摘要
目的通过研究细菌脂多糖(lipopolysaccharide,LPS)对人角膜上皮细胞系(hunman corneal epithelial cell line,HCEC)中Toll样受体(toll-like receptors,TLR)的表达和功能的影响,以及糖皮质激素对这种影响的调控,探讨TLR及糖皮质激素在角膜细菌感染中的作用。方法通过RT-PCR及实时PCR检测角膜上皮细胞中TLRmRNA的表达;并在细胞培养液中加入3种浓度LPS(1μg.L-1、10μg.L-1、100μg.L-1)共培养,检测TLR的表达;以及在此基础上,选择在10μg.L-1LPS刺激的细胞培养液中分别加入氢化可的松(1×103μg.L-1、10×103μg.L-1、100×103μg.L-1)进行干预,并检测TLRmRNA的表达;再采用酶联免疫吸附实验检测细胞培养液中IL-6、IL-8的释放;并通过免疫荧光组织化学检测TLR蛋白的表达。结果 TLR1-TLR10mRNA在HCEC中均有表达,且表达量各不相同,其中TLR2、TLR3、TLR4、TLR5、TLR6高表达。LPS能增加细胞TLRmRNA的表达,且当LPS为10μg.L-1时,TLRmRNA的表达量最高,同时IL-6、IL-8的释放也相应增加。加入氢化可的松干预后,随着激素浓度的增高,HCEC中TLRmRNA的表达也随之增加,此时IL-6的释放减少,IL-8的释放增加。免疫荧光组织化学也显示了3种浓度激素干预下HCEC的TLR2蛋白、TLR4蛋白在胞内均有表达,且强度不同,趋势与分子水平的表达一致。结论 LPS能改变角膜上皮细胞TLRmRNA的表达,提示上皮细胞表达的TLR与角膜抵御细菌的固有免疫密切相关;糖皮质激素可以调控LPS刺激的HCEC中TLR的表达,即轻度促进LPS刺激的HCEC中TLR的表达,在细菌侵袭角膜的早期,糖皮质激素可能具有一定的免疫促进作用。
Objective To investigate the role of toll-like receptors（TLR）and glucocorticoids in corneal bacterial infections by studying the effects of lipopolysaccharide（LPS）on expression and function of TLR of human corneal epithelial cell line（HCEC）and the regulation effects of glucocorticoids.Methods TLR mRNA levels were detected by RT-PCR and real-time PCR.Expression of TLR was detected after co-culturing with adding 1 μg·L-1,10 μg·L-1 and 100 μg·L-1 LPS into cell culture fluid,and TLR mRNA levels were detected after adding 1×103 μg·L-1,10×103 μg·L-1,100×103 μg·L-1 hydrocortisone into 10 μg·L-1 LPS culture fluid.IL-6 and IL-8 levels at cell culture fluid were measured by enzyme-linked immunosorbent assays,and TLR protein levels were examined by immunofluorescence histochemistry method.Results The expression levels of TLR1 mRNA to TLR10 mRNA were all found and varied in HCEC,and TLR3,TLR4,TLR5 and TLR6 with high expression.LPS could increase expression of TLR mRNA.When LPS was 10 μg·L-1,TLR mRNA was with the highest expression level,both IL-6 and IL-8 levels increased relatively.After adding hydrocortisone into cell culture fluid,TLR mRNA expression increased,IL-6 level decreased and IL-8 levels increased in HCEC with the increasing of hormone concentrations.Immunofluorescence histochemistry method showed both TLR2 protein and TLR4 protein were found in HCEC in above three conditions,the expression levels were different and the expression tendency was consisted with molecular levels.Conclusions LPS can change the expression of TLR mRNA in corneal epitehlial cells,which indicate that TLR in corneal epithelial cells has close relation with immunity of cornea to resist bacteria,glucocorticoids can regulate expression of TLR in HCEC induced by LPS that is to say glucocorticoids can slightly improve expression of TLR in HCEC induced by LPS.Glucocorticoids has a certain immunological enhancement at early stage when bacteria invading corneas.
出处
《眼科新进展》
CAS
北大核心
2010年第7期637-643,共7页
Recent Advances in Ophthalmology
基金
浙江省自然科学基金资助(编号:Y205672)~~
