【作者】 孙传宾；

【导师】 姚克；

【作者基本信息】 浙江大学 ， 眼科学， 2012， 博士

【摘要】 第一部分：表面自组装TGF-β2抗体多层膜的新型人工晶状体研制及其理化性质和分子生物学指标检测目的：评价应用静电层层自组装技术在疏水性丙烯酸酯人工晶状体(IOL)表面构建TGF-β2抗体多层膜的可行性,并检测表面自组装TGF-β2抗体多层膜的新型IOL的理化性质和分子生物学指标。方法：首先应用大气压下辉光放电等离子体预处理疏水性丙烯酸酯IOL,使其表面带上足量负电荷。然后应用静电层层自组装技术在IOL表面逐层沉积聚乙烯亚胺(PEI)、TGF-β2抗体(anti-TGF-β2)和聚赖氨酸(PLL),从而在IOL表面构建PEI-(anti-TGF-β2/PLL)4-(anti-TGF-β2)多层膜。以石英晶体微天平(QCM)模拟并实时监测静电层层自组装过程,了解自组装膜的增长方式,自组装膜中每层沉积的TGF-β2抗体的平均质量、厚度和密度,以及自组装膜中TGF-β2抗体的免疫活性。以静态水接触角(WCA)检测新型IOL的亲水性。以X-射线光电子能谱分析(XPS)检测新型IOL表面化学元素组成变化。以场发射扫描电子显微镜(FESEM)和原子力显微镜(AFM)观察新型IOL的表面形态变化。应用SFDA关于IOL质量检测国家标准(YY-0290)对新型IOL进行光焦度、像质、光谱透过率和动态疲劳耐久性测试,以观察新型IOL的光学和力学特性有无改变。应用免疫荧光和激光扫描共焦显微镜观察新型IOL表面自组装膜的稳定性,以及自组装膜中TGF-β2抗体的免疫活性。结果：应用QCM模拟静电层层自组装过程,发现PEI、TGF-β2抗体和PLL可以线性增长的方式逐层沉积在IOL表面,自组装膜中每层沉积的TGF-β2抗体的平均质量、厚度和密度分别为(77.45±8.00)ng,(1.42±0.15)nm和(0.48±0.05)μg/cm2。QCM检查同时发现自组装膜中的TGF-β2抗体可以0.82：1的比例结合人重组TGF-β2因子,提示自组装膜中TGF-β2抗体保持良好的免疫活性,而且主要以侧面向上的方式分布。新型IOL的WCA为73.2°,较未处理IOL的WCA为92.1°明显降低,提示新型IOL的亲水性明显改善。XPS检查发现新型IOL表面化学成分中氧和氮元素含量明显升高,进一步证实TGF-β2抗体成功自组装到IOL表面。AFM检查发现新型IOL的表面粗糙度较未处理组IOL稍有增加,FESEM检查发现新型IOL表面形态光滑、平整,与未处理组IOL无差别。新型IOL的光焦度、像质、光谱透过率和动态疲劳耐久性测试均符合国家标准。免疫荧光和激光扫描共焦显微镜检查发现TGF-β2抗体多层膜可在新型IOL表面持续稳定存在至少3个月,自组装膜中TGF-β2抗体可以与外加TGF-β2因子结合,形成均匀、密集分布的免疫荧光沉积颗粒,提示自组装膜中TGF-β2抗体保持良好的免疫活性。结论：本研究应用大气压下辉光放电等离子体和静电层层自组装技术首次在IOL表面成功构建了TGF-β2抗体多层膜,TGF-β2抗体多层膜可以在IOL表面长期稳定存在,并保持良好的抗体免疫活性,而且对IOL表面形态、光学和力学特性无明显影响。第二部分：表面自组装TGF-β2抗体多层膜的新型人工晶状体抑制后发性白内障的研究目的：评价表面自组装TGF-β2抗体多层膜的新型IOL在体外和活体兔眼内预防后发性白内障的有效性和安全性。方法：将人晶状体上皮细胞(LECs)接种到表面自组装TGF-β2抗体多层膜的新型IOL和未处理IOL的后表面,LECs接种后6h、24h和48h,分别应用倒置相差显微镜观察LECs在实验组和对照组IOL表面的粘附和增殖情况。应用划痕实验观察并比较新型IOL和未处理IOL表面LECs在10ng/ml TGF-β2因子作用下的迁移情况。以a-SMA作为上皮-间质转分化标记蛋白,应用免疫荧光显微镜观察新型IOL和未处理IOL表面LECs在10ng/ml TGF-β2因子作用下的转分化情况。在活体兔眼内行晶状体超声乳化吸除并植入新型IOL(实验组)或未处理IOL(对照组),制备兔眼后发性白内障模型,在术后1d、3d、1w、1mon、3mon,应用裂隙灯显微镜观察实验组和对照组兔眼前房闪辉、前方细胞和虹膜后粘连程度等前房炎症反应情况,并按照相应评价标准进行评分；术后1mon和3mon应用裂隙灯后照法拍摄后囊膜照片,以自行设计的PCO评价软件测量并比较实验组和对照组兔眼的后囊膜浑浊(PCO)严重程度。术后3mon处死兔,以Miyake-Apple后照法拍照进行PCO评分。摘取兔术眼眼球,取出IOL,应用免疫荧光联合激光扫描共焦显微镜检查了解新型IOL表面TGF-β2抗体多层膜的完整性。将晶状体囊膜以石蜡包埋切片后行HE染色,观察囊膜表面LECs增殖和细胞外基质沉积情况；同时应用免疫组织化学法观察a-SMA、 Collagen Ⅰ和Collagen Ⅲ在囊膜表面的表达情况。结果：表面自组装TGF-β2抗体多层膜的新型IOL可以短暂(细胞接种后6h内)抑制LECs的粘附,但最终对LECs的粘附和增殖无明显影响。划痕实验发现LECs在新型IOL表面的迁移能力较在未处理IOL表面明显下降。免疫荧光显微镜检查发现新型IOL表面LECs可以保持上皮细胞形态,而且a-SMA表达很少；而未处理IOL表面LECs呈纤维细胞形态,而且大量表达a-SMA.直接免疫荧光联合激光扫描共焦显微镜检查显示,IOL表面的TGF-β2抗体多层膜在活体兔眼内可以较长时间稳定存在。兔眼后发性白内障模型研究发现,除术后1w和1mon时实验组的前房闪辉和前房细胞较对照组有所减轻外,两组之间的术后前房炎症反应无明显差别。裂隙灯后照法与Miyake-Apple后照法PCO评分均显示对照组后囊膜PCO评分显著高于实验组。HE染色显示实验组囊膜较薄,表面LECs数量较对照组明显为少。免疫组织化学检查发现实验组囊膜表面a-SMA和Collagen I表达量较对照组明显为少。结论：表面自组装TGF-β2抗体多层膜的新型IOL可以显著抑制晶状体后囊膜浑浊和纤维化,而且眼内使用的安全性良好。更多还原

【Abstract】 Part I Fabrication of TGF-β2antibody multilayers on acrylic intraocular lens surfaces by layer-by-layer self assembly techniqueObjective:To fabricate TGF-β2antibody (anti-TGF-β2) multilayers on the surfaces of hydrophobic acrylic intraocular lens (10L) by layer-by-layer self assembly (LBL) technique, and test the physico-chemical and molecular biological properties of the surface modified IOL.Methods:Hydrophobic acrylic IOL was pretreated by atmospheric pressure glow discharge plasma, then polyethylenimine (PEI), anti-TGF-(32and poly (L-lysine)(PLL) were sequentially deposited onto IOL surfaces by LBL technique until multilayers of PEI-(anti-TGF-β2/PLL)4-(anti-TGF-β2) was completed. Quartz Crystal Microbalance (QCM) was used to monitor assembly process of anti-TGF-β2multilayers, and test the immunological activity of the deposited anti-TGF-β2on IOL surfaces, as well as evaluate the orientational aspect of anti-TGF-β2immobilized onto IOL. Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS) were used to test the hydrophilicity and surface element changes of the surface modified IOL. Atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM) were used to characterize the surface roughness of the surface modified IOL. The optical and mechanical properties of surface modified IOL were tested on the basis of National Quality Test Standard of IOL (YY-0290). Immunofluorescence combined with laser scanning confocal microscopy (LSCM) was used to test the long-term stability of anti-TGF-β2multilayers and immunological activity of the immobilized anti-TGF-(32on IOL surfaces.Results:QCM showed linear growth of the multilayers when PEI, anti-TGF-(32and PLL sequentially deposited onto IOL surfaces, and confirmed good immunological activity of the immobilized anti-TGF-β2on IOL surfaces. QCM also revealed that mean mass, thickness, and density of anti-TGF-β2monolayer was (77.45±8.00) ng,(1.42±0.15) nm, and (0.48±0.05)μg/cm2, respectively, which confirmed that anti-TGF-β2was deposited on the IOL surfaces with a side-on orientation in the multilayers. WCA revealed an improved hydrophilicity of surface modified IOL. XPS revealed an significantly elevated percentage of Oxygen and Nitrogen element on the surface of surface modified IOL. AFM revealed a slightly elevated roughness of surface modified IOL. FESEM showed no difference in smoothness between surface modified10and control IOL. The optical and mechanical properties of surface modified IOL were proved to be qualified by National Quality Test Standard of IOL. Immunofluorescence combined with LSCM revealed that anti-TGF-β2multilayers could keep stable on IOL surfaces for at least3months, and that the immobilized anti-TGF-β2on surface modified IOL kept good immunological activity, based on the fact that it could combine with applied TGF-β2factor and form immunoflurescence deposite particles.Conclusions:Anti-TGF-β2multilayers was firstly fabricated onto IOL surfaces via atmospheric pressure glow discharge plasma pretreatment and LBL technique in this study. Anti-TGF-β2multilayers showed long-term stability and good immunological activity of antibody on IOL surfaces, while had no side effects on the smoothness, optical and mechanical properties of IOL. Part Ⅱ Prevention of posterior capsule opacification by surface modified acrylic intraocular lens with TGF-β2antibody multilayersObjective:To evaluate the efficacy and safety of surface modified acrylic intraocular lens (IOL) with TGF-β2antibody (anti-TGF-β2) multilayers in preventing posterior capsule opacification (PCO).Methods:Human lens epithelial cells (LECs) were cultured onto surface modified acrylic