热休克因子1基因转染对烧伤血清刺激的巨噬细胞炎性介质表达的影响

Influence of heat shock factor 1 gene transfection on the expression of inflammatory mediators in macrophages induced by bum serum

目的了解外源性热休克因子1(HSF 1)对烧伤血清刺激的巨噬细胞中相关炎性介质基因表达的影响。方法制备严重烧伤和正常大鼠血清;构建pcDNA 3.1/HSF 1重组载体。将培养的RAW 264.7巨噬细胞株转染(具体分为:未转染、转染空载体、转染重组载体)后再分别使用前述2种血清刺激。另取部分未行血清刺激的转染重组载体的巨噬细胞,用蛋白质印迹法检测其HSF 1蛋白表达水平;反转录-PCR法检测经血清刺激的细胞中肿瘤坏死因子α(TNF-α)、高迁移率族蛋白B1 (HMGB 1)和白细胞介素10(IL-10)基因表达水平。结果已转染重组载体的细胞株较稳定,检出有HSF 1部分活化。反转录-PCR检测到,未转染的正常血清刺激的巨噬细胞中TNF-α、HMGB 1、IL-10的基因仅有少量表达,而烧伤血清刺激能明显上调其基因表达(分别为0.910±0.100、0.860±0.020、0.430±0.010);与转染空载体+烧伤血清组(上述3项指标分别为0.800±0.050、0.880±0.030、0.420±0.010)比较,转染重组载体+烧伤血清组可明显抑制巨噬细胞中TNF-α和HMGB 1的基因表达并上调IL-10的基因表达(分别为0.130±0.100、0.450±0.020、0.450±0.020)。结论严重烧伤后给予HSF 1可以抑制巨噬细胞产生的某些促炎介质的表达,适当上调某些抗炎介质的表达,对机体发挥保护作用。

Objective To investigate the influence of heat shock factorl （ HSF1 ） on gene expression of inflammatory mediators in RAW264.7 murine macrophage cells induced by burn serum. Methods Sera were separated from blood of normal rats and rats with severe burns, and the recombinant vector pcDNA3. 1/HSF1 was constructed. RAW264.7 macrophages were divided into non-transfection group, vacant vector group （with burn and normal sera stimulation, respectively after vacant vector transfection） and recombinant vector group （ with burn and normal sera stimulation, respectively after recombinant vector transfection）. Some recombinant vector transfected macrophages without serum stimulation were prepared for the determination of HSF 1 expression with Western blotting. The mRNA expressions of TNF-α, HMGB 1 and IL-IO were determined with RT-PCR. Results The cell line attained after recombinant vector transfection was comparatively stable,with partial activation of HSF 1. Burn sera markedly upregulated TNF-α, HMGB1 mRNA expression （0.910 ±0. 100, 0. 860 ±0. 020, respectively ） , but downregulated IL-10 expression （ 0. 430 ± 0.010, respectively） in normal macrophages,while these genes maintained in a very low level in normal macrophages with normal serum stimulation, macrophages with recombinant vector transfection and burn serum stimulation could obviously inhibit the expression of TNF-α and HMGB 1, but enhance the IL-IO gene expression （0. 130 ±0. 100, 0. 450 ±0. 020 , 0. 450 ±0. 020, respectively ） when compared with that with vacant vector transfection and burn serum stimulation （ 0. 800 ±0. 050,0. 880 ±0. 030,0. 420 ±0. 010, respectively）. Conclusion HSF1 can inhibit the expression of some pro-inflammatory mediators in macro- phages after a severe burns, indicating that appropriate upregulation of anti-inflammtory mediators might exert protective effects on the organism.