非甲基化的CpG寡核苷酸对鼠巨噬细胞Toll样受体9及Th1/Th2类细胞因子分泌的影响

Effects of CpG-ODN on TLR-9 expression and Th1/Th2 cytokines secretion from mouse macrophages

目的探讨非甲基化的CpG寡核苷酸(CpG-ODN)对鼠单核-巨噬细胞系RAW264.7 Toll样受体9(TLR-9)的表达及Th1/Th2类细胞因子分泌的影响。方法体外培养小鼠单核-巨噬细胞系RAW264.7,置24孔培养板中,随机将细胞分为四组,分别为CpG-ODN组,Non-CpG-ODN组,CpG-ODN+氯喹组和对照组,分别添加CpG-ODN(0.3μg/ml),Non-CpG-ODN(0.3μg/ml),CpG-ODN+氯喹组(CpG-ODN和氯喹各0.3μg/ml),培养基。逆转录聚合酶链反应及Western blot检测24h后巨噬细胞TLR-9的表达,放免法及ELISA法检测刺激前后巨噬细胞上清液中肿瘤坏死因子α(TNF-α)、白介素6(IL-6)及IL-12的表达水平。结果刺激24h后,巨噬细胞TLR-9mRNA的表达,CpG-ODN组与Non-CpG-ODN组比较,差异有统计学意义(t值为7.12,P<0.01);CpG-ODN组与对照组比较,差异有统计学意义(t值为8.04,P<0.01);CpG+氯喹组与Non-CpG-ODN组比较,差异有统计学意义(t值为4.25,P<0.05);CpG+氯喹组与对照组比较,差异有统计学意义(t值为5.58,P<0.01)。TLR-9蛋白,CpG-ODN组为1.35±0.18,高于Non-CpG(1.01±0.04)及对照组(0.91±0.10),差异有统计学意义(t值分别为3.21、3.67,P值均<0.05)。CpG-ODN+氯喹组为1.34±0.15,高于Non-CpG及对照组,差异有统计学意义(t值分别为3.71、4.10,P值均<0.05),而CpG-ODN组及CpG-ODN+氯喹组比较,差异无统计学意义(t值为0.10,P>0.05)。细胞上清液中TNF-α水平CpG-ODN组[(0.48±0.12)ng/ml]高于Non-CpG-ODN组[(0.29±0.23)ng/ml]、CpG-ODN+氯喹组[(0.32±0.24)ng/ml]和对照组[(0.29±0.19)ng/ml],差异有统计学意义(t值分别为2.84、2.38、3.25,P值均<0.05);IL-6,IL-12水平:CpG-ODN组分别为[(27.69±15.61)pg/ml和(48.59±25.17)pg/ml]与Non-CpG-ODN组分别为[(17.02±10.66)pg/ml和(59.58±32.89)pg/ml]、CpG-ODN+氯喹组分别为[(28.58±24.08)pg/ml和(41.30±13.17)pg/ml]和对照组分别为[(16.12±17.70)pg/ml和(61.54±39.69)pg/ml],差异无统计学意义(t值分别为1.94、0.75,P值均>0.05)。结论CpG-ODN刺激可引起鼠巨噬细胞TLR-9表达增高,Th1类细胞因子分泌增加。

Objective To investigate the effects on Toll like receptor-9(TLR-9) expression and Th1/Th2 cytokines secretion from mouse macrophages(RAW264.7) induced by CpG motifs.Methods RAW264.7 cells grown in 24-well plates in vitro were randomly divided into 4 groups,namely CpG-ODN group,Non-CpG-ODN group,CpG-ODN+chloroquine group and control group.CpG-ODN group was treated with CpG-ODN (0.3 μg/ml).Non-CpG-ODN group was treated with Non-CpG-ODN (0.3 μg/ml).CpG-ODN+chloroquine group was treated with CpG-ODN (0.3 μg/ml) and chloroquine (0.3 μg/ml).Control group was treated with DMEM.After 24 hours,the expressions of TLR-9 mRNA and protein were analyzed by RT-PCR and Western blot respectively.The concentration of TNF-αand IL-6 in the supernatants of each group were examined by Radioimmunoasssy(RIA) kits and the level of IL-12 was analyzed by ELISA.Results After treatment for 24 hours,the expression of TLR-9 mRNA in CpG-ODN group was statistically different as compared with the Non-CpG-ODN group(t=7.12,P<0.01).The CpG-ODN group was statistically different as compared with the control group(t=8.04,P<0.01);The CpG-ODN+chloroquine group was statistically different as compared with the Non-CpG-ODN group(t=4.25,P<0.05);The CpG-ODN+chloroquine group was statistically different as compared with the control group (t=5.58,P<0.01).The expression of TLR-9 protein in CpG-ODN group (1.35±0.18) was statistically different as compared with the Non-CpG group (1.01±0.04) and control group(0.91±0.10),(t=3.21、3.67,all P<0.05).CpG-ODN+chloroquine group (1.34±0.15),was statistically different as compared with the Non-CpG group and control group(t=3.71、4.10,all P<0.05).But CpG-ODN group was not statistically different as compared with the CpG-ODN+chloroquine group(t=0.10,P>0.05).The level of TNF-α in the supernatant in the CpG-ODN group [(0.48±0.12)ng/ml] was higher than Non-CpG-ODN group [(0.29±0.23)ng/ml ]、CpG-ODN- chloroquine group [(0.32±0.24)ng/ml] and control group [(0.29±0.19)ng/ml,P<0.05].(t=2.84、2.38、3.25 respectively at Non-CpG-ODN group,CpG-ODN-chloroquine group,control group,all P<0.05)The levels of IL-6 and IL-12 in the supernatants in the CpG-ODN group [(27.69±15.61)pg/ml and(48.59±25.17)pg/ml] had no significant difference compared with that in Non-CpG-ODN group [(17.02±10.66)pg/ml and (59.58±32.89)pg/ml]、CpG-ODN+chloroquine group [(28.58±24.08)pg/ml and(41.30±13.17)pg/ml] and control group [(16.12±17.70)pg/ml and (61.54±39.69)pg/ml](t=1.94、0.75 all P>0.05).Conclusions CpG-ODN can enhance the expressions of TLR-9 and secretion of Th1 cytokines from macrophages.