核转录因子抑制剂对脂多糖诱导角膜基质细胞分泌细胞因子的影响  

The effects of the inhibitor of nuclear factor on lipopolysaccharide-induced cytokine expression in cultured human corneal fibroblasts

作　　者：陈国玲[1] 吴欣怡[2] 

机构地区：[1]山东大学第二医院眼科,济南250033 [2]山东大学齐鲁医院眼科

出　　处：《中华眼科杂志》2008年第1期61-66,共6页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目（0328026）

摘　　要：目的探讨二硫代氨基甲酸吡咯烷（PDTC）对绿脓杆菌脂多糖（LPS）诱导的人角膜基质细胞核因子κB（NF—κB）活化与细胞因子释放的干预作用及其机制。方法为实验研究。原代培养人眼角膜基质细胞（HCFs），选取生长良好的传代HCFs分为对照组、LPS刺激组及PDTC预处理组。LPS组给予单纯LPS刺激，PDTC预处理组则在LPS刺激前先加PDTC培养30min，再给予相同浓度的LPS。在LPS刺激1、2、4及8h时，分别收集各组细胞和上清液，Western blot检测HCFs NF—κB的活化表达；酶联免疫吸附实验（EHSA）检测HCFs培养上清液中自细胞介素6（IL-6）和IL-8的分泌情况；逆转录聚合酶链反应（RT—PCR）检测HCFs IL-6和IL-8 mRNA的表达。结果与对照组相比较，LPS刺激后，HCFs胞核NF—κB p65的表达明显升高，HCFs中IL-6与IL-8蛋白的分泌和mRNA的表达均显著升高。PDTC预处理30min后再给予LPS刺激，可部分抑制HCFs胞核中NF—κB p65的活化表达（t1h=9．3766，t2h=15．9011，t4h=12．5851，t8h=10．8346；均P〈0．01）；PDTC预处理可不同程度的下调LPS诱导的HCFs分泌IL-6、IL-8蛋白及IL-6、IL-8 mRNA的表达，二者均明显低于同时间点LPS组HCFs的表达，差异均有统计学意义（IL-6蛋白：t1h=7．9154，t2h=10．8630，t4h=8．2451，t8h=13．5063；IL-8蛋白：t1h=8．5663，t2h=20．5169，t4h=25．1580，t8h=34．8699；IL-6 mRNA：t1h=12．0235，t2h=13．2894，t4h=24．0799，t8h=27．2261；IL-8 mRNA：t1h=20．9424，t2h=24．1314，t4h=29．8580，t8h=47．9442；均P〈0．01）。结论绿脓杆菌LPS可引起HCFs NF—κB活化，促进细胞因子的表达，PDTC可部分抑制LPS对NF-κB的激活，下调相应细胞因子的表达。（中华眼科杂志，2008，44：6146）Objective To investigate the effects of pyrrolidine dithiocarbamate （PDTC） on Lipopolysaccharide（LPS） -mediated activation of nuclear factor kappa B（NF-κB） and cytokine expression in cultured human corneal fibroblasts. Methods It was a experimental study. A completely random design was employed in this research. Human corneal fibroblasts （HCFs） were obtained from human specimen. HCFs were divided into three groups, control group（ group 0 h） ,LPS alone group and PDTC treatment group. Cells were incubated with PDTC for 30 min in the PDTC pretreatment group before LPS challenged. At different time after LPS challenged, the activities of NF-κB were assessed by Western Blot analysis, the secretion of IL-6 and IL-8 from cultured corneal fibroblasts was measured with enzyme-linked immunosorbent assays （ELISA） ; the mRNAs expression of IL-6 and IL-8 was determined by reverse transcription polymerase chain reaction （RT-PCR）. The effects of PDTC on activation of NF-κB and the expression of IL-6 and IL-8 were also assessed in HCFs challenged with LPS. Results Compared with control group, NF-κB level was significantly enhanced in the nucleus in the LPS alone group, indicating that LPS mediated the activation of NF-κB in HCFs. The activation of NF-κB by LPS was markedly inhibited by PDTC （ t1h = 9. 3766, t2h = 15. 9011, t4h = 12. 5851, t8h = 10. 8346,P 〈 0. 01 ）. Compared with control group, LPS increased IL-6 and IL-8 expression both mRNA and protein in HCFs. At the same time, PDTC partly inhibited the expression of IL-6 and IL-8 in corneal fibroblasts induced by LPS. These inhibitory effects were significant at both the mRNA and protein levels （ protein of IL-6 ：t1h = 7. 9154, t2h = 10. 863, t4h = 8. 2451, t8h = 13. 5063. protein of IL-8 ： t1h = 8. 5663, t2h = 20. 5169, t4h = 25. 1580, t8h = 34. 8699. mRNA of IL-6 ： t1h = 12. 0235, t2h = 13. 2894, t4h = 24. 0799, t8h = 27. 2261. mRNA of IL-8 ： t1h = 20. 9424, t2h = 24. 1314, t4h = 29. 8580, t8h = 47. 9442. P 〈 0. 01 ）. C

关 键 词：角膜基质 硫代氨基甲酸酯类 吡咯烷类 NF—κB 细胞因子类 

分 类 号：R686[医药卫生—骨科学]