【作者】 陈博；

【导师】 胡义珍；

【作者基本信息】 华中科技大学 ， 眼科学， 2009， 博士

【摘要】 目前白内障的主要治疗方法是手术治疗，通过手术能够获得良好的术后视力。然而后发性白内障（after cataract）（主要表现在后囊膜混浊（posterior capsule opacification，PCO））作为白内障术后最常见的并发症会导致很大一部分患者术后视力的下降，这样需要进一步的激光手术治疗，但有一定风险。白内障术后，残留的晶状体体上皮细胞（lens epithelial cells，LECs）转分化为肌成纤维细胞（myofibroblast），增殖、移行到位于光学通路上的晶体后囊膜表面，并引起后囊发生皱缩。转分化的LECs表达myofibroblast的表型标志α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）；大量异常的细胞外基质（extracellular materials，ECM），例如纤维连接蛋白（fibronectin，FN）、（Ⅰ型胶原collagen-Ⅰ，CA-Ⅰ）等聚集于转分化细胞周围，最终导致PCO的发生。CTGF（connective tissue growth factor，CTGF）是一大小为36～38kDa的富含半胱氨酸多肽生长因子，第一次在人的脐带内皮细胞中发现。作为转化生长因子-β（transforming growth factor-β，TGF-β）的下游调控因子，在细胞增殖、细胞移行、细胞外基质合成、细胞表型转化等方面发挥重要作用。研究表明，CTGF能够上调肺成纤维细胞以及血管平滑肌细胞中CA-Ⅰ以及FN的表达。CTGF的过量表达能够引起肺以及肾的纤维化。同时，有报道证实CTGF在PCO的发生中起着一定作用。因此为了进一步研究CTGF在PCO中的作用，我们进行了本项试验工作，共分为四部分。第一部分CTGF对体外培养牛眼晶体上皮细胞转分化及迁移的作用研究目的观察结缔组织生长因子（connective tissue growth factor，CTGF）在体外培养牛眼晶体上皮细胞（bovine lens epithelial cells，BLECs）转分化和迁移中发挥的作用。方法将体外培养的第2～3代牛眼晶体上皮细胞分为对照组和实验组，实验组分别经0.1×10⁶ng·L^-1、0.5×10⁶ng·L^-1、1.0×10⁶ng·L^-1CTGF处理24h后，采用半定量RT-PCR和Western blotting技术检测α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）mRNA以及蛋白表达的变化。迁移实验中，利用transwell小室对晶体细胞的移行能力进行检测。结果与空白对照组相比，CTGF能够明显促进α-SMA mRNA以及蛋白表达（P＜0.01），并且呈现剂量依赖性。迁移实验发现，CTGF能够促进晶体上皮细胞的移行，且差异有显著意义（P＜0.01）。结论CTGF作为TGF-β的下游调节因子，可能在后囊膜混浊（posterior capsuleopacification，PCO）中晶状体上皮细胞移行、转分化以及细胞外基质沉积的过程中发挥重要的作用。第二部分CTGF SiRNA转染体外培养牛眼晶体上皮细胞的研究目的观察结缔组织生长因子（connective tissue growth factor，CTGF）干扰RNA（shortinterfering RNA，SiRNA）对牛眼晶体上皮细胞（bovine lens epithelial cells，BLECs）的CTGF、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）及纤维连接蛋白（fibronectin，FN）表达的影响。方法将体外培养的牛眼晶体上皮细胞分为空白对照组、阴性对照组、刺激组、SiRNA₁组、SiRNA₂组共5组，各实验组先经50nm·L^-1浓度的SiRNA处理后再加入转化生长因子-β₁（transforming growth factor-β，TGF-β₁），然后采用实时荧光定量PCR（Real-Time PCR）检测FN、α-SMA和CTGF mRNA的表达，同时采用Western blotting方法分别检测相应蛋白的表达。结果Real-Time PCR结果显示，实验组FN、α-SMA和CTGF mRNA的表达与刺激组相比均有不同程度的降低，差异有统计学意义（P＜0.05）。Western blotting结果显示，与刺激组相比，SiRNA对FN、α-SMA和CTGF表达有明显的抑制作用，差异有统计学意义（P＜0.05）。结论CTGF SiRNA能够明显下调其目标蛋白的表达，并可以抑制α-SMA和细胞外基质的表达。CTGF作为后囊膜混浊（posterior capsule opacification，PCO）中晶体上皮细胞移行、转分化以及细胞外基质沉积的过程中发挥一定作用的细胞因子，可能可以通过干扰CTGF的表达来预防PCO的发生。第三部分CTGF在牛眼晶体上皮细胞中促进FN表达的机制研究目的观察JNK和PI3K／Akt信号通路在牛眼晶体上皮细胞（bovine lens epithelial cells，BLECs）中结缔组织生长因子（connective tissue growth factor，CTGF）促进纤维连接蛋白（fibronectin，FN）表达的作用研究。方法取2～3代细胞分别加入JNK抑制剂SP-600125、Akt抑制剂LY-294002、JNKSiRNA和Akt SiRNA，之后加入CTGF（1μg／ml）孵育24h。采用Western blotting技术检测FN、p-JNK和p-Akt蛋白表达的变化。结果SP-600125和JNK SiRNA能够抑制CTGF诱导FN的表达（P＜0.05）。然而LY-294002和Akt SiRNA对CTGF诱导FN的表达没有影响（P＞0.05）。结论CTGF在BLECs中诱导FN的表达是通过JNK信号通路介导的，然而Akt信号通路没有影响。这样对CTGF诱导后囊膜混浊发生机制有了更进一步认识。第四部分大鼠PCO模型中CTGF和α-SMA表达的研究目的后囊膜混浊（posterior capsule opacification，PCO）作为后发性白内障（aftercataract）的主要表现形式，是白内障术后最常见的并发症，其严重影响术后视力。本部分主要研究大鼠后发性白内障模型形成过程中结缔组织生长因子（connective tissuegrowth factor，CTGF）和α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）的表达变化及其关系。方法对50只Sprague Dawley（SD）大鼠行晶状体囊外摘除术（extracapsular lensextraction，ECLE），术后不同时间点进行裂隙灯检测，western blot法检测各时间点后囊膜CTGF和α-SMA表达。结果通过光镜的检查，在大鼠的PCO模型中可以观察到典型的PCO病理特征。在PCO的发展过程中，CTGF和α-SMA表达发生了变化。α-SMA作为成纤维母细胞的标志，于术后0小时几乎检测不到，3天可检测到表达，7天达到高峰，14天下降，28天明显下降。这一变化说明了晶体上皮细胞（lens epithelial cells，LECs）发生了转分化。CTGF作为转化生长因子-β（transforming growth factor-β，TGF-β）下游的重要调节因子，与α-SMA具有同样的表达趋势。回归分析显示，α-SMA的表达与CTGF的表达密切相关（R²=0.893，P＜0.05）。结论结果表明，CTGF可能参与了PCO模型中α-SMA表达的调节，同时α-SMA可以作为PCO的形成过程中晶体上皮细胞转分化的重要标志。更多还原

