目的:探讨轴突导向因子3A（Sema3A）对脂多糖（LPS）诱导的CD4n +CD25n +调节性T细胞（Tregs）稳定性的作用及机制。n 方法:采用体外免疫磁珠法分离和培养C57BL/6J小鼠脾脏CD4n +CD25n + Tregs，将分离的细胞按随机数字表法分为对照组（仅给予抗小鼠CD3e和CD28诱导细胞处于激活状态）、LPS组（在对照组的基础上给予LPS 100 μg/L）、LPS+核转录因子-κB（NF-κB）抑制剂吡咯烷二硫代氨基甲酸酯（PDTC）组（给予LPS 100 μg/L+PDTC 25 mg/L）、LPS+磷酸盐缓冲液（PBS）组（给予LPS 100 μg/L+PBS 10 μL）、LPS+PDTC+重组Sema3A（rSema3A）组（给予LPS 100 μg/L+PDTC 25 mg/L+rSema3A 300 μg/L）和LPS+PBS+rSema3A组（给予LPS 100 μg/L+PBS 10 μL+rSema3A 300 μg/L）。各组培养24 h后，采用反转录-聚合酶链反应（RT-PCR）和免疫荧光法检测CD4n +CD25n + Tregs特异性标志物叉头翼状转录因子-3（Foxp-3）、细胞毒性T淋巴细胞相关抗原-4（CTLA-4）和膜相关转化型生长因子-β1（TGF-β1n m+）的基因及蛋白表达，采用酶联免疫吸附试验（ELISA）检测细胞上清液中白细胞介素-10（IL-10）和分泌型转化生长因子-β1（sTGF-β1）的水平，采用免疫荧光法检测细胞凋亡，采用甲基化特异性聚合酶链反应（MSP）检测Foxp-3-Tregs特异性去甲基化区（Foxp-3-TSDR）的去甲基化程度，以反映CD4n +CD25n + Tregs的稳定性，采用电泳迁移率分析（EMSA）检测NF-κB信号通路的DNA结合活性，采用蛋白质免疫印迹试验（Western blotting）检测NF-κB信号通路活性。n 结果:与对照组比较，LPS能够增加细胞稳定性，表现为Foxp-3、CTLA-4和TGF-β1n m+的基因及蛋白表达上调，IL-10和sTGF-β1分泌增加，细胞凋亡减少，Foxp-3-TSDR去甲基化程度增加；同时LPS可增加细胞内NF-κB信号通路的DNA结合活性，以及主要分子NF-κB抑制蛋白激酶β（IKKβ）和p65的磷酸化水平，说明LPS增加细胞稳定性的机制与NF-κB信号通路有关。与LPS组比较，PBS并未对细胞稳定性和NF-κB信号通路产生影响；但添加rSema3A后能进一步增加细胞稳定性，并激活NF-κB信号通路。而PDTC能够抑制rSema3A增加细胞稳定性的功能，表现为：与LPS+PBS+rSema3A组比较，LPS+PDTC+rSema3A组Foxp-3、CTLA-4和TGF-β1n m+的基因及蛋白表达明显下调〔Foxp-3基因（2n -ΔΔCt）：8.092±1.117比18.509±1.068，Foxp-3蛋白（相对荧光强度）：1.224±0.033比1.826±0.181；CTLA-4基因（2n -ΔΔCt）：3.254±0.760比11.840±0.827，CTLA-4蛋白（相对荧光强度）：1.305±0.058比1.842±0.111；TGF-β1n m+基因（2n -ΔΔCt）：3.589±1.180比8.509±0.472，TGF-β1n m+蛋白（相对荧光强度）：1.319±0.033比1.822±0.063，均n P<0.01〕，IL-10和sTGF-β1分泌减少〔IL-10（ng/L）：445.33±54.08比992.67±83.10，sTGF-β1（ng/L）：1 116.67±65.25比1 494.67±94.45，均n P<0.01〕，细胞凋亡明显增加（荧光强度：0.398±0.031比0.268±0.046，n P<0.01），Foxp-3-TSDR去甲基化程度明显降低（灰度值：0.467±0.048比1.780±0.119，n P<0.01），NF-κB信号通路的DNA结合活性被明显抑制（灰度值：1.23±0.02比3.95±0.06，n P<0.01），IKKβ和p65的磷酸化水平降低〔p-IKKβ表达（p-IKKβ/IKKβ）：0.97±0.07比1.97±0.04，p-p65（p-p65/p65）：0.95±0.08比1.93±0.06，均n P<0.01〕。n 结论:LPS能通过NF-κB信号通路增加CD4n +CD25n + Tregs稳定性；Sema3A能够进一步增加CD4n +CD25n + Tregs稳定性，并且与NF-κB信号通路有关。n “,”Objective:To investigate the effect and mechanism of semaphorin-3A (Sema3A) in maintaining the cellular stability of CD4n + CD25n + regulatory T cells (Tregs) induced by lipopolysaccharide (LPS).n Methods:In vitro, using immunomagnetic beads, splenic CD4n + CD25n + Tregs of C57BL/6J mice were isolated and cultured. According to the random number table, the isolated cells were divided into control group (treated with anti-CD3e and anti-CD28 for polyclonal activation), LPS group (on the basis of control group, treated with LPS at the dose of 100 μg/L), LPS + nuclear factor kappa B (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC) group (treated with LPS at the dose of 100 μg/L and PDTC at the dose of 25 mg/L), LPS + phosphate buffer solution (PBS) group (treated with LPS at the dose of 100 μg/L and PBS at the volume of 10 μL), LPS + PDTC + recombinant Sema3A (rSema3A) group (treated with LPS at the dose of 100 μg/L, PDTC at the dose of 25 mg/L and rSema3A at the dose of 300 μg/L), and LPS + PBS + rSema3A group (treated with LPS at the dose of 100 μg/L, PBS at the volume of 10 μL and rSema3A at the dose of 300 μg/L). mRNA and protein expressions of the specific markers of CD4 n + CD25n + Tregs, including forkhead