中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2011年
12期
1117-1122
,共6页
娄秉盛%袁钊辉%罗益文%林晓峰
婁秉盛%袁釗輝%囉益文%林曉峰
루병성%원쇠휘%라익문%림효봉
眼内炎%葡萄球菌,金黄色%色素上皮,眼%中性白细胞%细胞因子类
眼內炎%葡萄毬菌,金黃色%色素上皮,眼%中性白細胞%細胞因子類
안내염%포도구균,금황색%색소상피,안%중성백세포%세포인자류
Endophthalmitis%Staphylococcus aureus%Pigment epithelium of eye%Neutrophils%Cytokines
目的 观察金黄色葡萄球菌培养上清液对中性粒细胞及视网膜色素上皮细胞(RPE)炎性相关因子IL-1β、TNF-α、IL-6表达水平的变化.方法 实验研究.分离入外周血中性粒细胞,与人RPE细胞株D407共培养,加入无菌体的金黄色葡萄球菌ATCC29213培养上清液.其中细菌上清液分为50μl、100μl、250μl、500μl以及空白组、脑心浸萃液态培养基对照组6组；中性粒细胞数量分为空白组、1 ×104、5 ×104、5×105,4组.在6、12、24、48、72 h等时间点搜集培养液上清,应用酶联免疫吸附试验检测培养液中IL-1β、TNF-α、IL-6表达水平的变化.结果 当不同剂量金黄色葡萄球菌上清液单独作用RPE细胞时,培养液中IL-1β水平随着细菌上清液剂量增加而上升、亦随着作用时间的延长而增加；但IL-6水平500μl组高于空白组,仅在24h[(23.17±3.16)ng/L；(7.61±1.53) ng/L]及48 h[ (35.00 ±4.37) ng/L；(13.17±3.27)ng/L]时差异具有统计学意义(P =0.001；P =0.026).RPE细胞在100μl细菌上清液作用下,加入不同数量中性粒细胞共培养,5×105组培养液中IL-1β水平在6 h[ (236.62±8.20) ng/L]及12 h[ (447.42±35.13)ng/L]均高于其余各组,差异具有统计学意义(6 h:P =0.000,P=0.000,P=0.002；12 h:P =0.000,P=0.000,P=0.000)；培养液中IL-6的水平在12 h时5×105组[( 46.96±2.72) ng/L]亦高于其余各组,差异具有统计学意义(P =0.000,P=0.000,P=0.000).在各时间点培养液中均检测不到明显的TNF-α表达.结论 中性粒细胞及RPE细胞共培养体系在金黄色葡萄球菌上清液的刺激下可检测到IL-1β和IL-6表达；其中IL-1β表达在早期出现,且高水平状态维持较长.金黄色葡萄球菌的毒力因子含量及中性粒细胞数量对引发IL-1β和IL-6的高表达起重要作用.
目的 觀察金黃色葡萄毬菌培養上清液對中性粒細胞及視網膜色素上皮細胞(RPE)炎性相關因子IL-1β、TNF-α、IL-6錶達水平的變化.方法 實驗研究.分離入外週血中性粒細胞,與人RPE細胞株D407共培養,加入無菌體的金黃色葡萄毬菌ATCC29213培養上清液.其中細菌上清液分為50μl、100μl、250μl、500μl以及空白組、腦心浸萃液態培養基對照組6組；中性粒細胞數量分為空白組、1 ×104、5 ×104、5×105,4組.在6、12、24、48、72 h等時間點搜集培養液上清,應用酶聯免疫吸附試驗檢測培養液中IL-1β、TNF-α、IL-6錶達水平的變化.結果 噹不同劑量金黃色葡萄毬菌上清液單獨作用RPE細胞時,培養液中IL-1β水平隨著細菌上清液劑量增加而上升、亦隨著作用時間的延長而增加；但IL-6水平500μl組高于空白組,僅在24h[(23.17±3.16)ng/L；(7.61±1.53) ng/L]及48 h[ (35.00 ±4.37) ng/L；(13.17±3.27)ng/L]時差異具有統計學意義(P =0.001；P =0.026).RPE細胞在100μl細菌上清液作用下,加入不同數量中性粒細胞共培養,5×105組培養液中IL-1β水平在6 h[ (236.62±8.20) ng/L]及12 h[ (447.42±35.13)ng/L]均高于其餘各組,差異具有統計學意義(6 h:P =0.000,P=0.000,P=0.002；12 h:P =0.000,P=0.000,P=0.000)；培養液中IL-6的水平在12 h時5×105組[( 46.96±2.72) ng/L]亦高于其餘各組,差異具有統計學意義(P =0.000,P=0.000,P=0.000).在各時間點培養液中均檢測不到明顯的TNF-α錶達.結論 中性粒細胞及RPE細胞共培養體繫在金黃色葡萄毬菌上清液的刺激下可檢測到IL-1β和IL-6錶達；其中IL-1β錶達在早期齣現,且高水平狀態維持較長.金黃色葡萄毬菌的毒力因子含量及中性粒細胞數量對引髮IL-1β和IL-6的高錶達起重要作用.