IOL with anti-TGF-β2multilayers and untreated acrylic IOL, inverted phase contrast microscope was used to compare LECs adhesion and proliferation on IOL surfaces at6h,24h and48h after LECs culture. Scratch test was used to compare LECs migration between surface modified IOL and untreated IOL group. α-SMA was uesed as an epithelial-mysenchymal transition indicator, and tested by immunoflurorescence microscope to compare LECs transition on IOL surfaces between surface modified IOL and untreated IOL group.20coloured rabbits were randomly devided into test group or control group, each group had10rabbits. The right eye of each rabbit was performed phacoemusification combined with implantation of a surface midified IOL (test group) or untreated IOL (control group). Slitlamp microscope was used to examine the anterior chamber inflammation including aqueous flare, cells, and iris synechia on lday,3days, lweek, lmonth and3months after operation. Scores of anterior chamber inflammation were calculated and compared between the test and control group. Standardized digital retroilluminated slitlamp images of posterior capsule was taken and PCO areas were calculated by software designed by ourselves. PCO scores were then compared between between the test and control group. All rabbits were killed and right eyes were enucleated3months after operation, Miyake-Apple posterior photography was used to evaluate PCO severity between the test and control group. IOLs were then extracted from rabbits eyes, tested by immunofluorescence and laser scanning confocal microscope to check the integrity of anti-TGF-β2multilayers on IOL surfaces. Lens capsule was embedded by paraffin and paraffin-cut sections were then dyed by hematoxylin-eosin (HE) staining, LECs proliferation and extracellular matrix deposition above posterior capsule were examined. Immunohistochemistry was used to test a-SMA, Collagen Ⅰ and Collagen Ⅲ expression above posterior capsule.Results:Short-terminhibition of adhesion to LECs on surface modified IOL was found at6h after LECs culture, while no difference in adhesion and proliferation of LECs between surface modified IOL and untreated IOL at24h and48h. LECs migration was dramatically decreased in surface modified IOL group, compared to untreated IOL group, immunoflurorescence microscopy showed much less a-SMA expression in surface modified IOL group, compared to untreated IOL group. In vivo PCO model showed no difference in anterior chamber inflammation was found between the test and control group except for less aqueous flare and cells in the anterior chamber in the test group at1w and1mon follow-up. Both retroillumination slitlamp photography and Miyake-Apple posterior photography revealed less PCO occurred in test group. HE staining revealed a thinner posterior capsule and less LECs above posterior capsule in test group, compared to the control group. Immunohistochemistry revealed less a-SMA and Collagen Ⅰ expression above posterior capsule in test group.Conclusions:Surface modified acrylic IOL with TGF-β2antibody multilayers is effective and safe in the prevetion of PCO.更多还原