【作者】 孙靖；

【导师】 袁佳琴；

【作者基本信息】 天津医科大学 ， 眼科学， 2003， 博士

【摘要】 白内障是世界范围内重要的致盲眼病，囊外摘除或超声乳化联合人工晶状体植入术是目前最有效的治疗手段，但术后的主要并发症后囊混浊，常引起术后视力再次下降，影响手术的远期效果。 研究表明手术引发的残留晶状体上皮细胞向后囊的增殖、移行、纤维化并分泌胶原等细胞外基质是后囊混浊形成的主要原因，因此防治的重点在于减少晶状体上皮细胞的残留并阻止其增殖和移行。大量的体外实验已经证实抗代谢药物如5-氟脲嘧啶、丝裂霉素、柔红霉素等，环孢霉素A等皆可抑制晶状体上皮细胞的增殖和分化，但这些药物对眼内其他正常组织的毒副作用限制了其临床应用。如何实现药物对晶状体上皮细胞的特异性杀伤作用，减少毒副反应，是后囊混浊药物防治的关键问题。因此制备特异性识别晶状体上皮细胞而不与眼部其他组织发生反应的单克隆抗体，并以其为导向载体合成免疫导向药物，实现后囊混浊的靶向治疗，具有重要的临床实用价值和意义。 本课题以原代培养的人晶状体上皮细胞作为免疫原，使用杂交瘤技术制备了一组抗人晶状体上皮细胞膜单克隆抗体，间接ELISA法测定免疫球蛋白的亚类，用间接免疫荧光法、免疫组化方法对单克隆抗体进行特异性鉴定。BALB／c小鼠体内诱生法扩增单克隆抗体，用羟基磷灰石色谱法加以纯化，SDS-PAGE测定抗体的纯度。MTT比色法及光镜和透射电镜下观察单克隆抗体对体外培养晶状体上皮细胞生长的影响。结果如下： 1．本实验采用改良的组织块培养法，成功地建立了胎儿、成人、老年性白内障患者、兔晶状体上皮细胞的培养体系，本法简单、易行，重复性好，适宜推广。 2.通过脾内注射培养的人晶状体上皮细胞免疫BALB/c小鼠，利用杂交瘤技术成功制备了2株抗人晶状体上皮细胞膜单克隆抗体，命名为HILE4、HILE6，免疫球蛋白亚类均为I目随。脾内注射细胞免疫法不仅抗原用量少，而且缩短了免疫时间，对于某些稀有抗原或者急需制备单克隆抗体时可考虑采用。 3.该组单克隆抗体与人晶状体上皮细胞膜呈特异性反应，与眼内其他组织如角膜、虹膜、睫状体、脉络膜、视网膜均为阴性反应，与兔晶状体上皮细胞膜有交叉反应。 4.小鼠腹腔接种HILE6杂交瘤细胞，制得大量高滴度腹水，效价为1:8 000。经两次经基磷灰石色谱法纯化，5%~15%SDS一队GE分析结果显示，抗体的纯度为90%。经基磷灰石色谱法是一种快速、简便、有效地纯化IgM单克隆抗体的方法。 5.一定浓度的HILE6对人和兔晶状体上皮细胞的增殖有抑制作用，可引起细胞形态学的改变。 本研究成功地制备了能与人晶状体上皮细胞膜特异性结合的单克隆抗体，为进一步制备能主动选择性结合、特异性杀伤晶状体上皮细胞的免疫导向药物，实现后囊混浊的靶向治疗奠定了实验基础。更多还原

【Abstract】 Cataract is an important ocular disease that induces blindness. To date, extracapsular cataract extraction (ECCE) or phacoemulsification combined with artificial intraocular lens (IOL) implantation is considered to be the best treatment. However, posterior capsular opacification (PCO), the most common postoperative complication, may decrease the visual acuity again.Some clinical and experimental studies suggest that PCO is mainly due to the proliferation and migration of postoperative remnants of lens epithelial cells (LECs) on the posterior capsule. The etiology of PCO is incompletely known. The surgical trauma might activate LECs in a wound-healing process. Several experimental studies showed that some agents have been effective in inhibiting LECs proliferation and differentiation, and also in development of PCO, such as anti-metabolites, nonsteroid antiphlogistic drugs, cyclosporin A. But their clinical use is limited by intolerable toxic side effects to other ocular tissues. Thus, it is imperative to produce a monoclonal antibody specific for LECs as carrier of the target-directing agents designated to identify and selectively kill LECs and to prevent them from subsequent proliferation.We produced monoclonal antibody (McAb) specific for human lens epithelial membrane with hybridoma technique. The subclass of the McAb was determined by indirect enzyme-linked immunosorbent assay (ELISA) method and the specificity was confirmed both by immunohistochemical analysis and by indirect immunofluorescence techniques. We prepared large quantities of the McAb by the hybridoma cells inoculated to the peritoneal cavity of BALB/c mouse. The McAb was purified by hydroxyapatite chromatography. Cell enzyme-linkedimmunosorbent assay (CELISA) and SDS-polyacrylamide electrophoresis were used to identify the purified antibody. We evaluated its effect on LECs proliferation by MTT colorimetry and observed the relevant morphological changes of LECs with light microscopy and transmission electron microscopy. The main results are the following:1. LECs from human fetus, adult, senile cataract patients and rabbit were successfully cultured using modified explant technique in vitro and established LECs culture system. The technique is simple, easy to perform and to popularize.2. Monoclonal antibodies, HILE4 and HILE6 specific for human lens epithelial membrane were developed successfully after two intrasplenic injections of cultured human LECs. The subclass of the monoclonal antibodies is IgM. The intrasplenic immunization procedure could facilitate monoclonal antibody production in some instances in which small quantities of immunogen or limited time is available.3. These antibodies reacted specifically with the cell membrane of LECs and cross-reacted with rabbit lens epithelial membrane. No other tissue in human eyes, such as the cornea, iris, ciliary body, choroid, retina, sclera were associated.4. We prepared large quantities of HILE6 by the hybridoma cells inoculated to the peritoneal cavity of BALB/c mouse. The titer is 1:8000. The mouse ascitic fluid was purified by hydroxyapatite chromatography twice and the purity was up to mass fraction 90%. Hydroxyapatite chromatography is a simple, rapid and efficient procedure for the purification of monoclonal IgM antibody.5. HILE6 at appropriate doses possessed prohibitive effects on the proliferation of LECs and induced the morphological changes of lens epithelium, even to death.We successfully prepared monoclonal antibody against human lens epithelial membrane, which can serve as a carrier of the target-directing agents designated to actively identify and selectively kill LECs and to prevent them from subsequent proliferation. The agents maybe more efficient in prevention and treatment of PCO.更多还原