【作者】 孙洁；

【导师】 谢立信；

【作者基本信息】 青岛大学 ， 眼科学， 2004， 博士

【摘要】 目的 后发性白内障(简称后发障)是白内障摘除术后影响视力的最主要并发症之一，虽经多年不懈努力研究，至今尚未解决。术后2～5年成人发生率高达50％，儿童更是高达100％，目前尚无有效的药物进行防治。因此，研究如何减少后发性白内障的发生，提高患者术后视力，具有重要的临床意义。 多年来，人们在不断改良人工晶状体的设计和手术技术的同时，还尝试了药物、激光、手术等多种治疗手段，但对儿童白内障和并发性白内障的效果均无法令人满意。基因治疗近年来在多种难治性疾病的防治方面取得了很大的发展，显示出其优越的先进性和较为令人满意的效果。目前，已有大量的体外实验显示，反义基因疗法可以抑制细胞增殖，促进细胞凋亡的发生。反义c-myc基因转染对人视网膜色素上皮细胞、乳腺癌细胞、淋巴瘤细胞、肝癌细胞等的增殖均有良好的抑制作用。基于反义c-myc基因治疗抑制上述细胞增殖研究的成功经验，我们认为通过转基因技术调控细胞周期，改变细胞增殖活性，很可能是一种极有前景的防治后发性白内障的途径。 本研究通过体外、体内实验，利用转基因技术在国内外第一次将反义c-myc基因转染晶状体上皮细胞，观察其对晶状体上皮细胞增殖和凋亡的影响，从细胞周期的改变、mRNA和蛋白质水平的变化等方面探讨其抑制后发性白内障发生的作用。反义c一myc基因转染抑制后发性白内障的实验研究2004孙洁博}论文材料和方法 一、体外实验 1.293细胞及永生化人晶状体上皮细胞(HLEC)的培养 一2.两种重组腺病毒(Ad一Lacz，Ad一AS一myc)的扩增、纯化与滴度测定 3.重组腺病毒转染效率的测定 将Ad一LaeZ按MOI(感染强度，Multiplieity ofxnfeetion)100，200感染HLEe，48h后进行X一Gal染色。显微镜下观察，计算蓝染细胞占所有细胞的百分率，即为转染效率。 4.Ad一AS一myc转染对HLEC影响的检测 (l)细胞形态学改变 将Ad一As一myc按Mol 100感染HLEc，于感染后每日观察并记录细胞的形态变化，并与正常HLEC进行比较。 (2)细胞生长曲线的变化 以5 x 103个细胞/孔接种HLEC至6孔板，培养过夜。按MOI 100将Ad一AS一myc转染细胞。定期(转染后24、48、72、96h)消化收集细胞，计算细胞密度。将实验结果与对照组进行比较。 (3) MTT比色法分析细胞增殖的变化 以103个细胞/孔接种HLEc至%孔板，培养过夜。按MOI 100将Ad一AS一myc转染细胞。定期(转染后24、48、72、%h)加入MTT溶液，4h后吸弃孔内上清液，加入DMSO，振荡，在酶联免疫测定仪上检测其在492nm处的吸光值。将实验结果与对照组进行比较。 (4)流式细胞仪的检测 l)细胞凋亡的测定(Annexin一v Kit法):按MOI 1 00将Ad一AS一mye感染HLEC，48、96h后采用Annexin一v Kit法检测，MultiGraph软件分析结果。将实验结果与对照组进行比较。 2)细胞周期分析:按MOI 100将Ad一AS一myc感染HLEC，48、96h后常规DNA倍体检测，Multicycte软件分析结果。将实验结果与对照组进行比较。 (5)Rl’- PCR检测c一mye mRNA的表达反义c一myc基因转染抑制后发性白内障的实验研究2004孙洁博!论文 按Mol 1 00将Ad一As一myc感染HLEc，48、96h后分别提取细胞总RNA，紫外分光光度计定量后进行逆转录，逆转录产物进行PCR，tubulin为内对照。琼脂糖凝胶电泳分离PCR产物，紫外灯下观察。图像分析系统分析底片的灰度。将实验结果与对照组进行比较。 (6)免疫组织化学法分析c一myc蛋白质的表达 按MOI 1 00将Ad一AS一myc感染HLEc，48h后进行免疫组化检查。将实验结果与对照组进行比较。 (7)Western Blotting分析e一mye蛋白质的表达 按MOI 1 00将Ad一As一mye感染HLEC，48、96h后提取细胞总蛋白质，用BCAProtein Assay Kit定量，蛋白质样品进行sDs一PAGE电泳，将电泳产物转至硝酸纤维膜上，按ECL蛋白分析试剂盒方法杂交。图像分析系统分析底片的灰度。将实验结果与对照组进行比较。 二、体内实验 1.实验动物 对38只新西兰白兔均行单眼超声乳化透明晶状体摘除术。选用Storz Premium超声乳化仪，全部手术均获成功。 2.实验分组:随机分为四组。 A组:空白对照组10只 术毕前房注射平衡盐溶液 B组:Ad一LacZ组8只 术毕前房注射Ad一LacZ C组:Ad一AS一myc注射一次组10只 术毕前房注射Ad一AS一myc D组:Ad一As一myc注射二次组10只 术毕及术后Zd，前房注射Ad一 AS一myc 3.术后检测项目 (l)裂隙灯检查 术后第一周的每一天及14、21、28d，行裂隙灯检查，观察后囊混浊出现的时反义c一myc幕因转染抑制后发性白内障的实验不J于究2004孙洁博士论文间、形态改变以及眼内其它部位的反应情况并记录。 (2)病毒转染效率的测定 术后Zd，取B组2眼，仔细分离晶状体囊袋，X一Gal染色，显微镜下观察晶状体上皮细胞中有无蓝染的阳性细胞，计算转染效率。 (3)非靶向性转染的测定 术后Zd，取B组2眼，x一Gal染色，观察有无角膜更多还原

