内皮素-1对人小梁细胞吞噬功能的影响

目的观察内皮素-1（ET-1）对人小梁细胞（TMCs）吞噬功能的影响及作用机制。方法取培养第3代人TMCs，与直径0．5μm荧光标记的乳胶微粒共孵育0、4、8、12、24、48、72h，荧光显微镜下动态定量观察人TMCs吞噬动力学。取6孔板培养3代人TMCs，随机分为4组，观察不同浓度ET-1对人TMCs吞噬功能的影响：对照组（0mol／LET-1）、低剂量组（10-mol／LET-1）、中剂量组（10-mol／LET-1）、高剂量组（10～mol／LET-1），分别与乳胶颗粒共孵育后观察各组细胞吞噬乳胶颗粒数。取6孔板培养人TMCs随机分为4组，观察内皮素受体对吞噬功能的影响：对照组（0mol／LET-1）、ET-1组（10～mol／LET-1）、ETA受体拮抗剂组（10-mol／LET-1＋10μmol／LBQl23）、ETB受体拮抗剂组（10mol／LET-1＋10。mol／LBQ788），并分别与乳胶颗粒共孵育，观察各组人TMCs吞噬乳胶颗粒数。结果人TMCs免疫组织化学鉴定纤维连接蛋白（FN）、层粘连蛋白（LN）、神经元特异性烯醇酶（NSE）染色均呈阳性；Ⅷ因子相关抗原（FⅧRAg）染色阴性；人TMCs吞噬动力学观察示，人TMCs与乳胶颗粒共孵育4h后可见明显的吞噬颗粒，随时间延长吞噬微粒数明显增多，24h吞噬微粒密度最佳，48h吞噬微粒达到饱和，细胞形态改变；加入ET-1干预后，细胞内吞噬乳胶微球数明显减少，其吞噬抑制效果呈ET-1浓度依赖性（F=28．91，P〈0．05）；ET-1组吞噬颗粒数较对照组减少（q=13．7228，P〈0．05），而ETA受体拮抗剂组吞噬颗粒较ET-1组明显增多（q=9．4312，P〈0．05），ETB受体拮抗剂组与ET—1组相比差异无统计学意义（q=1．1600，P〉0．05）。结论ET-1能抑制体外培养人TMCs的吞噬能力；ET-1主要通过ETA受体发挥抑制人TMCs吞噬功能的作用。

The effect of endothelin-1 on the phagocytic function of human trabecular meshwork cells in vitro

Background Endothelin-1 (ET-1) is an active regulator of intraocular pressure.The ET-1 level in aqueous humor is elevated in primary open-angle glaucoma,normal intraocular tension glaucoma and the animal model of glaucoma.There is now accumulating evidence for a role of ET-1 in the pathogenesis of glaucoma.However,the effect of ET-1 on the phagocytic function in trabecular meshwork cells (TMCs) is unclear.ObjectiveThis study is to observe the effect of ET-1 on the phagocytic function in cultured human TMCs.Methods Human trabecular meshwork tissue was obtained from healthy donator and cultured and subcultured in vitro by the explant culture method.The third passage of human TMCs were incubated with fluoresent red-labeled latex beads for 0,4,8,12,24,48 and 72 hours.The phagocytic kinetics of human TMCs were continuously evaluated by counting the number of latex beads in TMCs using a fluorescence microscope.Depending on the concentrations of ET-1 in culture medium,the TMCs were divided into control group (without ET-1),low-dose ET-1 (10~(-9)mol/L) treatment group,middle-dose ET-1 (10~(-8)mol/L) treatment group and high-dose ET-1 (10~(-7) mol/L) treatment group.In addition,based on the addition of endothelin receptor (ETAR) antagonist,the TMCs were divided into control group (without ETAR antagonist),ET-1 (10~(-8)mol/L) treatment group,ETAR antagonist (1×10~(-7)mol/L BQ123+10~(-8)mol/L ET-1) treatment group and ETBR antagonist(1×10~(-7) mol/L BQ788+10~(-8) mol/L ET-1)treatment group.TMCs of each group were incubated with latex beads,and the numbers of latex beads in TMCs were counted under a fluorescent microscope.Results Cultured HTM cells showed positive reactions for FN,LN,NSE and negative response for FⅧRag.The phagocytic kinetics test revealed that the latex beads were detected 4 hours after incubation.The density of latex beads was gradually increased with the delay of incubation duration and peaked at 24 hours.The number of the latex beads saturated after 48 hours of incubation.However,the number of latex beads in TMCs was significantly reduced after the addition of ET-1 in a dose-dependent manner (F=28.91,P＜0.05).The number of latex beads in the ET-1 group was less than that in the control group and the ETAR receptor antagonist group (q=13.7228,q=9.4312,P＜0.05).No significant difference was found in latex beads number between the ET-1 group and the ETBR antagonist group (q=1.1600,P＞0.05).Conclusion ET-1 inhibits the phagocytic function of human TMCs and ETAR plays a partial role in the phagocytic function of human TMCs.