Blockade of OX40/OX40L pathway promotes CD4 + CD25 + T regulatory cells proliferation / 中华普通外科杂志

ABSTRACT

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.