摘要
为了解B7共刺激对细胞因子 ,特别是对IL 2mRNA及转录因子NF κB和AP 1的影响 ,探讨B7介导的IL 2调节的分子机制 ,在异基因混合淋巴细胞反应 (MLR)体系中分别或联合加入抗B7 1、抗B7 2单克隆抗体和CTLA 4Ig以阻断B7/CD2 8信号传导 ,通过竞争性PCR定量检测其对IL 2和IL 4mRNA的影响 ,并初步测定IFN γmRNA的改变 ,同时用转染MHCⅡ类分子及联合转染等量B7 1或B7 2的NIH3T3转基因细胞tDR7,tDR7/B7 1和tDR7/B7 2刺激CD2 8+ T细胞 ,通过DNA 蛋白结合实验观察B7对IL 2转录因子NF κB和AP 1的影响。结果表明 :抗B7 2单抗和CTLA 4Ig可明显抑制B7介导的IL 2和IL 4mRNA合成 ,而抗B7 1单抗仅有轻度抑制作用 ,2种或 3种抗体联合应用时抑制作用相加。MLR 1 - 6小时 ,单独tDR7即可诱导NF κB的表达 ,联合转染B7早期对其结合活力无明显影响 ,6小时后tDR7诱导作用减弱 ,B7却可显著延长tDR7的诱导作用至 72小时。tDR7早期同样可诱导AP 1的表达 ,联合转染B7分子在 2 4小时内对其有一定的抑制作用 ,而在反应后期可延长tDR7对AP 1的上调作用 ,B7 1与B7 2间作用未见明显不同。结论 :B7通过减少IL 2mRNA降解和影响基因转录而上调IL 2分泌 ,并可同时影响多种细胞因子分泌 ;在转录水平B7 1与B7 2作用未见明显不同 。
To detect effects of B7 co stimulation on cytokines, especially on IL 2 mRNA and transcription factors NF κB and AP 1, antiB7 1 McAb, antiB7 2 McAb and CTLA 4 Ig were added into mixture lymphocyte reaction (MLR) system to block B7/CD28 signal transduction, IL 2 mRNA and IL 4 mRNA were determined by using competitive PCR and IFN γ mRNA by using semi quantitative PCR. MHC class Ⅱ molecules and B7 transfectants were used to stimulate CD28 + T cell, effects of B7 on NF κB and AP 1 were detected by DNA protein binding assay. The results showed that IL 2, IL 4 and IFN γ mRNA were significantly lower when blockade of B7 2 in MLR than blockade of B7 1. Synergistic effects could be seen with combination of two or three antibodies. One to six hours after MLR, tDR7 alone induced NF κB binding activity; cotransfecting B7 no significantly difference at early time point. After 6 hours, induction of tDR7 was decreased whereas B7 prolonged the induction of NF κB till 72 hours. tDR7 alone also upregulated AP 1 binding activity, on the contrary to NF κB, co transfecting B7 1 and B7 2 decreased AP 1 binding activity within 24 hours. But during 48-72 hours, B7 also prolonged the AP 1 binding activity. It is concluded that after MLR, B7 increased IL 2 secretion by decreasing the degradation of IL 2 mRNA and upregulating IL 2 transcription factors. B7 also induced several kinds of cytokines secretion. Effects of B7 1 and B7 2 had no significant difference on transcription factors. It is suggested that the different functions between B7 1 and B7 2 maybe related to the difference of cell expression and kinetics. To study the molecular mechanism of B7 mediated T cell immune tolerance can help us to design a better clinic schema to prevent transplantation rejection and GVHD.
出处
《中国实验血液学杂志》
CAS
CSCD
2002年第6期512-518,共7页
Journal of Experimental Hematology
