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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 a human conjunctival goblet cell line in vitro and to observe the biological characteristics of its morphology and structure and to observe the goblet cell ultrastructure. Design experimental research. Study Objective To culture human conjunctival goblet cells in vitro. Methods Normal ocular conjunctiva tissues were obtained during the operation of external ophthalmology. The cells were placed in RPMI1640 medium containing 10% fetal bovine serum and cultured with tissue culture method. Goblet cells were identified by inverted phase contrast microscope under cell morphology, special histochemical staining and immunohistochemistry. The ultrastructure was observed by transmission electron microscope. The main indicators of cell morphology, structure. Results After culturing for 24 hours, the cells grew out from the tissue mass, forming cell monolayers after 6 ~ 10 days and transferring 3 to 5 generations. Inverted phase contrast microscope shows goblet cells were oval, cytoplasm containing mucin particles. Positive staining was performed on alitake blue / periodic acid-Schiff reagent (AB-PAS staining), and positive for goblet cell-specific middle silk keratin-7 (CK-7) and mucin MUC5AC monoclonal antibody , While negative for the non-keratinocyte epithelial-specific intermediate filament keratin-13 (CK-13). Transmission electron microscopy shows that the goblet cells in vitro are larger in volume and oval in shape, with the nucleus located at one end of the cell, and the cytoplasm is filled with round sticky particles with different sizes. Pass three generations after the goblet cell purity up to about 80%. CONCLUSION: Human conjunctival goblet cells can be cultured and passaged in vitro and maintained in somatic cell characteristics, providing a laboratory basis for further study of goblet cell-related ocular surface diseases. (Ophthalmology, 2006,15: 172-176)