尼古丁对卵蛋白致敏大鼠CD4＋T细胞Thl／Th2细胞因子和T-bet／GATA-3表达的影响

Effect of nicotine on the expression of Thl/Th2 cytokines and transcription factor T-bet/GATA-3 incultured CD4＋ T cell of rat sensitized by ovalbumin

目的研究尼古丁对卵白蛋白致敏大鼠CD4T淋巴细胞Thl／Th2平衡的影响。方法卵清白蛋白（OVA）腹腔注射致敏Wistar大鼠，CD4’T淋巴细胞纯化柱分离大鼠脾脏CD4T细胞，体外培养，将细胞随机分为4组：对照组、1μg∥ml尼古丁组、10μg∥ml尼古丁组、100μg∥ml尼古丁组（各组加等浓度的OVA），不同浓度尼古丁刺激24h，酶联免疫吸附试验（ELISA）检测细胞培养上清液IFN-y和IL4含量，实时荧光定量PCR检测培养细胞T-bet和GATA．3mRNA的表达。结果（1）不同尼古丁干预组IFN-1的表达分别为（113．78±6．06）rig／I．、（70．31±7．26）ng／L、（20．00±2．14）ng／L，均较对照组[（142．30＋5．89）ng／I．]明显减少，差异有统计学意义（F=265．52，P〈0．01）；不同尼古丁干预组IL-4的表达分别为（50．97＋3．07）ng／L、（69．49±3．91）ng／L、（93．63±4．56）ng／L，均较对照组[（36．91±3．24）ng／L]明显增加，差异有统计学意义（F=128．67，P〈0．01）。（2）不同尼古丁干预组T-betmRNA分别为0．73±0．03、0．57±0．04、0．31±0．00，均较对照组（0．98±0．09）明显减少，差异有统计学意义（F=75．76，P〈0．01）；不同尼古丁干预组GATA-3tuRNA的表达分别为4．31±0．26、5．16＋0．23、1．56±0．14，均较对照组（1．00±0．07）明显增加，差异有统计学意义（F：348．41，P〈0．01）。结论尼古丁可能通过促进转录因子GATA-3mRNA的表达同时抑制T．betmRNA的表达，在哮喘Th2过敏性气道炎症中具有一定的作用。

Objective To investigate the effects of nicotine on the expression of Thl/Th2 cytokines and transcription factor T-bet/GATA-3 in cultured CIM＋ T of rat sensitized by ovalbumin. Methods Two weeks after immunization by ovalbumin, splenic CIM＋ T cells of Wistar rat were purified using CIMT cell enrichment kit. Purified CIMT cells of rat were cultured and divided into 4 groups： a control group, 1 pLg/ml nicotine stimulated group, 10 μg/ml nicotine stimulated group, 100μg/ml nicotine stimulated group. These cells, in their groups, were stimulated with or without nicotine and were all challenged simultaneously with OVA. Supernatants and cell pellets were harvested after being stimulated for 24 h. The concentration of IFN-y and IL-4 in supernatants were measured by enzyme-linked immunosorbent assay （ELISA）. Real-time PCR was used to determine the mRNA expression of T-bet and GATA-3 in CD4T cells. Results （ 1 ） IFN-y production was significantly decreased in all nicotine treated groups [ （ 113.78±6.06） ng/L, （70.31±7.26） ng/L, （20.00±2.14） ng/L] compared with the control group[ （ 142.30±5.89） ng/L] , and the level of IL-4 was significantly increased in all nicotine treated groups[ （69.49±3.91 ） ng/L, （93.63±4.56 ） ng/L, （ 50.97±3.07 ） ng/L ] compared with the control group [ （ 36.91 ±3.24） ng/L]. （2） Expression of T-bet mRNA in all nicotine treated groups（0.73±0.03, 0. 57± 0. 04 , 0. 31±0. 00 ） was lower than that in the control group（0.98±0.09） , but expression of GATA-3 mRNA in all nicotine treated groups（4.31±0.26, 5.16±0.23, 1.56±0.14） was significantly higher than that in the control group （1.00±0.07）. Conclusion Nicotine may play a key role in the development of Th2-type allergic inflammation in asthma by promoting over-expression of GATA-3 mRNA and downregulating the expression of T-bet mRNA.