목적 관찰금황색포도구균배양상청액대중성립세포급시망막색소상피세포(RPE)염성상관인자IL-1β、TNF-α、IL-6표체수평적변화.방법 실험연구.분리입외주혈중성립세포,여인RPE세포주D407공배양,가입무균체적금황색포도구균ATCC29213배양상청액.기중세균상청액분위50μl、100μl、250μl、500μl이급공백조、뇌심침췌액태배양기대조조6조；중성립세포수량분위공백조、1 ×104、5 ×104、5×105,4조.재6、12、24、48、72 h등시간점수집배양액상청,응용매련면역흡부시험검측배양액중IL-1β、TNF-α、IL-6표체수평적변화.결과 당불동제량금황색포도구균상청액단독작용RPE세포시,배양액중IL-1β수평수착세균상청액제량증가이상승、역수착작용시간적연장이증가；단IL-6수평500μl조고우공백조,부재24h[(23.17±3.16)ng/L；(7.61±1.53) ng/L]급48 h[ (35.00 ±4.37) ng/L；(13.17±3.27)ng/L]시차이구유통계학의의(P =0.001；P =0.026).RPE세포재100μl세균상청액작용하,가입불동수량중성립세포공배양,5×105조배양액중IL-1β수평재6 h[ (236.62±8.20) ng/L]급12 h[ (447.42±35.13)ng/L]균고우기여각조,차이구유통계학의의(6 h:P =0.000,P=0.000,P=0.002；12 h:P =0.000,P=0.000,P=0.000)；배양액중IL-6적수평재12 h시5×105조[( 46.96±2.72) ng/L]역고우기여각조,차이구유통계학의의(P =0.000,P=0.000,P=0.000).재각시간점배양액중균검측불도명현적TNF-α표체.결론 중성립세포급RPE세포공배양체계재금황색포도구균상청액적자격하가검측도IL-1β화IL-6표체；기중IL-1β표체재조기출현,차고수평상태유지교장.금황색포도구균적독력인자함량급중성립세포수량대인발IL-1β화IL-6적고표체기중요작용.
Objective To evaluate the effects of staphylococcus aureus supernatant on the levels of interleukin-1β (IL-1β),tumor necrosis factor-α (TNF-α) and interleuin-6 (IL-6) in co-cultures of neutrophils and retinal pigment epithelium (RPE) cells.Methods The co-culture system was established by co-culturing of human RPE cell line D407 and human peripheral blood neutrophils. Bacterium-free supernatant of staphylococcus aureus ATCC29213 was added to the co-culture system for studying its' effects.The volume of bacterium-free supernatant was divided into six groups:negative control,brain-heart infusion control,50 μl,100 μl,250 μl and 500 μl group.The number of neutrophils was divided into four groups:negative control,1 × 104,5 × 104 and 5 × l05 group.The supernatants was collected at 6 h,12 h,24 h,48 h and 72 h later and the levels of IL-1β,TNF-α and IL-6 were measured by ELISA kits.Results When RPE cells were cultured with different doses of bacterium-free supernatant (0,50,100,250 and 500 μl),the levels of IL-1β was positively correlated with the volume of bacterium-free supernatant and the duration.The levels of IL-6 were significantly higher than that of the control group in the 500 μl group at 24 h[ (23.17 ± 3.16) ng/L vs (7.61 ±1.53)ng/L] and 48 h[(35.00±4.37)ng/L vs (13.17 ±3.27 )ng/L] duration( P =0.001 and P =0.026,respectively).When RPE cells were co-cultured with the bacterium-free supernatant and the neutrophils (1 × 104,5 × 104 and 5 × 105 cells) for 6 and 12 h,the levels of IL-1β in the 5 × 105 group at both 6 h[ (236.62 ± 8.20) ng/L ] and 12 h [ (447.42 ± 35.13 )ng/L]was statistically higher than that in other groups (6 h:P =0.000,P =0.000,P =0.002 ; 12 h:P =0.000,P =0.000,P =0.000,respectively ).The levels of IL-6 in the 5 × 105 group [ (46.96 ± 2.72 ) ng/L ] was significantly higher than that in the other groups at 12 h ( P =0.000,P =0.000,P =0.000,respectively).TNF-α could not be detected in the conditioned media from all cultures.Conclusions Both IL-1β and IL-6 are expressed in the co-cultures of neutrophils and RPE cells with staphylococcus aureus supernatant.IL-1 β is upregulated at the early stage and the high level is maintained longer than that of IL-6.The virulence factor of bacteria and the neutrophil may play a role in the expression of IL-1β and IL-6 in the RPE cells.
