EJ, Lubsen NH. Insulin and IGF-1 affect the protein composition of the lens fiber cell with possible consequences for cataract[J]. Exp Eye Res,2000;70(6):785-794
35. Mosmann T. Rapid colorimetric assay for cellular growth and Survival: application to proliferation and cytotoxicity assays [J]. Immunology Method, 1993; 65:55-58.
36. Kraff MC, Sanders DR. Inhibition of blood-aqueous humor barrier breakdown with diclofenac [J]. Arch Ophthalmology, 1990;108:380-383.
37. Wormstone IM, Tamiya S, Marcantonio JM, Reddan JR. Hepatocyte growth factor function and c-met expression in human lens epithelial cells[J]. Invest Ophthalmol Vis Sci,2000;41(13):4216-4222.
38. Weng J, Liang Q, Mohan RR, Li Q,Wilson SE. Hepatocyte growth factor, keratinocyte growth factor, and other growth factor-receptor systems in the lens[J]. Invest Ophthalmol Vis Sci, 1997;38(8):1543-1554.
39.黄玲.肝细胞生长因子的眼组织表达及作用.国外医学眼科学分册,2000;24(4):236 -240.
40.莫宾,刘武.结缔组织生长因子与眼病.国外医学眼科学分册,2005;29(2):128-131.
41. Nishi O, Nishi K, Ohmoto Y. Synthesis of interleukin-1, interleukin-6, and basic fibroblast growth factor by human cataract lens epithelial cells[J]. Cataract Refract Surg,1996;22(1):852-858.
42. Nishi O, Nishi K, Fujiwara T, Shirasawa E, Ohmoto Y. Effect of the cytokines on the proliferation and collagen synthesis by human cataract lens epithelial cells[J]. Br J Ophthalmol, 1996;80(1):63-68.
43. Aslam TM, Aspinall P, Dhillon B. Posterior capsule morphology determinants of visual function [J] .Graefes Arch Clin Exp Ophthalmol, 2003; 241 (3) : 208～212.
44. Latina MA , Belmonte SJ . Gamma interferon effects on human fibroblasts from Tennon’s capsule [J]. Invest Ophthalmol Vis Sci, 1991; 32:2806-2815.
45. Granstein RD, Rook A. A controlled trial of intralesional recombinant interferon -gamma in the treatment of keloidal scarring :clinical and histological findings[J]. Arch Dermatal, 1990;126:1295-1302.
46. Findl O, Buehl W, Siegl H, et al. Removal of reflections in the photographic assessment of PCO by fusion of digital retro illumination images[J]. Invest Ophthalmol Vis Sci, 2003; 44 (1) : 275～280.
47.张晓红,李筱荣.肿瘤坏死因子和表皮生长因子对晶状体上皮细胞增殖的作用[J].中华眼科杂志,1998;16(3):90-92.
48.张凤妍,陆道炎,王丽天,等. IL-1和TNF-α对晶体上皮细胞增殖及胞浆内游离Ca2+浓度的影响.中华眼科杂志,1998;34(2):106-108.
49. Wang E, Zhao M, Forrester JV, et al. Electric fields and MAP kinase signaling can regulate early wound healing in lens epithelial [J].Invest Ophthalmol Vis Sci, 2003; 44 (1) : 244～249.