摘要
目的 研究表面活性蛋白D（SP-D）高表达对脂多糖诱导人肾小管上皮细胞（HK-2）单核细胞趋化蛋白1（MCP-1）表达的影响及其机制.方法 间接免疫荧光、Western印迹及RT-PCR检测SP-D蛋白及mRNA在HK-2细胞内的表达.脂多糖在不同浓度（0、0.1、1、2、5、10 mg/L）作用8 h及5 mg/L脂多糖作用HK-2细胞不同时间（0、2、4、8、16、24 h）,采用Western印迹和实时定量PCR检测SP-D表达,ELISA和实时定量PCR检测MCP-1表达.脂质体转染法将pEE14-hSP-D质粒转染HK-2细胞,筛选稳定转染细胞株,Westem印迹检测转染后SP-D蛋白表达.采用5 mg/L脂多糖刺激pEE14-hSP-D稳定转染的HK-2细胞8 h,ELISA和实时定量PCR检测HK-2细胞MCP-1表达.结果 HK-2细胞表达SP-D.脂多糖可诱导MCP-1 mRNA及蛋白表达显著增高（P〈0.05）;并町诱导SP-D mRNA及蛋白表达显著降低（P〈0.05）.转染pEEl4-hSP-D质粒使HK-2细胞高表达SP-D,并可下调脂多糖诱导的MCP-1表达（P〈0.01）.结论 SP-D可通过下调脂多糖诱导的HK-2细胞MCP-1的表达,从而在肾脏炎性反应中起重要作用.
Objective To investigate the effect of surfactant protein D（SP-D）overexpression on lipopolysaccharide（LPS）-induced monocyte chemoattractant protein-1（MCP-1）expression in human renal proximal tubular epithelial cells（HK-2）and its mechanism. Methods HK-2 cells were treated with LPS at various concentrations （0, 0.1, 1, 2, 5, 10 mg/L）for 8 h and at 5 mg/L for various time points（0, 2, 4, 8, 16, 24 h）. Expression of SP-D was detected by Western blotting and real-time PCR. Expression of MCP-1 was determined by ELISA and real-time PCR. Human SP-D cDNA eukaryotic expression vector pEE14-hSP-D was transfected to HK-2 cells. The changes in transfected cells of SP-D protein were observed by Western blotting. Expression of MCP-1 was detected by ELJSA and real-time PCR. Results SP-D was expressed in HK-2 cells. The levels of SP-D protein and mRNA in HK-2 cells were significantly decreased after treatment with LPS（P〈0.05）. Expression of MCP-1 protein and mRNA was increased remarkably after treatment with LPS（P〈0.05）. HK-2 cells transfected with pEE14-hSP-D showed up-regulated expression of SP-D. The overexpression of SP-D inhibited the LPS-inducedexpression of MCP-1（P〈0.01）. Conclusions SP-D inhibits LPS-induced expression of MCP-1 in HK-2 cells. SP-D may play an important role in the modulation of renal inflammation.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2010年第8期609-613,共5页
Chinese Journal of Nephrology
基金
国家自然科学基金（30670985）
