抑制性寡脱氧核苷酸对小鼠CD4+T细胞T-bet、STAT4及STAT6表达的影响

Effect of suppressive oligonucleotides on mice CD4+T cell transcription factor and its significance

目的探讨体外实验中,抑制性寡脱氧核苷酸(Sup ODN)对小鼠脾脏CD4+T细胞转录因子T细胞表达的T盒(T-bet)、信号转导子和转录激动子(STAT)4及STAT6表达的影响。方法分离小鼠脾脏淋巴细胞,免疫磁珠阳选法纯化CD4+T细胞,流式细胞仪分析分选纯度,在抗CD3ε抗体、抗-CD28抗体、白细胞介素(IL)12作用下,加入Sup ODN或对照寡脱氧核苷酸(ConODN)培养72 h后,常规提取核蛋白。Western-blot测定核蛋白中磷酸化的STAT(pSTAT)4、pSTAT6和T-bet蛋白表达。结果与ConODN组和PBS组比较,Sup ODN组中T-bet和pSTAT4的表达水平明显降低(P<0.05),而pSTAT6表达水平明显升高(P<0.05)。结论 SupODN可能通过下调pSTAT4和T-bet的表达,体外抑制小鼠CD4+T细胞向Th1细胞的功能性分化,上调pSTAT6的表达,促进Th2细胞的分化。

Objective To evaluate the effect of suppressive oligodeoxynucleotides （Sup ODN） on expression of T- bet, pSTAT4 and pSTAT6 of CD4 ＋ T spleen lymphocytes in vitro. Methods The spleen lymphocytes of BALB/c mice were isolated and then CD4 ＋ T cells were purified with immunomagnetic microbeads（positive selection）, the purification was analysed by flow cytometry. The co-culture of CD4 ＋ ceils, anti CD3ε, anti-CD28, ILl2 and Sup ODN or control oligodeoxynucleotides（Con ODN） was performed for 72 hours, the nucleoprotein was extracted with regular procedure. Expression of pSTAT4（phosphorylated STAT4）, pSTAT6, and T bet in nucleoprotein were detected by Western blot. Results Compared with Con ODN and PBS treated group, expression of T-bet and pSTAT4 in Sup ODN treated group decreased significantly （P〈0. 05）, while expression of pSTAT6 in Sup ODN treated group increased significantly （P〈0. 05）. Conclusion Sup ODN may inhibit Thl differentiation of mice CD4 ＋ T cell by down-regulating expression of pSTAT4 and Tbet, and promote Th2 differentiation by up-regulating expression of pSTAT6.