【作者】 孙正太；

【导师】 张晓峰；

【作者基本信息】 苏州大学 ， 眼科学， 2012， 硕士

【摘要】 目的：研究聚乙二醇400对体外培养人角膜上皮细胞生长的影响及其分子机制。方法：制备含0.1%聚乙二醇400的DMEM/F12培养基培养角膜上皮细胞，无聚乙二醇400的DMEM/F12培养基作为对照组。倒置显微镜观察并照相记录角膜上皮细胞培养24、48、72h时形态；划痕实验检测角膜上皮细胞迁移能力；四甲基偶氮唑蓝法检测角膜上皮细胞生长曲线；流式细胞仪检测角膜上皮细胞细胞周期；RT-PCR检测角膜上皮细胞中bFGF、EGF基因表达水平。结果：0.1%聚乙二醇400对角膜上皮细胞细胞形态无明显影响，实验组及正常对照组细胞形态均呈典型的多角形。划痕实验中，0.1%聚乙二醇400可以提高角膜上皮细胞迁移能力。MTT提示：0.1%聚乙二醇400对体外培养人CECs的生长有促进作用（P=0.0072）。细胞周期结果显示：0.1%聚乙二醇400体外培养人CECs后细胞增殖指数较对照组明显增高（P=0.0034）。0.1%聚乙二醇400培养人CECs后bFGF和EGF的基因表达水平比对照组高（P<0.05；P<0.01）。结论：聚乙二醇400可缩短人CECs的细胞周期，上调人CECs bFGF、EGF基因的表达，加速CECs增殖。目的：研究0.4%聚乙二醇滴眼液对小鼠角膜上皮机械损伤愈合的影响。方法：选择昆明小鼠30只，实验分为0.4%聚乙二醇滴眼液组、生理盐水组、空白对照组，每组各10只昆明小鼠。用角膜上皮刮匙刮除整个角膜上皮构建角膜上皮机械损伤动物模型，分别于建模后6h、12h、24h、48h及72h裂隙灯下拍照观察角膜上皮愈合情况。结果：角膜上皮机械损伤6h时，各实验组有效率均为0。12h时，0.4%聚乙二醇滴眼液组有效率为75%，空白对照组和生理盐水组均为0，0.4%聚乙二醇滴眼液组促进上皮愈合有效率明显高于生理盐水组和空白对照组（P=0.001）。24h时，0.4%聚乙二醇滴眼液组有效率为100%，空白对照组和生理盐水组均为16.7%，0.4%聚乙二醇滴眼液组上皮愈合有效率明显高于生理盐水组和空白对照组（P=0.001）。48、72h时，各实验组有效率均为100%。结论：0.4%聚乙二醇滴眼液能促进角膜上皮机械损伤修复的速度。目的：研究0.4%聚乙二醇滴眼液对小鼠角膜20%乙醇灼伤愈合的影响。方法：选择昆明小鼠30只，实验分为0.4%聚乙二醇滴眼液组、生理盐水组、空白对照组，每组各10只昆明小鼠。用沾有20%乙醇的滤片灼伤小鼠角膜构建角膜灼伤动物模型,分别在建模后1d、3d、7d处死各实验组中昆明小鼠3只，摘除眼球，角膜行HE染色以及连接黏附分子、成纤维细胞生长因子、表皮生长因子免疫组织化学研究。结果：20%乙醇灼伤角膜1d，0.4%聚乙二醇滴眼液组角膜组织中bFGF、EGF的表达较生理盐水组、空白对照组均增高（F=29.853，P=0.001；F=123.965，P=0.000），JAM的表达不增高（F=0.735，P=0.589）；20%乙醇灼伤角膜3d，各实验组角膜组织中bFGF、EGF、JAM的表达无明显差异（F=3.694，P=0.062；F=1.639，P=0.256；F=1.307，P=0.332）；20%乙醇灼伤角膜7d，各实验组角膜组织中bFGF、EGF、JAM的表达无明显差异（F=0.644，P=0.608；F=0.413，P=0.679；F=0.207，P=0.929）。结论：0.4%聚乙二醇滴眼液在20%乙醇灼伤早期能上调EGF和bFGF的表达，但对JAM的表达无明显影响。更多还原

【Abstract】 Objective To investigate the effects of polyethylene glycol400on the biologicalcharacteristics of corneal epithelial cells (CECs) and molecular mechanisms thereof.Methods Polyethylene glycol400was diluted to0.1%with DMEM/F12medium.HCECs were cultured by these medium. DMEM/F12medium without polyethylene glycol400was control group. The effects were tested by four ways. Scarification was used to testthe ability of cell migration; the MTT assay and flow cytometer were respectively used totest the effect on growth curve and cell cycle; RT-PCR was used to measure the expressionof basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in Humancorneal epithelial cells (HCECs).Result0.1%Polyethylene glycol400has no discernable effects in the morphologyof CECs. Cell migration of CECs was improved by0.1%Polyethylene glycol400. CECscultured in the dilution of polyethylene glycol400solutions and cell cyle measurementsshow that0.1%polyethylene glycol400promotes in vitro HCEC growth(P=0.0072;P=0.0034) and up-regulates the expression of bFGF and EGF in cultured CECs(P<0.05;P<0.01).Conclusion Polyethylene glycol400does not affect cell morphology and it promotesCEC proliferation via reducing cell cycle and increasing bFGF and EGF expression. Objective To investigate the effects of0.4%polyethylene glycol eye drop on thehealing of mechanical wound corneal epithelium of mice. Methods30KM mice were selected for this test. Experiment contained three groupsincluded0.4%polyethylene glycol eye drop group, NS group and control group. And eachgroup had10KM mice.we built the structure of defection of corneal epithelium by curette.We observed corneal healing from shooting under slit lamp after6h,12h,24h,48h and72h.Result The valid rate of all groups were zero in6h. In12h this rate of0.4%polyethylene glycol eye drop group was75%and the other groups were still zero, the rateof0.4%polyethylene glycol eye drop group became100%and the other groups were both16.7%, both differeces of these times had statistical meaning(P=0.001; P=0.001). And in48and72h all of groups were100%.Conclusion0.4%polyethylene glycol eye drop could speed repair of cornealepithelium after injury. Objective To investigate the effects of0.4%polyethylene glycol eye drop on thehealing of burning of corneal epithelium with20%alcohol.Methods30KM mice were selected for this test. Experiment contained three groupsincluded0.4%polyethylene glycol eye drop group, NS group and control group. And eachgroup had10KM mice.We built the structure of burning of corneal epithelium withfilter-paper contain20%alcohol. We executed three mice in each group after1d,3d and7d.We picked their eyes with HE stain and immunohistochemistry in JAM, bFGF andEGF.Result The expression of bFGF and EGF in the group of0.4%polyethylene glycoleye drop was more higher than the group of NS and control group after wound in one day.The difference had statistical meaning(F=29.853, P=0.001; F=123.965, P=0.000). Theexpression of JAM had no difference among three groups(F=0.735, P=0.589). After woundin three days the expression of bFGF, EGF and JAM all had no difference among threegroups(F=3.694, P=0.062; F=1.639, P=0.256; F=1.307,P=0.332) as same as in seven days(F=0.644, P=0.608; F=0.413, P=0.679; F=0.207, P=0.929).Conclusion It could up-regulate the expression of bFGF and EGF after20%alcoholburning. But it had no effects on the expression on JAM.更多还原