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目的利用siRNA沉默THP-1源性巨噬细胞(Mφ)的髓样分化因子88(MyD88)基因表达,论证MyD88在介导重组人热休克蛋白60(rhHSP60)诱导Mφ炎症反应中的作用及益气活血方宁心痛颗粒含药血清的抗炎机制。方法 THP-1细胞诱导分化为Mφ,分为空白对照组、模型组、阴性对照组、RNAi组(MyD88~(-/-)Mφ)、含药血清组、RNAi+含药血清组共6组,荧光定量PCR法测定MyD88基因表达,免疫印迹法测定MyD88及磷酸化核因子κB(p-NF-κB)的蛋白表达,酶联免疫法测定TNF-α、IL-6的含量。结果 1模型组MyD88基因表达升高,与空白对照组相比,差异有统计学意义(P<0.01);RNAi组、含药血清组分别与模型组相比表达均下降,差异有统计学意义(P均<0.01);含药血清可增强siRNA的抑制作用,RNAi+含药血清组与RNAi组相比,差异有统计学意义。2模型组MyD88及p-NF-κB蛋白表达升高,与空白对照组相比,差异有统计学意义(P均<0.01);RNAi组二者表达均下降,与模型组相比,差异有统计学意义;含药血清可增强siRNA的抑制作用,RNAi+含药血清组与空白对照组相比,差异无统计学意义。3模型组TNF-α、IL-6含量升高,与空白对照组相比,差异有统计学意义(P均<0.01);转染MyD88 siRNA可抑制二者升高,RNAi组与模型组相比,差异具有统计学意义(P均<0.01);含药血清单独使用可抑制二者升高,并可增强siRNA的抑制效果,RNAi+含药血清组与RNAi组相比差异有统计学意义(P均<0.01)。结论 MyD88/NF-κB是人热休克蛋白60诱导THP-1源性Mφ发生炎症反应的关键信号通路,宁心痛颗粒含药血清可通过调控该信号通路发挥抗炎作用。
Objective To investigate the role of MyD88 in inducing Mφ-induced inflammation in THP-1-derived macrophages (Mφ) induced by recombinant human heat shock protein 60 (rhHSP60) Anti-inflammatory mechanism of qihuoxue fangning heartache granule containing serum. Methods THP-1 cells were induced to differentiate into Mφ and divided into blank control group, model group, negative control group, RNAi group (MyD88 ~ (- / -) Mφ), drug-containing serum group and RNAi + drug- The expression of MyD88 gene was detected by real-time quantitative PCR, the protein expressions of MyD88 and phosphorylated nuclear factor κB (p-NF-κB) were measured by Western blotting, and the levels of TNF-α and IL-6 were determined by enzyme-linked immunosorbent assay. Results 1 MyD88 gene expression in model group increased, compared with the blank control group, the difference was statistically significant (P <0.01); RNAi group, drug-containing serum group compared with the model group, the expression was decreased, the difference was statistically significant (All P <0.01). The serum containing siRNA enhanced the inhibitory effect of siRNA. The difference between RNAi + RNAi group and RNAi group was statistically significant. 2 model group increased the expression of MyD88 and p-NF-κB protein, compared with the blank control group, the difference was statistically significant (P <0.01); RNAi group both decreased, compared with the model group, the difference was Statistical significance; containing serum can enhance the inhibition of siRNA, RNAi + drug-containing serum group compared with the control group, the difference was not statistically significant. 3 and TNF-α, IL-6 in model group were significantly higher than those in control group (all P <0.01). MyD88 siRNA inhibited the expression of TNF-α and IL-6, (P <0.01). Compared with RNAi group, the RNAi + drug-containing serum group had significant difference (P <0.01). The drug-containing serum alone could inhibit both of them and enhance the inhibitory effect of siRNA. P <0.01). Conclusions MyD88 / NF-κB is the key signal pathway of THP-1-derived Mφ-induced inflammatory reaction in human Hsp90. Ning-Ning-Tong Granules containing serum can exert anti-inflammatory effects by regulating this signaling pathway.