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目的:观察炎调方药物血清对离体巨噬细胞中热休克蛋白70(HSP70)及其他相关细胞因子的调控效应,以验证炎调方治疗脓毒症的作用途径和位点。方法:培养大鼠巨噬细胞RAW 264.7,将培养后的巨噬细胞RAW 264.7分为空白对照组(RAW 264.7+培养液)、正常血清组(RAW 264.7+正常大鼠血清)、炎调方药物血清组(RAW 264.7+炎调方药物血清),每组设4个平行孔。采用MTT比色法测定细胞增殖,确定炎调方药物血清的无毒性剂量范围;采用qRT-PCR方法检测细胞HSP70、核因子-κB(NF-κB)、肿瘤坏死因子(TNF)-α、白介素(IL)-1β、IL-8的mRNA表达;采用Western blot方法检测细胞HSP70、NF-κB蛋白表达,以ELISA方法检测细胞TNF-α、IL-1β、IL-8蛋白表达。结果:炎调方药物血清的无毒性浓度范围包括10%~30%,故后续实验选取20%浓度的炎调方药物血清进行。正常血清组的各项指标与空白对照组比较,其差异均无统计学意义(P>0.05);与空白对照组和正常血清组比较,炎调方药物血清组RAW 264.7细胞的HSP70 mRNA和蛋白表达均明显升高(P<0.01),NF-κB、TNF-α、IL-1β、IL-8的mRNA和蛋白表达均明显降低(P<0.01)。结论:炎调方治疗脓毒症的作用机制可能是上调HSP70的表达,进而抑制NF-κB的活化和相关细胞因子的表达。
Objective: To observe the regulation effect of Yanjianfang serum on heat shock protein 70 (HSP70) and other related cytokines in ex vivo macrophages in order to verify the action pathway and site of Yanjianfang recipe in treating sepsis. Methods: RAW 264.7 rat macrophages were cultured in vitro. The cultured macrophages RAW 264.7 were divided into control group (RAW 264.7+ medium), normal serum group (RAW 264.7 + normal rat serum) Serum group (RAW 264.7 + Inflammatory drug serum) with 4 parallel wells in each group. The cell proliferation was determined by MTT colorimetric assay, and the non-toxic dose range of the serum was determined. The expression of HSP70, NF-κB, TNF-α, interleukin (IL) -1β and IL-8. Western blot was used to detect the expression of HSP70 and NF-κB, and the protein expressions of TNF-α, IL-1β and IL-8 were detected by ELISA. Results: The non-toxic concentration range of Yanjianfang drug serum ranged from 10% to 30%. Therefore, 20% concentration of Yanfangfang serum was chosen for follow-up experiment. Compared with the blank control group and the normal serum group, the indexes of the normal serum group had no significant difference compared with the blank control group (P> 0.05); compared with the blank control group and the normal serum group, the HSP70 mRNA and protein (P <0.01). The mRNA and protein expressions of NF-κB, TNF-α, IL-1β and IL-8 were significantly decreased (P <0.01). Conclusion: The mechanism of Yan-ton prescription in treating sepsis may be to up-regulate the expression of HSP70 and then inhibit the activation of NF-κB and the expression of related cytokines.