胸腺素β4对体外培养的人晶状体上皮细胞增殖、迁移及上皮间质转分化的影响

发布时间：2018-04-12 00:32

  本文选题：胸腺素β4 + 后发性白内障　； 参考：《华中科技大学》2013年硕士论文

【摘要】：目的：后囊混浊（Posteriorcapsuleopaciftcation，PCO）主要由术后残留的晶状体上皮细胞（lensepithelialcells，LECs）的增殖、迁移及上皮间质转分化（epithelial mesenchymaltransition，EMT）引起。胸腺素β4（thymosinβ4，Tβ4）是一种重要的肌动蛋白耦联蛋白，参与介导多种生物学反应。本实验通过研究Tβ4对体外培养的人晶体上皮细胞增殖、迁移及上皮间质转分化的影响，探讨Tβ4在PCO防治中的可能作用。 方法 1.用MTT法检测Tβ4对LECs增殖的影响。用含不同浓度Tβ4（0ng/ml、1ng/ml、10ng/ml、100ng/ml、1000ng/ml）无血清培养基处理LECs，分别于24h、48h、72h行MTT检测，测量OD值。 2.细胞划痕实验及Transwell小室迁移模型观察Tβ4对LECs迁移的影响。 （1）划痕后细胞培养基中加入不同浓度Tβ4（0ng/ml、1ng/ml、10ng/ml、100ng/ml、1000ng/ml）的0.1%牛血清白蛋白DMEM培养基，分别于划痕后0h、24h、48h选取10个随机视野拍照记录划痕间距（前后拍照位置须一致），比较不同时间点不同浓度的平均迁移距离。 （2）种细胞于上室，下室为不同浓度Tβ4（0ng/ml、1ng/ml、10ng/ml、100ng/ml、1000ng/ml）的20%胎牛血清DMEM培养基，作用24h后比较不同浓度穿过小室的平均细胞数目。 3.实时定量PCR技术比较处理72h后Tβ4处理组和正常对照组中E-钙粘蛋白及α-SMAmRNA表达水平的变化以检测Tβ4对LECs的转分化作用的影响。 结果 1.与对照组相比，当浓度为100ng/ml、1000ng/ml时Tβ4能够明显促进LECs的增殖，差异有显著性(P0．05)。相同药物浓度组随着作用时间的延长促进作用加强，各时间点相互比较差异亦有显著性(P0．05)。 2.24h各浓度组（0~1000ng/ml）平均迁移距离分别为：329.6621±47.43715μm,354.2761±52.5730μm,453.5897±46.3792μm,499.6127±46.0270μm,555.4763±71.6129μm;48h各浓度组（0~1000ng/ml）平均迁移距离为：614.6647±97.2274μm,636.1453±649.7268μm,720.5249±41.9510μm,739.9166±85.3627μm,770.9152±62.3144μm。10ng/ml、100ng/ml、1000ng/ml处理组相对于对照组而言，差异有统计学意义（P＜0.05），且细胞迁移距离随时间和浓度递增。 Tβ4处理24h后，穿过小室的细胞数各组（0~1000ng/ml）分别为82±3个,109±6个,128±10个，163±5个,204±11个。10ng/ml、100ng/ml、1000ng/ml处理组相对于对照组而言，差异有统计学意义（P＜0.05），且细胞迁移能力随浓度递增。 3.Tβ_4加药处理72h后LECs表达α-SMA、E-钙粘蛋白mRNA水平的变化：加药处理后，α-SMA表达量升高（151.7±5.0）%，E-钙粘蛋白表达量下降(68.0±8.1)%，差异有统计学意义（P＜0.05）。 结论：胸腺素β4可促进人晶状体上皮细胞增殖、迁移及上皮间质转分化，其在PCO防治中的可能作用值得作进一步研究。
[Abstract]:Aim: the posterior capsular opacification (PCOs) is mainly caused by the proliferation, migration and epithelial mesenchymal transition of lens epithelial cells (LECs).Thymosin 尾 4(thymosin 尾 4 (T 尾 4) is an important actin coupling protein involved in many biological responses.In this study, we studied the effects of T 尾 4 on the proliferation, migration and epithelial stromal transdifferentiation of cultured human lens epithelial cells in vitro, and explored the possible role of T 尾 4 in the prevention and treatment of PCO.Method1.The effect of T 尾 4 on the proliferation of LECs was detected by MTT assay.LECs were treated with 10ng / ml 10ng / ml 10ng / ml 10ng / ml 10ng / ml 10ng / ml 10ng / ml 10 ng / ml 10 ng / ml 10 ng / ml T 尾 4 / ml 0 ng / ml of different concentrations of T 尾 4mg / ml respectively. The MTT was measured at 24 h / 48h / 72 h, respectively, and OD value was measured.2.The effect of T 尾 4 on LECs migration was observed by cell scratch test and Transwell compartment migration model.After scratch, 0.1% bovine serum albumin (BSA) DMEM medium was added to the cell culture medium with different concentrations of T 尾 4 0 ng / ml 1 ng / ml 10 ng / ml 10 ng / ml 10 ng / ml 100 ng / ml 100 ng / ml 1 000 ng / ml.Ten random visual fields were selected to record the scratch spacing at 0 h 24 h and 48 h after scratch (the position before and after taking pictures should be consistent to compare the average migration distance of different concentrations at different time points.(2) A 20% fetal bovine serum DMEM medium with different concentrations of T 尾 4 0 ng / ml 1 ng / ml 1 ng / ml 10 ng / ml 10 ng / ml 10 ng / ml 100 ng / ml 100 ng / ml 1 000 ng / ml) was used to compare the average number of cells passing through the cell at different concentrations after 24 hours of exposure.3.The changes of Ecadherin and 伪 -SMA mRNA expression in T 尾 4 treated group and normal control group were compared by real time quantitative PCR technique in order to detect the effect of T 尾 4 on the transdifferentiation of LECs.Result1.Compared with the control group, T 尾 4 could significantly promote the proliferation of LECs when the concentration was 100ng / ml or 1000ng / ml, and the difference was significant (P 0.05).The effect of the same drug concentration group was enhanced with the prolongation of the action time, and there were significant differences among the different time points (P 0.05).2.24h鍚勬祿搴︾粍(0~1000ng/ml)骞冲潎杩佺Щ璺濈�诲垎鍒�涓，

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