核因子κB顺式“诱骗”元件对支气管哮喘患者T淋巴细胞功能的干预作用

In vivo transfection of cis element ‘decoy’ against nuclear factor-κB binding site modulates function of Tlymphocytes in asthma

目的 探讨核因子κB(NF κB)顺式诱骗寡脱氧核苷酸 (ODN)对体外支气管哮喘 (简称哮喘 )患者T淋巴细胞增殖 /凋亡功能和细胞因子合成功能的干预作用。方法 将T淋巴细胞分为四组 :正常对照组 (A组 )、哮喘空白组 (B组 )、哮喘NF κB顺式诱骗ODN组 (B1组 )和无序哮喘ODN组(B2 组 )。两种ODN分别经脂质体转染至后两组细胞。通过流式细胞术、四甲基偶氮唑盐微量比色法检测T淋巴细胞的凋亡和增殖 ;通过原位细胞杂交和酶联免疫吸附试验检测T淋巴细胞白细胞介素5(IL 5)mRNA和蛋白质的表达 ;通过逆转录 聚合酶链反应 (PT PCR)和Westernblot检测T淋巴细胞诱导型一氧化氮合酶 (iNOS)mRNA和蛋白质的表达。结果 B1组T淋巴细胞增殖率为 0 2 2 0± 0 0 2 0 ,与B组 (0 3 4 0± 0 0 3 0 )比较差异有显著性 (P <0 0 5) ;凋亡率为 (10 8± 1 3 ) % ,与B组 [(8 1± 1 2 ) % ]比较差异也有显著性 (P <0 0 5) ;同时B1组IL 5mRNA和蛋白质的表达分别为 2 1± 4、2 4± 4,与B组 (3 3± 4、54± 10 )比较 ,差异有显著性 (P <0 0 5) ;B1组iNOSmRNA和蛋白质的表达分别为 0 3 3± 0 0 5、782± 117,与B组 (0 75± 0 13、1185± 2 3 0 )比较 ,差异有显著性 (P <0 0 5)。而B2 组与B组比较 ,上述指标的差异无显著性 (P?

Objective To investigate the effect of nuclear factor-κB(NF-κB) decoy oligodeoxynucleotide(ODN) on proliferation, apoptosis and cytokine synthesis of T lymphocytes from asthmatic patients Methods T lymphocytes were divided into four groups,a normal control group (A group), an asthma control group (B group), a NF-κB cis decoy ODN group (B 1 group) and a scrambled ODN group (B 2 group) B 1 and B 2 groups were transfected by cationic lipofectamine to the latter two groups respectively The proliferation of T lymphocytes was measured by MTT and the apoptosis of them was measured by flow cytometry The expression levels of interleukin 5(IL-5) mRNA and protein were detected with cell hybridization in situ and enzyme-linked immunosorbent assay(ELISA) The expression levels of inducible nitric oxide synthase(iNOS) were measured by reverse transcription-poly merase chain reaction(RT-PCR) and Western blot Results The difference of proliferation rate between B 1 group (0 220±0 020) and B group(0 340±0 030) was significant ( P< 0 05) The difference of apoptosis rate between B 1 group(10 8±1 3) and B group(8 1±1 2) was also significant( P< 0 05) The expression levels of IL-5 mRNA and protein in B 1 group(21±4,24±4)were significantly different from those in B group (33±4,54±10, P< 0 05) The differences of iNOS mRNA and protein levels between B 1 group (0 33±0 05, 782±117)and B group (0 75±0 13,1185±230) were significant ( P< 0 05) However, these indices in B 2 group showed no difference to those in B group( P> 0 05) Conclusions NF-κB decoy ODN can reduce the abnormally increased proliferation of T lymphocytes in asthma and increase the abnormally decreased apoptosis of these cells, while decrease the abnormally increased levels of cytokine and enzyme in asthmatic T lymphocytes These may be the mechanisms underlying its potential therapeutic effects in asthma