DHHCs家族成员在小鼠CD4^+CD25^-T细胞和CD4^+CD25^+调节性T细胞活化中的表达及意义

The expression of DHHCs family members in murine activated CD4^+CD25^-T cells and activated CD4^+CD25^+ regulatory T cells

目的观察DHHCs家族成员在小鼠CD4+CD25-T细胞和CD4+CD25+调节性T细胞活化前后的表达情况并探讨其意义。方法磁珠法从小鼠脾脏分选CD4+CD25-T细胞和CD4+CD25+调节性T细胞,流式细胞术检测纯度后,体外用anti-CD3e/CD28抗体分别刺激其活化。TRIzol法提取细胞总RNA,RT-PCR逆转录成cD-NA,分别用24个DHHCs家族成员特异性引物进行PCR反应,对产物进行琼脂糖凝胶电泳分析。结果活化前CD4+CD25-T细胞高表达DHHC5、DHHC14和DHHC18,活化后其上调表达DHHC2、DHHC4、DHHC6、DHHC8、DHHC9、DHHC12、DHHC15和DHHC16;活化前CD4+CD25+调节性T细胞仅表达DHHC9,活化后其上调表达DH-HC18,而下调DHHC9的表达。结论 DHHCs家族成员在CD4+CD25-T细胞和CD4+CD25+调节性T细胞中表达存在差异,活化后表达谱发生显著变化,提示DHHCs家族成员的表达与CD4+T细胞的活化有关,可能参与CD4+T细胞不同亚群活化的调控。

Objective To detect the expression of DHHCs family members in murine activated CD4 ＋ CD25 - T cells and CD4 ＋ CD25 ＋ regulatory T cells and further explore the significance. Methods CD4＋ ＋ CD25 ＋ regulatory T cells （Tregs） and CD4 ＋ CD25- T cells were purified by Magnetic activated cell sorter （MACS） from murine splenocytes and identified by flow cytometry. Tregs and CD4 ＋ CD25- T cells were stimulated by anti -CD3e and CD28 antibodies in the presence of recombinant interleukin - 2, respectively. Total RNA of activated cells was extracted by TRIzol and reversely transcribed using First Strand cDNA Synthesis Kit. DHHCs family members expression was detected by PCR and analyzed by electrophoresis. Results Unactivated CD4 ＋ CD25 - T cells expressed high level of DHHC5, DHHC14 and DHHC18. However, the expression of DHHC2, DHHC4, DHHC6, DHHC8, DHHC9, DHHC12, DHHC14, DHHC15, DHHC16 and DHHC18 were significantly up -regulated in activated CD4＋ CD25-T cells. Unactivated CD4＋ CD25 ＋ Tregs only expressed DHHC9, whereas activated CD4＋ CD25 ＋ Tregs expressed high level of DHHC18. Furthermore, DHHC9 expression in activated CD4 ＋ CD25 ＋ Tregs was remarkably decreased. Conclusion DHHCs family members expression in unactivated CD4 ＋ CD25 ＋ Tregs and unactivated CD4 ＋ CD25 - T cells were different from those in activated - CD4 ＋CD25 ＋ Tregs and activated -CD4 ＋ CD25- T cells, respectively, suggesting DHHCs famlily members might be involved in the regulation of TCR signaling pathway in different CD4＋ T cell subsets.