摘要
目的 通过研究细菌脂多糖（LPS）对人角膜基质成纤维细胞（HCF）Toll样受体（TLR）表达的影响,以及糖皮质激素对这种影响的调控作用,探讨TLR及糖皮质激素在角膜细菌感染中的可能作用.方法 选取人供体角膜细胞,对其进行分离、培养和鉴定,通过RT-PCR及quantitative Real-time PCR检测角膜基质成纤维细胞中TLR在mRNA水平的表达;并在细胞培养液中加入3种浓度LPS（1、10、100 ng/ml）共培养,检测TLR的表达.在此基础上,选择在10 ng/ml LPS的细胞培养液中加入糖皮质激素氢化可的松（1、10、100 μg/ml）进行干预,并通过Real-time PCR检测此种情况下HCF中TLR的表达.再通过免疫荧光组化来检测TLR2、TLR4在蛋白水平的表达.各组数据的差异性采用Student t检验和非参数秩和检验,Real-time PCR的倍数间的比较采用非参数统计.结果 TLR1-10 mRNA在HCF中均有表达,且表达量各不相同,其中TLR2、TLR4、TLR5、TLR7、TLR8、TLR10呈高表达.LPS能增加HCF中TLR mRNA的表达,且当LPS为10 ng/ml时,TLR的表达量最高.加入氢化可的松干预后,随着激素浓度的增加,HCF中TLR mRNA的表达却随激素浓度的增高而下降.免疫荧光组化结果显示,在10 ng/ml LPS刺激、10 ng/ml LPS＋10 μg/ml氢化可的松共同刺激以及正常条件培养3种情况下,TLR2、TLR4在HCF内均有表达,且强度不同,趋势与分子水平的表达一致.结论 TLR1～10 mRNA在HCF中均有表达.LPS能改变角膜基质成纤维细胞TLR mRNA的表达,提示该细胞表达的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HCF TLR的表达,即对LPS刺激的HCF TLR的表达表现出抑制;提示当细菌的侵袭突破角膜上皮到达基质时,糖皮质激素起到了免疫抑制的作用.
Objective To evaluate the role of Toll-like receptors （TLR） in host responses to lipopolysaccharide （LPS） by the use of cultured immortalized human comeal fibroblasts （HCF） and to determine whether LPS can induce an antibacterial response in these cells; to evaluate if glucocorticoids can change this response; to investigate the relationship between glucocorticoids and TLR when comeal bacterial infection occurs. Methods The donor＇s corneal cell was separated, cultured and identified. Cultured HCF were stimulated with LPS （1, 10, 100 ng/ml） from bacteria, and the effect on the expression of TLR was determined by RT-PCR, Real-time polymerase chain reaction （PCR） and immunofluorescence. Cells were also co -cultured with LPS （10 ng/ml） and hydrocortisone （1, 10, 100 μg/ml） to determine whether hydrocortisone modulates the transcription of TLR. Results Incubation of HCF with LPS （1,10,100 ng/ml） up-regulated the expression of all TLR mRNA （TLR2 and TLR4 most noticeably）, and when LPS was 10 ng/ml, the expression of TLR mRNA was the highest. Immunofluorescence staining confirmed that expression of TLR2 and TLR4 was up -regulated in response to LPS. This up -regulation was inhibited by co -treatment with hydrocortisone, the expression of TLR mRNA decreased. Conclusion HCF is involved in the corneal immune response to LPS. TLR2 and TLR4 may play a crucial signaling role in response to LPS in HCF. Glucocorticoids can reduce the expression of the TLR on the corneal stroma and thus may reduce the resistance to LPS in the cornea. These findings may provide crucial information for understanding the immune mechanisms of bacterial keratitis and promote the design of new immune therapeutic approaches to bacterial keratitis.
出处
《中华眼视光学与视觉科学杂志》
CAS
2010年第2期104-109,共6页
Chinese Journal Of Optometry Ophthalmology And Visual Science
基金
浙江省自然科学基金资助项目（Y205672）
