蛋白激酶C和激活蛋白-1信号级联对哮喘大鼠IL-5基因转录的调控作用

The role of protein kinase C and activator protein-1 signal cascade in the regu lation of interleukin-5 gene transcription in asthmatic rat

目的 探讨蛋白激酶C(PKC)和激活蛋白 -1(AP- 1)信号转导级联在支气管哮喘(简称哮喘)大鼠外周血T淋巴细胞白细胞介素5 (IL -5 )基因转录中的作用。方法 建立大鼠哮喘模型。从每只大鼠外周血中分离T淋巴细胞,并随机分为空白对照组、PKC激动剂12 肉豆蔻酰 13 乙酸佛波酯(PMA)组、PMA和AP -1顺式诱骗元件(CDODNs)组及PMA和AP -1突变顺式诱骗元件(突变CDODNs)组进行培养。两种CDODNs分别经脂质体转染至后两组细胞。用电泳迁移率改变分析(EMSA)检测AP -1活化,用逆转录聚合酶链反应(RT- PCR)检测IL -5mRNA表达。结果 (1)PMA组的AP -1活化(86 777.2 2±2 2 5 7.79)和IL 5mRNA表达(0 .810 0±0 .0 316 )增加,与空白对照组(分别为2 0 70 8.5 4±96 8.0 4和0 .30 5 0±0 .0 12 9)相比较,差异有统计学意义(P <0 .0 1)。(2 )PMA和AP -1顺式诱骗元件组的AP -1活化(2 3475 .90±75 7.99)被抑制,IL -5mRNA表达(0 .345 0±0 .0 12 9)亦减少,与PMA组相比较,其差异有统计学意义(P <0 .0 1)。PMA和AP 1突变顺式诱骗元件组的AP 1活化(86 95 5 .71±16 0 0 .38)和IL- 5mRNA表达(0 .8175±0 .0 2 2 2 )与PMA组相比较,差异无统计学意义(P >0 .0 5 )。(3)T淋巴细胞AP -1活化和IL 5mRNA表达呈显著正相关(P <0 .0 1)。结论 哮?

Objective To explore the role of protein kinas e C (PKC) and activator protein-1 (AP-1) signal transduction cascade in the tran scription of interleukin-5 (IL-5 ) gene by peripheral blood T lymphocytes from asthmatic rat. Methods T lymphocytes were isolated and p urified from peripheral blood of each asthmatic Wistar male rat, and randomly di vided into 4 groups for culture. Group A, blank control; group B, PKC agonist ph orbol 12-myristate 13-acetate (PMA); group C, PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs); group D, PMA and AP-1 mutant decoy ODNs. T he decoy ODNs were transfected into the T cells of group C and D by cation lipos ome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift as says (EMSA) for the activation of AP-1. Results The acti vation of AP-1 (86 777.22±2257.79) and the expression of IL-5 mRNA (0.8100±0 .0316) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 708.54±968.04 and 0.3050±0.0129 respectively,P<0.01 ), while the indexes (23 475.90±757.99 and 0.3450±0.0129 respectively) of T ly mphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P<0.01). The indexes (86 955.71±1600.38 and 0.81 75±0.0222 respectively) in T lymphocytes stimulated with PMA and AP-1 mutant d ecoy ODNs did not exhibit significant changes, by contrast with group B (P>0 .05). The significant positive correlation was found between the activation of A P-1 and the expression of IL-5 mRNA (P<0.01). Conclusion AP-1 may participate in the signal transduction of PKC-triggered transcr iption of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important ro le in the pathogenesis of asthma.