【Abstract】 Psterior capsule opacification （PCO） is the the most common complication of catraractsurgery. At present the only means of treating cataract is by surgical intervention, and thisinitially restores high visual quality. Unfortunately, PCO develops in a significantproportion of patients to such an extent that a secondary loss of vision occurs, whichconsequently required further corrective laser surgery which is not without risk.After cataract surgery, residual LECs transdifferentiate into myofibroblast, and oftenmigrate and proliferate on the posterior capsule, induce capsule wrinkling. Thetransdifferentiated cells expressα-smooth muscle actin （α-SMA） which is the mark ofmyofibroblast; abnormal extracellular materials （ECM）, including collagen-Ⅰ（CA-Ⅰ）and fibronectin （FN）, accumulate around the transdifferentiated cells, that will induce PCO.Connective tissue growth factor （CTGF） is a 36 to 38kDa cysteine-rich peptide, and isfirst identified from human umbilical endothelial cells. As the downstream regulatory factorof transforming growth factor-β（TGF-β）, CTGF could promote cells proliferation,migration, adhesion, and ECM production. As reported by previous research, CTGFupregulates the expression CA-Ⅰand FN in human lung fibroblasts and vascular smoothmuscle cells. Overproduction of CTGF therefore has been implicated as an importantpathway leading to lung fibrosis and kidney fibrosis. It has been reported that CTGF playsan important role in pathogenesis of PCO. In order to research the effect and possible mechanism of the action about the CTGF on the PCO, we are going to this reseach project,which includes four sections.PartⅠThe research of migration and transdifferentiation effects ofconnective tissue growth factor in bovine lens epithelial cellsObjective To observe the migration and transdifferentiation effects of connective tissuegrowth factor （CTGF） in bovine lens epithelial cells （BLECs）.Methods Cultured 2～3 passage BLECs were divided into control group and experimentalgroup with 0.1×10⁶ng·L^-1, 0.5×10⁶ng·L^-1, 1.0×10⁶ng·L^-1 CTGF for 24h, the mRNA andprotein ofα-smooth muscle actin （α-SMA） in BLECs were examined respectively bysemiquantitative RT-PCR and Western blotting. In migration experiment, transwell insertswere used to evaluate migration ability of BLECs.Results In comparison with control group, experimental group could promote theexpression ofα-SMA at mRNA and protein level with dose dependent obviously inBLECs （P＜0.01）. In migration experiment, CTGF could significantly promote BLECsmigration （P＜0.01）.Conclusion As the downstream regulatory factor of TGF-β, CTGF play an important rolein lens epithelial cells migration, transdifferentiation and deposition of ECM in posteriorcapsule opacification （PCO）.PartⅡEffect of connective tissue growth factor SiRNA inbovine lens epithelial cellsObjective To observe the effects of connective tissue growth factor （CTGF） SiRNA on the expression of CTGF、α-smooth muscle actin （α-SMA） and fibronectin（FN） in bovine lensepithelial cells （BLECs）.Methods Cultured 2～3 passage BLECs were divided into control group,TGF-β₁+negative SiRNA group, TGF-β₁ group, TGF-β₁+CTGF SiRNA₁ group andTGF-β₁+CTGF SiRNA₂ group. The experimental group was treated by 50nm·L^-1 SiRNA₁and SiRNA₂, and then incubation with 10.0×10³ng·L^-1 TGF-β₁. The mRNA and proteinexpression