金黄色葡萄球菌外毒素及中性粒细胞对视网膜色素上皮细胞损伤作用的初步研究

Preliminary study of the damage effects of staphylococcus aureus exotoxins and neutrophils on retinal pigment epithelium cells

目的观察金黄色葡萄球菌外毒素及中性粒细胞对视网膜色素上皮（RPE）细胞的毒性作用。方法分离人外周血中性粒细胞，和人RPE细胞株D407（RPED407）共培养，并加入金黄色葡萄球菌ATCC29213外毒素建立细菌性眼内炎体外模型。通过观察RPE细胞培养液中乳酸脱氢酶（LDH）水平、流式细胞仪分析RPE细胞存活状况改变、以及Hoechst 33342／Propidium Iodide（PI）染色观察细胞核的荧光染色等方法，验证金黄色葡萄球菌外毒素及中性粒细胞埘RPE细胞的损伤作用。结果金黄色葡萄球菌外毒素单独作用RPE细胞时，细胞外LDH水平及RPE细胞的坏死细胞比率随着外毒素剂量增加而升高，但仅250、500μl组差异有统计学意义。当加入中性粒细胞共同培养后，100μl外毒素作用下，仅6h，RPE细胞外LDH水平即高于无外毒素绀，差异具有统计学意义；在6h时，流式细胞仪分析湿示，RPE细胞发生了明显的细胞凋亡，并且随着中性粒细胞数量增多而加剧，差异具有统计学意义。结论金黄色葡萄球菌外毒素对RPE细胞具有毒性作用，其机制主要以细胞坏死的方式进行。中性粒细胞超过一定数量后对RPE细胞具有损伤作用。外毒素和中性粒细胞共间参与时，对RPE细胞的毒性作用大于两者单独对RPE细胞的损害。

Objective To evaluate the toxic effects of staphylococcus aureus exotoxins and neutrophils on retinal pigment epithelium （RPE） cells （RPEC）. Methods An in vitro model of baeteroidal endophthalmitis was established by co culturing of human RPE cell line D407 and human peripheral blood neutrophils in the present of staphylococcus aureus exotoxins ATCC29213. The level of lactate dehydrogenase hydroxide （I.DH）in the cuture supernant was measured, and the viability of RPE was evlauated by flow cytometry and Hoechst 33342/propidium iodide （PI） staining. Results When RPE cells were cultured with the exotoxin ATCC29213, the LDH level and necrotic RPE cells were positive proportional to the dosage of exotoxin, but only 250 μl or 500 μl of ATCC29213 had a statistical significant effect. When RPE cells were co cultured with neutrophils in the present of ATCC29213 for 6 hours, 100μl of ATCC29213 already had a statistical significant effect on LDH level and necrotic RPEC, and the effect was proportional to the amount of neutrophils in the culture. Conclusion Both staphylococcus aureus exotoxins and neutrophils can damage the RPEC by inducing necrosis, and their function had synergetic effect.