重组蛋白酶抑制蛋白拮抗中性粒细胞释放炎性介质的作用  

Role of Recombination Protein of Secretory Leukocyte Protease Inhibitor Suppressing the Release of Inflammatory Mediators from Neutrophilic Leukocyte

作　　者：胡毓华[1] 朱亮华[1] 陆超[1] 

机构地区：[1]南京医科大学第一附属医院儿科,南京210029

出　　处：《实用儿科临床杂志》2012年第10期760-762,共3页Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金(81170487);江苏省卫生厅医学科技发展基金(H201022)

摘　　要：目的克隆分泌型白细胞蛋白酶抑制(SLPI)蛋白基因并表达人SLPI重组蛋白,探讨其拮抗中性粒细胞释放炎性介质的作用。方法抽提人外周血中性粒细胞mRNA,克隆SLPI基因,构建质粒,转染后抽提纯化蛋白,实验分组后,分别进行不同剂量的SLPI和内毒素(LPS)共处理中性粒细胞。A组:正常对照组(不予LPS和重组SLPI蛋白处理);B组:LPS处理组(加终质量浓度0.001 g·L-1的LPS);C组:低剂量重组SLPI蛋白干预组(加重组SLPI蛋白0.010 g·L-1和终质量浓度0.001 g·L-1的LPS);D组:中剂量重组SLPI蛋白干预组(加重组SLPI蛋白0.050 g·L-1和终质量浓度0.001 g·L-1的LPS);E组:高剂量重组SLPI蛋白干预组(加重组SLPI蛋白0.100 g·L-1和终质量浓度0.001 g·L-1的LPS)。然后进行中性粒细胞弹性蛋白酶(NE)活性实验、炎性介质释放实验以及转录因子STAT3活性实验,分别测定NE活性、炎性介质IL-6/IL-8表达量及转录因子STAT3的DNA结合活性。结果成功构建SLPI基因全长cDNA的真核表达载体,发现重组SLPI蛋白呈剂量依赖性地抑制LPS诱导的NE活性[(17.78±0.54)nmol·L-1vs(5.46±0.12)nmol·L-1,P<0.05]。重组SLPI蛋白也显著抑制LPS诱导的炎性介质IL-6[(179.51±6.61)ng·L-1vs(63.13±4.55)ng·L-1,P<0.05]和IL-8[(192.98±7.65)ng·L-1 vs(103.42±4.76)ng·L-1,P<0.05]的释放及转录因子STAT3的DNA结合力升高。结论 SLPI可有效抑制NE活性,减轻炎性损伤。Objective Through cloning the secretory leukocyte protease inhibitor （SLPI） gene, and SLP1 preparation of recombinant protein, explore the role of SLPI on antagonisting the release of inflammation from neutrophils. Methods mRNA was extracted from peripheral blood, SLPI gene was cloned into plasmid and protein was expressed. Experimental groups were divided with different dosages of SLPI and LPS. Group A ： normal control group without treatment of LPS and restructured SLPI protein. Group B ： neutrophils were treated with LPS at the concentration of 0. 001g·L^-1. Group C ：neutrophils were treated with 0. 010g·L^-1 SLPI protein and 0. 001g·L^-1 LPS. Group D：neutro- phils were treated with 0. 050 g·L^-1 SLPI protein and 0. 001 g·L^-1 LPS. Group E ： neutrophils were treated with 0. 100ng·L^-1 SLPI protein and 0. 001g·L^-1 LPS. Then the activity experiment of neutrophil elastase （NE） was performed, and releases of inflammatory factors IL-6/ IL-8 were determined. The DNA binding activity of transcription factor STAT3 was detected. Results The eukaryotic expression vector contai- ning was successfully constructed. Restructured SLPI protein inhibited LPS - induced activation of NE [ （ 17.78 ± 0.54） nmol·L^-1 vs （5.46 ± 0.12） nmol·L^-1,P〈0.05],the releases of inflammatory factors IL-6[（179.51 ±6.61） ng·L^-1 vs （ 63.13±4.55） ng·L^-1,P〈 0.05 ] and IL-8 [ （ 192.98 ± 7.65 ） ng·L^-1 vs （ 103.42± 4.76 ） ng·L^-1, p 〈 0.05] , and the DNA binding activity of transcription factor STAT3 in neutrophils were in a way of dose - dependent. Conclusion SLPI suppressed the activity of NE and relieved the damage of inflammation.

关 键 词：分泌型白细胞蛋白酶抑制蛋白 弹性蛋白酶 炎性介质 转录因子 

分 类 号：R363[医药卫生—病理学]