box protein P-3 (Foxp-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and membrane-associated transforming growth factor-β1 (TGF-β1n m+) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence methods after 24 hours. The supernatant interleukin-10 (IL-10) and secretory type TGF-β1 (sTGF-β1) were detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic level was detected by immunofluorescence. The demethylation of Foxp3-Tregs-specific demethylated region (Foxp-3-TSDR) was detected by methylation specific PCR (MSP) in order to reflect the cellular stability of CD4n + CD25n + Tregs. DNA binding activity of NF-κB signaling pathway was determined by electrophoretic mobility shift assay (EMSA), and activity of NF-κB signaling pathway was determined by Western blotting.n Results:Compared with control group, LPS could increase the cellular stability, including an increase in the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1n m+ and secretion of IL-10 and sTGF-β1, decrease in the level of apoptosis and increase in the methylation of Foxp-3-TSDR. At the same time, LPS increased DNA binding activity of NF-κB signaling pathway and phosphorylation levels of the major molecules of NF-κB, including inhibitory protein kinaseβ (IKKβ) and p65, suggesting that the mechanism of enhancing cellular stability by LPS was related to the NF-κB signaling pathway. Compared with LPS group, PBS had no effect on cellular stability and NF-κB signaling pathway. However, administration of rSema3A further promoted cellular stability and activated NF-κB signaling pathway. Compared with LPS + PBS + rSema3A group, PDTC inhibited the function of rSema3A to increase cellular stability, as the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1n m+ were significantly down-regulated in the LPS + PDTC + rSema3A group [Foxp-3 mRNA (2n -ΔΔCt): 8.092±1.117 vs. 18.509±1.068, Foxp-3 protein (relative fluorescence intensity): 1.224±0.033 vs. 1.826±0.181; CTLA-4 mRNA (2n -ΔΔCt): 3.254±0.760 vs. 11.840±0.827, CTLA-4 protein (relative fluorescence intensity): 1.305±0.058 vs. 1.842±0.111; TGF-β1n m+ mRNA (2n -ΔΔCt): 3.589±1.180 vs. 8.509±0.472, TGF-β1n m+ protein (relative fluorescence intensity): 1.319±0.033 vs. 1.822±0.063, all n P < 0.01], the secretion of IL-10 and sTGF-β1 were significantly decreased [IL-10 (ng/L): 445.33±54.08 vs. 992.67±83.10, sTGF-β1 (ng/L): 1 116.67±65.25 vs. 1 494.67±94.45, both n P < 0.01], the apoptosis was significantly increased (fluorescence intensity: 0.398±0.031 vs. 0.268±0.046, n P < 0.01), the methylation of Foxp-3-TSDR was significantly decreased (grey value: 0.467±0.048 vs. 1.780±0.119, n P < 0.01), the DNA binding activity of NF-κB signaling pathway was significantly inhibited (grey value: 1.23±0.02 vs. 3.95±0.06, n P < 0.01), and the phosphorylation levels of IKKβ and p65 were significantly decreased [p-IKKβ (p-IKKβ/IKKβ): 0.97±0.07 vs. 1.97±0.04, p-p65 (p-p65/p65): 0.95±0.08 vs. 1.93±0.06, both n P < 0.01].n Conclusion:LPS increases the cellular stability of CD4n + CD25n + Tregs through the NF-κB signaling pathway, and Sema3A further increases the cellular stability of CD4n + CD25n + Tregs, and is related to the NF-κB signaling pathway.n