ofα-SMA, FN and CTGF in BLECs were examined respectively by Real-TimePCR and Western blotting.Results Real-Time PCR indicated that in comparison with TGF-β₁ group, mRNAexpression of FN,α-SMA and CTGF decreased in tranfection group （P＜0.05）. Westernblotting indicated that in comparison with TGF-β₁ group, CTGF SiRNA could inhibitedthe expression of FN,α-SMA and CTGF notablely （P＜0.05）.Conclusion The data suggested that CTGF SiRNA could inhibit the expression of CTGF,α-SMA and ECM. Because CTGF play an essential role in lens epithelial cells migration,transdifferentiation and deposition of ECM in posterior capsule opacification （PCO）, thatinterferring of CTGF could prevent PCO.PartⅢThe research of mechanism at connective tissue growth factorinduced fibronectin expression in bovine lens epithelial cellsObjective To observe the effect of JNK and PI3K/Akt on connective tissue growth factor（CTGF）-induced fibronectin （FN） expression in bovine lens epithelial cells （BLECs）.Methods The Cultured 2～3 passage BLECs were respectively treated by JNK inhibitorSP-600125, Akt inhibitor LY-294002, JNK SiRNA and Akt SiRNA, and then incubationwith 1μg/ml CTGF for 24h. The protein of FN, p-JNK and p-Akt were examined by western blotting.Results In the presence of JNK inhibitor （SP-600125） or transfected by JNK SiRNA,CTGF-induced protein expression of FN increase was completely inhibited （P＜0.05）. Aktinhibitor （LY-294002） and Akt SiRNA had no effect on CTGF-stimulated proteinexpression of FN （P＞0.05）.Conclusion FN expression which induced by CTGF is mediated through the JNK pathway,but not affected by PI3K/Akt pathway. From these, we could know more about theCTGF-induced PCO.PartⅣThe expression of connective tissue growth factor andα-smoothmuscle actin in rat posterior capsule opacification modelsObjective Posterior capsule opacification （PCO）, the major complication of cataractsurgery, would induce visual loss. In this study, the expression of connective tissue growthfactor （CTGF） andα-smooth muscle actin （α-SMA） in rat posterior capsule opacificationmodels has been studied.Methods An extracapsular lens extraction （ECLE）, was performed in 50 Sprague-Dawley（SD） adult male rats. The pathological characteristics of PCO were observed in rat modelswith slit lamp at each time point. The posterior capsule was taken out for examining theexpression of CTGF andα-SMA.Results A classical pathological characteristics of posterior capsule opacification could beobsrved in PCO models with slit lamp. In this process, we detected the expression ofα-SMA and CTGF in the posterior capsules of models,α-SMA, a special hallmark formyofibroblast, increased significantly at 3 day, and reached the peak level at 7 day aftersurgery. However, the expression decreased at 14 day after surgery, and further decreased at28 day. It demonstrated that the lens epithelial cells transdifferentiated into mesenchyme-like cells. CTGF, a major downstream regulatory factor of transforminggrowth factor-β（TGF-β）, also showed a significant increase after surgery. The regressionanalysis strongly indicated thatα-SMA expression was associated with CTGF expression（R²=0.893, P＜0.05）.Conclusions These results suggest that CTGF may be involved in regulatingα-SMAexpression, which is the important marker of transdifferentiation of lens epithelial cells inrat PCO models.更多还原