【Abstract】 PurposeThe most common complication of cataract surgery is the development of posterior capsule opacification (PCO), which is a problem that has not been well solved. Generally, the occurrence rate of PCO 2-5 years after surgery is as high as 50% in adults and 100% in children [1]. Hyperplasia of the lens epithelial cells is one of the main cellular events following phacoemulsification and is found to be an important feature contributing to PCO [2].Attempts have been made to prevent PCO by modifying the designs of intraocular lenses and the surgical procedures, but PCO still occurs. Chemicals and antimitotics (such as 5-FU, MMC) have been used to inhibit the proliferation of lens epithelial cells, but the efficiency and the tolerance are not satisfying especially because of their toxic effects [3,4]. Nd:YAG laser capsulotomy has also been used. However, it can lead to severe complications such as macular edema and retinal detachment [2,5].Recent advances in science and technology have provided opportunities to develop new treatment strategies using gene therapy. Among them, antisense strategies have proved efficient in numerous studies with different kinds of cells [12-15].In this study, we explored the feasibility of adenovirus-mediated transfer of an antisensec-myc protooncogene into human lens epithelial cells in vitro and in a rabbit model of PCO after phacoemulsification in vivo.MethodsI. In vitro experimentsRecombinant adenovirusesAd-LacZ and Ad-AS-myc were kindly provided by Professor Chen Lin (Cancer Institute, Chinese Academy of Medical Science, Peking Union Medical College). These two replication-defective recombinant adenoviruses (E1-E3 deleted) were expanded in the 293 cell line (Fig. 2) and purified by centrifugation. Virus titer was determined by standard assay in 293 cells.Cell cultureImmortalized human lens epithelial cell (HLEC) line (HLE-B3, ATCC, USA) was kindly provided by Dr. Youhai Chen (Department of Molecular and Cellular Engineering, University of Pennsylvania, School of Medicine)(Fig.3). The cells were cultured in Eagle’s minimum essential medium (MEM, Sigma, USA) containing 10% fetal bovine serum (FBS, Hyclone, USA), 1.0mM sodium pyruvate (Hyclone, USA), 100IU/ml penicillin G and 100 u g/ml streptomycin sulphate. The incubation condition was 37C in a humidified atmosphere of 95% air and 5% carbon dioxide. The cells were subcultured at a ratio of 1: 3.Determination of transduction efficiencyHLECs were seeded at a density of 2x104 cells/well in six-well plates. After overnight attachment, the cells were transduced with 1ml of Ad-LacZ suspension (MOI of 100 or 200) and incubated for 1hr at 37C in 5% CO2. Then 3ml of fresh MEM containing 2% FBS was added to each well. Forty-eight hours after transduction, the cells were washedtwice with warm PBS and fixed for 10min in 1% formaldehyde-0.05% glutaraldehyde-PBS. After fixation, the cells were washed twice again with PBS and stained with X-Gal (5-bromo-4-chloro-3-indolyl-B-D-galactosidase, Shanghai Sangon Biological Engineering Technology and Service Co., China).X-Gal-positive cells, as well as total cells were counted in the microscopic field at a magnification of x20 using a phage-contrast microscope (IMT-2, Olympus, Japan). This procedure was performed for five random fields and the transduction efficiency was calculated.Morphological changes of transduced cellsThe transduced cells were pictured at daily intervals (0, 24, 48, 72 and 96 hrs) afterAd-AS-myc transduction (MOI of 100).HLECs proliferation of transduced cellsThe inhibition of Ad-AS-myc transduction on HLECs proliferation was evaluated bymaking cell growth curve and MTT colorimetric assay.i. Cell growth curveHLECs were seeded at a density of 5x103 cells/well in six-well plates. The next day, thecells were transduced with Ad-AS-myc (MOI of 100). Then the cells were counted atdaily intervals (0, 24, 48, 72 and 96 hrs) and the cell growth curve was made.Non-transduced HLECs were taken as control group. The procedures were performed更多还原