【作者】 张光红；

【导师】 王智；

【作者基本信息】 华中科技大学 ， 眼科学， 2007， 硕士

【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。更多还原

【Abstract】 【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.更多还原