人眼小梁细胞体外培养与细胞鉴定

Human Trabeculer Cells in Culture and Cell Identification

目的:建立人眼小梁细胞体外培养方法和确定该细胞的鉴定要点。方法:小梁网和角巩膜组织来源于除眼部疾患以外各种原因死亡的6岁以下儿童,在24小时内取材的34人68只正常眼球。组织块原代培养和细胞传代培养。用光镜和电镜观察培养细胞;用免疫组织细胞化学S-P法染色培养细胞的细胞外基质包括纤粘连蛋白(fibronectin,FN)、层粘连蛋白(laminin,LN)和Ⅳ型胶原(Ⅳ collagen,ⅣC)。结果:供者年龄小、取材距死亡时间短者小梁细胞易生长;分离小梁网必须准确;原代培养的接种与换液和传代培养的时机与比例应适当;实验研究选用传3～5代细胞。培养的小梁细胞生长特性和形态特征不同于角巩膜组织的细胞;超微结构使其与角膜基质细胞相鉴别;细胞外基质染色使其与巩膜成纤维细胞相鉴别。结论:本研究表明,只要掌握培养方法和要点,人眼小梁细胞体外培养在常规培养条件下并不困难。对培养的人眼小梁细胞鉴定至少从其生长特性,形态特征和细胞外基质(FN,LN和ⅣC)免疫组织细胞化学S-P法染色特点三方面相结合方能确定小梁细胞。建立人眼小梁细胞体外培养方法与细胞鉴定是进一步研究原发性开角型青光眼病因和发病机制的必要条件和重要保证。眼科学报1998;14:190～194。

Purpose :To establish the method of culturing human trabecular cells(HTCs) in vitro and to determine identifing main points concerning cultured HTCs. Methods:Trabecular meshwork,cornal and scleral tissue were collected from sixty-eight eyeballs of thirty-four human donors less than six years of age within 24 hours after death. The tissues were primarily cultured and the cells subcultured. Cultured cells were observed by light and electron microscopes. Fibronectin(FN) ,laminin(LN) and IV collagen (IVC)in extracellular matrix (ECM) of the cultured cells were im-munohistocytochemically stained by S-P method. Results: HTCs easily grew in the event of young donors and short-time drawing materials. Trabecuiar meshwork should be accurately separated. Techniques in primary culture,and time and proportion in subculture should be suitabled. Experimental studies should select the third to fifth passages. The growing characteristics,morphological features of cultured HTCs under light and electron mircoscopes differed from those of corneal and scleral cells adjoining them. Ultramicrostucture distinguished HTCs from corneal interstitial cells. ECM stained by S-P method distinguished HTCs from scleral f ibroblast. Conclusions .-These studies suggest that it is not difficult to culture HTCs in general culturing condition as long as the main points about culturing them are known-well. The identificastion of cultured HTCs must be combined with three aspects: the growing characteristics and morphological features of cultured cells under light and electron microscopes,and the immunohistochemical stained peculiarties of FN,LN and IVC in ECM of cultured cells. To establish the method of culturing HTCs in vitro and to determine identifing main points conerning cultured HTCs become an essential condition and an important guarntee in order to investigate the pathogenesis and mechanism on primary open angle glaucoma(POAG)deeply. Eye Science 1998 ; 14 ; 190 - 194 .