RNA干扰阻断OX40/OX40L共刺激通路对CD4＋CD25＋T调节细胞的影响

Blockade of OX40/OX40L pathway promotes CD4 ^＋ CD25^ ＋ T regulatory cells proliferation

目的 探讨慢病毒介导的RNA干扰技术诱导小鼠树突状细胞OX40L基因沉默对调节性T细胞的影响. 方法 设计针对小鼠OX40L基因的RNA于扰序列,通过外源筛靶筛选出干扰效果最佳的序列.以293T为包装细胞,制备含OX40L的siRNA序列的慢病毒载体OX40L-RNAi-LV和阴性对照载体NC-GFP-LV.采用磁式分选器分离培养骨髓来源的小鼠树突状细胞（DCs）,以MOI为25进行转染,分别将转染OX40L-RNAi-LV（实验组）、转染NC-GFP-LV（阴性对照组）和未转染（空白对照组）的DCs与磁式分选器分选得到的CD4^＋CD25^＋T调节细胞共培养,6 d后通过流式细胞仪检测T调节细胞的增殖和凋亡情况. 结果外源筛靶筛选出干扰效果最佳的RNA干扰序列（靶序列为GCTCATACAAGAATGAGTA）,OX40L蛋白表达的抑制率为73.1%.感染复数为25时,慢病毒载体感染DCs的效率为86.4%.DCs与CD4^＋CD25^＋T调节细胞共培养后,实验组的凋亡细胞比例为8.7%,显著低于阴性对照组（20.1%）和空白对照组（19.8%）,F=244.22,P=0.000;而增殖细胞前体频率为38.3%,明显高于阴性对照组（24.5%）和空白对照组（22.9%）,F=95.40,P=0.000.结论 小鼠OX40L的siRNA慢病毒载体可以有效降低树突状细胞OX40L的表达,对体外培养的CD4＋CD25＋T调节细胞具有显著地促进增殖、减少凋亡的作用.

Objective To evaluate the feasibility of OX40L gene silence in dentritic cells by RNAi through lentiviral vector, so that to explore the influence of CD4^ ＋ CD25^ ＋ T regulatory cells by blocking OX40/OX40L costimulation signals. Methods Serial plasmids were constructed, consisting of lentiviral vector framework, containing different OX40L siRNA sequences. The most effective packaged one by 293T cells to be the operating siRNA vector named OX4OL-RNAi-LV was chosen. The negative comparing vector was NC-GFP-LV. DCs isolated from C57BL/6 mice by magnetic selecting system were cultured in vitro and divided into 3 groups. Experimental group and negative control group were transfected with OX40L-RNAi-LV and NC-GFP-LV, respectively, and the blank control group was given the same volume culture solution. The MOI was 25. The status of transfection and GFP expression was monitored by GFP fluorescence. 6 days later, CD86 positive DCs were isolated and were co-cultured with CD4 ^＋ CD25 ^＋ T regulatory cells isolated from na（y）ve BALB/C. 6 days later, the proliferation and apoptosis of Tregs were evaluated by flow cytometry.Results The most effective siRNA sequence targeted the C,CTCATACAAGAATGAGTA episode of OX40L gene. The suppressive ratio of the OX40L protein expression was 73.1%. While the MOI was 25, the DCs transfected ratio by lentiviral vectors was at the level of 86. 4%. After co-cultured for 6 days, the CD4＋CD25 ＋T regulatory cells of experimental group have a higher proliferation index （respectively, 38.3% vs.24.5 % ,22. 9%, F = 95.40, P = 0. 000） and lower apoptosis percentage （ respectively, 8.7 % vs. 20. 1%,19.8%, F=244.22,P=0. 000） than negative group and blank group. Conclusions The OX40L siRNA lentiviral vetor was constructed. This vector could effectively transfect DCs and block OX40/OX40L pathway, so to promote CD4^ ＋ CD25^ ＋ T regulatory cells proliferation in vitro.