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目的:分析第10号染色体缺失的磷酸酶和张力蛋白同源基因(PTEN)对氧化低密度脂蛋白(ox-LDL)诱导的巨噬细胞炎症因子影响及其作用机制。方法:将构建pc DNA3.1(+)-PTEN(r PTEN)重组表达载体及PTEN siRNA转染小鼠巨噬细胞系RAW 264.7,用制备的50 mg/L的ox-LDL孵育24 h,RT-PCR及Western blot分析PTEN的mRNA及蛋白表达水平;ELISA检测炎性因子TNF-α和IL-6的水平;同时Western blot分析对Toll样受体4(TLR4)及转录因子-κB(NF-κB)的影响及其作用机制。结果:PTEN过表达增高了ox-LDL诱导的TNF-α和IL-6的水平;PTEN沉默抑制了ox-LDL诱导的TNF-α和IL-6炎性因子水平。进一步分析表明,PTEN过表达加强了巨噬细胞中ox-LDL诱导的TLR4及其下游NF-κB通路的活化,而抑制其表达后,TLR4-NF-κB通路明显受到抑制;用TLR4特异性抗体预处理后,PTEN过表达诱导的TNF-α和IL-6的水平明显下降。进一步机制分析证实,磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)抑制剂LY294002(1μmol/L)预处理后,PTEN沉默抑制的TLR4-NF-κB通路的活性明显增加,且伴随有TNF-α和IL-6水平的上调。结论:PTEN有可能通过负向调节PI3K/AKT抗炎通路来影响TLR4-NF-κB炎性通路,进而参与巨噬细胞介导的炎症进程。因此,本研究将为心脑血管疾病的防治提供新的靶标。
OBJECTIVE: To analyze the effect of PTEN on the inflammatory cytokines induced by ox-LDL on chromosome 10 and its mechanism of action. Methods: RAW 264.7 cells were transfected with recombinant plasmid pcDNA3.1 (+) - PTEN (r PTEN) and PTEN siRNA and incubated with 50 mg / L ox-LDL for 24 h. RT The mRNA and protein expression of PTEN were analyzed by PCR and Western blot. The levels of TNF-α and IL-6 were detected by ELISA. The expressions of TLR4 and NF- κB) and its mechanism of action. Results: Overexpression of PTEN increased the levels of TNF-α and IL-6 induced by ox-LDL; PTEN silencing inhibited the levels of TNF-α and IL-6 induced by ox-LDL. Further analysis showed that overexpression of PTEN enhanced ox-LDL-induced activation of TLR4 and its downstream NF-κB pathway in macrophages, and inhibition of TLR4-NF-κB pathway was significantly inhibited by PTEN overexpression; TLR4-specific antibody Pretreatment, PTEN overexpression induced TNF-α and IL-6 levels were significantly decreased. Further mechanism analysis confirmed that PTEN silencing-inhibited TLR4-NF-κB pathway activity was significantly increased following pretreatment with PI3K / Akt inhibitor LY294002 (1 μmol / L) There is upregulation of TNF-α and IL-6 levels. CONCLUSION: PTEN may affect the inflammatory pathway of TLR4-NF-κB through negative regulation of PI3K / AKT anti-inflammatory pathway, which may be involved in the process of macrophage-mediated inflammation. Therefore, this study will provide a new target for the prevention and treatment of cardiovascular and cerebrovascular diseases.