【摘要】 目的研究靶向趋化因子受体CCR7的反义肽核酸(PNA)对树突状细胞(DC)CCR7蛋白表达及凋亡的影响,并探讨其凋亡机制。方法体外培养大鼠骨髓来源的DC,脂多糖(LPS)诱导成熟。设计靶向CCR7 mRNA翻译起始区的反义PNA,以随机PNA和空白组为对照处理体外培养7d的未成熟DC,脂多糖诱导48h后收集成熟DC,免疫细胞化学染色法检测CCR7蛋白表达,流式细胞术检测细胞凋亡率,Western blot检测磷酸化Akt和Akt蛋白表达。应用渥曼青霉素(wortmannin)抑制磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)/Akt信号通路进一步观察磷酸化Akt蛋白表达和细胞凋亡率的变化。结果反义PNA组DC CCR7蛋白表达明显低于随机PNA组和空白组,而细胞凋亡率却明显增高,差异有统计学意义(P<0.05),Western blot检测显示反义PNA组Akt蛋白磷酸化水平明显低于随机PNA组和空白组,差异有统计学意义(P<0.05)。DC经PI3K抑制剂预处理后,CCR7介导的Akt蛋白磷酸化及抗凋亡作用被阻断。结论CCR7反义PNA能够有效抑制体外培养的大鼠DC趋化因子受体CCR7的表达并导致其发生凋亡,其机制可能是阻断了CCR7介导P13K/Akt信号转导通路的活化。更多还原

【Abstract】 Objective To investigate anti-apoptotic mechanism through which CCR7 regulates the sur- vival of mature dendritic cells(DC)by blocking the expression of CCR7 in DC.Methods Antisense peptide nu- cleic acid(PNA)was designed targeting at the AUG coding region of CCR7 mRNA and incubated with immature DC for 7 days.Expression of CCR7 and apoptosis of the incubated mature DC samples were observed with immuno- cytochemical staining and flow cytometric analysis.Western blot was applied to assess the levels of phosphorylated Akt and total Akt proteins.Wortmannin was used to study if inhibition of this kinase affected the survival of DC. Results Compared with mismatch PNA and blank control groups,mature DC treated by antisense PNA showed a significantly lower expression of CCR7 protein with higher apoptosis rate shown by flow cytometric analysis.West- em blot results showed that phosphorylation of Akt in DC treated by antisense PNA was significantly decreased. Pretreatment of DC with inhibitor abrogated PI3K activity,the phosphorylation of Akt,and the effects of CCR7 on the survival of the cells.Conclusion Antisense PNA targeting CCR7 can suppress the expression of CCR7 in DC, and the absence of CCR7 may contribute to apoptosis of these cells in vitro.CCR7 antisense PNA can demonstrate the proapoptotic results through blocking the PI3K/Akt cell signal transduction mediated by CCR7.更多还原