表面活性蛋白A在脂多糖诱导的人肾小管上皮细胞中的表达

Lipopolysaccharide-induced expression of surfactant protein A subtypes in human renal tubular epithelial cells

目的 研究表面活性蛋白A（SP-A）及其亚型在人肾组织和人肾小管上皮细胞（HK-2）的表达和分布,同时分析脂多糖（LPS）对HK-2细胞中SP-A亚型的mRNA和蛋白表达的影响.方法 收集10例人的肾组织,以5例人的肺组织作为对照,同时培养HK-2细胞.免疫组化法检测SP-A在人肾组织的表达部位;RT-PCR法检测SP-A mRNA在人肾组织和HK-2细胞中的表达;限制性内切酶片段长度多态性（RFLP）和测序的方法分析SP-A亚型在HK-2细胞的表达;实时定量PCR法比较SP-A mRNA在人肾组织和在人肺组织中的相对含量;Western 印迹法检测人肾组织和HK-2细胞中SP-A蛋白的表达;Western印迹和ELISA法检测人的尿液和HK-2细胞培养上清液中SP-A的分泌量.RT-PCR和Western印迹检测LPS在不同浓度（0、0.1、1、2、5、10 mg/L）作用8 h及5 mg/L LPS在不同时间（0、2、4、8、16、24 h）作用HK-2细胞后,SP-A mRNA和蛋白表达的变化情况.结果 免疫组化结果显示SP-A主要表达在肾皮质的远曲和近曲小管的肾小管上皮细胞.RFLP和测序的方法均证实HK-2细胞可同时表达SP-A1和SP-A2亚型.实时定量PCR证实SP-A mRNA在人肾组织的表达量仅为肺组织的30%（n=5）.Western印迹检测到人肾组织和HK-2细胞可表达SP-A蛋白.同时在人的尿液和HK-2细胞培养上清液中也检测到SP-A的分泌,其分泌量分别为（106.614±72.772）nmol/L（n=30）和（85.533±58.622）nmol/L（n=10）.应用1、2、5、10 mg/L的LPS刺激HK-2细胞8 h后,SP-A1和SP-A2 mRNA及SP-A蛋白表达较0、0.1 mg/L显著升高（P＜0.05）;同时,应用5 mg/L的LPS作用HK-2细胞4、8、16、24 h后,SP-A1和SP-A2 mRNA及SP-A蛋白表达较LPS作用0、2 h显著升高（P＜0.05）.结论 HK-2细胞能同时表达SP-A1和SP-A2亚型,能产生和分泌SP-A蛋白.SP-A1和SP-A2可能在肾脏的天然免疫和炎性反应调节方面起重要作用.

Objective To determine the surfactant protein A （SP-A） subtype distribution and expression in human renal tissue and cultured human renal tubular epithelial cells（HK-2）, and to explore the influence of Lipopolysaccharide （LPS） on the expression of SP-A subtypes mRNA and SP-A protein. Methods lmmunohistochemical staining was performed using SP-A polyclonal antibodies. RT-PCR was performed with mRNA from HK-2 cells and normal human kidney.Restriction fragment length polymorphism（RFLP） and sequencing were used to evaluate the subtypes of SP-A. The relative content of SP-A mRNA in human kidney and human lung was compared by real-time PCR. Western blotting analysis for SP-A was performed on protein from renal tissue and cultured HK-2 cells SP-A protein in human urine and culture supernatant of HK-2 cells was measured by enzyme-linked immunosorbent assay （ELISA） and Western blotting respectively. HK-2cells were treated with LPS at various concentrations （0,0.1,1,2,5,10 mg/L） for 8 h and at 5mg/L for various time points （0,2,4,8,16,24 h）. Expression of SP-A mRNA and protein was analyzed by RT-PCR and Western blotting. Results SP-A was localized in renal tubular epithelial cells of both proximal and distal convoluted tubules. SP-A1, SP-A2 mRNA and protein could be detected in normal HK-2 cells and human kidney. The significant secretion of SP-A [urine： （106.614172.772） nmol/L, n=30; culture supernatant： （85.533±58.622） nmol/L, n=10] was shown. The levels of SP-A1, SP-A2 mRNA and Sp-A protein in HK-2 cells were significantly decreased after treatment with LPS. Conclusions Human renal tubular epithelial cells can express both SP-A1 and SP-A2 genes which may play an important role in inflammation modulation of kidney.