反义肽核酸抑制树突细胞趋化因子受体CCR7表达的实验研究

目的探讨靶向趋化因子受体CCR7的反义肽核酸(PNA)对树突细胞(DCs)CCR7的表达及趋化活性的影响。方法体外培养大鼠骨髓来源DCs,设计靶向CCR7mRNA翻译起始区的反义PNA,以随机PNA、空白组为对照处理体外培养7d的DCs,分别于24h,48h,72h后收集细胞,利用Western-blot和逆转录聚合酶链反应(RT-PCR)检测反义PNA对CCR7分子表达的影响,通过趋化实验检测DCs的趋化活性。结果DCs经PNA处理后24h,各组DCs其CCR7表达无明显差别,在48h,Western-blot检测显示反义PNA组CCR7蛋白表达明显低于对照组,而RT-PCR检测在mRNA水平却无明显差别。在72h,反义PNA组CCR7的表达在不同水平均明显低于对照组。经趋化实验证实,在48h以后反义PNA组DCs趋化活性受到明显抑制(P<0.05)。结论CCR7反义PNA能够有效抑制体外培养的大鼠树突细胞趋化因子受体CCR7的表达及其趋化活性,为进一步研究体内应用反义PNA抑制DCs的定向迁移和抗原递呈,调节免疫反应和诱导免疫耐受提供了新的策略。

Inhibitation of gene expression in rat dendritic cells by antisense peptide nucleic acid against chemokine receptor CCR7

Objective To block the expression of CCR7 in dendritic cells(DCs) by using antisense peptide nucleic acid (PNA) so that a suppression of chemotaxis activity can be achieved. Methods DCs from rat bone marrow cells were induced and cultured. Antisense PNA were designed for targeting at the AUG coding region of CCR7 mRNA and incubated with rat bone marrow derived DCs,and setting random PNA groups and blank control groups for comparison. At 24,48,and 72 h,the expression of CCR7 in incubated DCs samples were observed with Western-blot and RT-PCR respectively, and the chemotaxis assays were performed on DCs in presence of secondary lymphoid tissue chemokine(SLC). Results Compared with blank control groups,at 48 h,DCs treated by antisense PNA showed significantly lower expression of CCR7 by Western-blot,but no difference in the mRNA level by RT-PCR.At 72 h,the expression levels of CCR7 were all decreased significantly. As a result, the chmotaxis activities of DCs were inhibited in response to CCR7 expression(P<0.05). Conclusion Antisense PNA targeting CCR7 can inhibit the expression of CCR7 in DCs so as to suppress the chmotaxis activity of DCs in vitro.The results indicate that the strategy of using PNA against the CCR7 gene in vivo may be a promising approach in trapping migratory capacity of DCs and inducing allogeneic tolerance.