神经生长因子对人结膜杯状细胞增殖与黏蛋白基因表达的影响

The effect of nerve growth factor on proliferation and expression of mucin gene in human conjunctival goblet cells

目的探讨神经生长因子（NGF）对体外培养人结膜杯状细胞增殖与黏蛋白MUC5AC基因表达的影响。方法利用抗NGF高亲和力受体（TrkA）的单克隆抗体进行免疫组织化学染色，初步检测杯状细胞膜表面是否存在此受体。在传代的人结膜杯状细胞中加入含20、40、60及80ng／ml NGF的RPMI-1640培养液，加入不含NGF的培养液作为阴性对照。于加药后1、3、5d分别用四甲基偶氮唑盐（MTT）法观察细胞增殖情况。加入含80ng／ml NGF的培养液后第5天，通过逆转录聚合酶链反应（RT-PCR）方法检测NGF对杯状细胞中MUCSAC基因表达的影响。结果人结膜杯状细胞膜表面存在NGF高亲和力受体（TrkA）。加入NGF后不同时间（F=12．766，P=0．000）、不同浓度组（F=892．406，P=0．000）之间的吸光度值比较，差异有统计学意义，时间和浓度这两个因素之间无交互作用（F=1．778，P=0．095）。NGF组MUC5AC的mRNA表达量明显高于对照组（t=4．963，P=0．000）。结论体外培养的人结膜杯状细胞膜表面存在NGF高亲和力受体；NGF有促进杯状细胞增殖的作用，并随浓度增加和时间的延长而促增殖作用增强；NGF能够促进杯状细胞黏蛋白MUC5AC基因表达。

Objective To study the effect of nerve growth factor （NGF） on the proliferation and the expression of MUC5AC gene in cultured human conjunctival goblet cells. Methods The presence of high affinity receptors of nerve growth factor （TrkA） in cultured human conjunctival goblet cells was identified with immunocytochemical analysis. Proliferation of goblet cells was determined by MTT proliferation assay after exposure to different concentrations of NGF, （20, 40, 60 and 80 ng/ml） in serum-free RPMI-1640 culture medium （RPMI） for 1, 3 and 5 days. The effect of NGF on gene expression of MUC5AC of goblet cells was determined by RT-PCR after exposing cells to NGF （80 ng/ml in serum-free RPMI） for 5 days. Results Human conjunctival goblet cells in subculture exhibited positive staining for TrkA. After exposure to NGF, the differenes in absorpation value of MTT test of goblet cells cultured with different levels of NGF （F = 12. 766, P = 0. 000） and at different time points （ F = 892. 406, P = 0. 000 ） were statistically significant, but the difference in interaction effects was not significant. MUC5AC gene expression of NGF group was higher than that of the control group （ t = 4. 963, P = 0. 000）. Conclusions NGF receptors （TrkA） are present in human conjunctival goblet cells. NGF causes a dose-dependent increase in the proliferation of goblet cells. The expression of MUC5AC gene in goblet cells is upgraded by NGF.