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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 To clone the secretory leukocyte protease inhibitory (SLPI) gene and express human SLPI recombinant protein and explore its antagonism of neutrophil release of inflammatory mediators. Methods Human peripheral blood neutrophil mRNA was extracted from human peripheral blood mononuclear cells (PBMCs). The SLPI gene was cloned and the plasmids were constructed. After transfection, the protein was extracted and purified. After the experimental grouping, SLPI and LPS co-treated neutrophils. Group A: normal control group (without LPS and recombinant SLPI protein treatment); Group B: LPS treatment group (LPS with final concentration of 0.001 g · L-1); Group C: low dose recombinant SLPI protein intervention group (P <0.05); Group D: Medium dose of recombinant SLPI protein intervention group (SLPI protein 0.050 g · L-1 and the final concentration of 0.001 g · L-1 LPS); Group E: high-dose recombinant SLPI protein intervention group (LPS with 0.100 g · L-1 SLPI protein and 0.001 g · L-1 final concentration). Neutrophil elastase (NE) activity test, inflammatory mediator release test and transcription factor STAT3 activity test were carried out. The activity of NE, the expression of IL-6 / IL-8 in inflammatory mediators and the DNA binding of STAT3 active. Results The eukaryotic expression vector of full-length cDNA of SLPI gene was successfully constructed. The recombinant SLPI protein inhibited the LPS-induced NE activity in a dose-dependent manner [(17.78 ± 0.54) nmol·L-1 vs (5.46 ± 0.12) nmol·L-1 , P <0.05]. The recombinant SLPI protein also significantly inhibited IL-6 [(179.51 ± 6.61) ng · L-1vs (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 capacity of STAT3 was increased. Conclusion SLPI can effectively inhibit NE activity and reduce inflammatory injury.