正常人结膜杯状细胞的体外培养

Culture in vitro of human conjunctival goblet cells

目的体外建立人结膜杯状细胞系,观察其形态结构等生物学特征,并观察杯状细胞超微结构。设计实验性研究。研究对象体外培养的人结膜杯状细胞。方法外眼手术中取正常球结膜组织,放入含10%胎牛血清的RPMI1640培养液中,以组织块培养法进行培养。通过倒置相差显微镜下细胞形态、特殊组化染色和免疫组化方法鉴定杯状细胞。利用透射电镜观察其超微结构。主要指标细胞形态、结构。结果培养24小时后细胞从组织块向外长出,6￣10天后可形成细胞单层,并可传3￣5代。倒置相差显微镜下可见杯状细胞呈长椭圆形,胞浆中含有黏蛋白颗粒。对阿利新蓝/过碘酸-希夫试剂染色(AB-PAS染色)呈阳性;对杯状细胞特异性中间丝角蛋白-7(CK-7)和黏蛋白MUC5AC单克隆抗体均呈阳性反应,而对复层非角化上皮细胞特异性中间丝角蛋白-13(CK-13)呈阴性反应。透射电镜下可见离体杯状细胞体积较大,呈长椭圆形,核位于细胞一端,胞浆中充满圆形黏蛋白颗粒,大小不一。传三代后杯状细胞纯度可达80%左右。结论人结膜杯状细胞可在体外培养并传代,并保持在体细胞特性,为进一步研究杯状细胞相关的眼表疾病提供实验室基础。(眼科,2006,15:172-176)

Objective To establish goblet cell line in vitro and observe its morphology and uhrastructure. Design Experimental study. Participants Cultured goblet cells. Methods Bulbar conjunctiva tissue was surgically removed from patients. Goblet cell was isolated from bulbar conjunctiva using explant cultures. Cells derived from the explants were propagated in RPMI medium supplemented with 10% fetal bovine serum and characterized morphologically. Goblet cells were identified with TEM, histochemistry and immunohistochemistry. Main Outcome Measures Cellular morphology and structure. Results Goblet cells were successfully isolated from conjunctiva explants. As early as 24 hours after establishment of the organ culture, adherent and round cells were visible around most sides of the tissue plug. Within 6 to 10 days of euhure, evenly spaced nodules were visible.forming a circular pattern around the plug of conjunctival tissue. Tiny droplets were visible on the surface of cultured goblet cells with inverted phase contrast microscope. Cultures have been passaged a minimum of three times without loss of their specific cellular markers. Human goblet cells exhibited positive reactivity with alcian blue-periodic acid Schiff （AB-PAS） reagent, goblet cell-specific cytokeratin-7 and MUC5AC, but negative reactivity to the stratified squamous epithelial cell marker, cytokeratin-13. Typical goblet cells were revealed as an elongated body containing an apically placed nucleus and several storage vesicles with transmission electron microscopy. After passaged 3 times, the purity of goblet cells was about 80%. Conclusion Human goblet cells that retain characteristics of goblet cells in vivo could be cultured. Cultured goblet cells may serve as a useful tool in the research of ocular surface diseases related to goblet cells. （ Ophthalmol CHN, 2006, 15： 172-176）