T-bet基因修饰树突细胞对哮喘模型小鼠气道炎症的阻抑和逆转作用

目的 探讨T-bet基因修饰树突细胞(DC)用作哮喘治疗策略的可行性和机制.方法 将从小鼠骨髓单个核细胞诱导培养获得的DC分2组,分别以腺病毒载体介导的方法 转染T-bet基因和β-半乳糖苷酶(LacZ)基因,另设空白对照组,酶联免疫吸附试验(ELISA)检测3组DC培养液中干扰素γ(IFN-γ)水平.以卵清蛋白(OVA)腹腔注射致敏(第1、15天)和雾化吸入激发(第29～31天)方法 制备哮喘小鼠模型和哮喘再激发模型(第45～47天雾化吸入OVA),分别用于气道炎症阻抑试验和气道炎症逆转试验,各设正常对照组、模型对照组、激发或再次激发前静脉注射T-bet基因修饰DC组(T-bet组)和静脉注射LacZ基因修饰DC组(LacZ组),每组8只小鼠.阻抑和逆转试验小鼠分别于建模第37和49天处死,ELISA检测血浆IFN-γ和白细胞介素4(IL-4)水平,取肺脏行常规组织病理学检查.结果 转染T-bet基因组DC培养液中IFN-γ水平(ng/m1)为15.24±4.75,明显高于转染LacZ组和空白对照组(分别为3.08±0.61、2.35±0.41,均P＜0.01).气道炎症阻抑和逆转试验中,T-bet组小鼠血浆IFN-γ水平(pg/ml)分别为130.2±10.5、145.7±16.7,均明显高于正常对照组(分别为25.0±6.5、24.6±5.9,均P＜0.01)、模型对照组(分别为20.7±4.5、16.5±7.0,均P＜0.01)和LacZ组(分别为17.6±7.0、24.2±9.0,均P＜0.01),而IL-4水平均低于模型对照组和LacZ组(均P＜0.01).组织病理学检查显示T-bet组小鼠气道炎症表现明显轻于模型对照组和LacZ组.结论 以T-bet基因修饰DC为基础的哮喘治疗策略是可行的,其机制可能与T-bet基因修饰可提高DC的IFN-γ表达水平从而在抗原呈递水平干预辅助性T细胞的分化有关.

T-bet gene modified dendritic cells abrogate and reverse the airway inflammation in allergic asthma: experiment with mice

Objective To explore the feasibility of the therapeutic strategy to use T-bet gene modified dendritc cells(DCs)to reverse the eourse of asthma.Methods(1)Mature DCs were derived from mononuclear cells obtained from femur of BALB/c mouse and divided into 3 groups,T-bet group transfected with recombinant adenovirus Ad-T-bet containing T-bet gene,LacZ group transfected with recombinant adenovirus Ad-LacZ containing LacZ gene,and control group.Seven days later ELISA was used to detect the interferon(IFN)-γ level in the culture fluid.(2)Airway inflammation abrogatingtrial.Twentyfour BALB/c mice were sensitized with intrapefitoneal injection of ovalbumin(OVA)(on day 1 and 15)to estabHsh asthma models,and then randomly divided into 3 equal groups:T-bet group injected intravenously with T-bet-modified Des on day 27,LacZ group injected with LacZ-modified DCs,and model control group without intravenous injeetion.Two days later the model mice began to undergo challenge by inhalation of OVA twice(on day 29-31).Eight mice were used as control group treated with PBS.On day 37 all mice were killed,ELISA was used to detect the blood intedeukin(IL)-4 and IFN-γ levels,and microscopy was conducted to observe the airway inflammation.(3)Airway inflammation reversing trial.Another 24 model mice were divided into 3 equal groups as well:re-challenged T-bet group injected intravenously with T-bet modified DCs on day 27 and 42,re-challenged LacZ group injected intravenously with LacZ-modified DCs on day 27 and 42,and model control group.Since the day 45 OVA inhalation was given once a day for successive 3 days.On day 49 these mice were all killed to undergo the tests as mentioned above.Results The IFN-γ level in the culture fluid of the T-bet gene modified DCs was(15.24±4.75)ng/ml,significantly higher than that of the LacZ gene modified DCs and control DCs[(3.08±0.61)and(2.35±0.41)ng/ml respectively,both P＜0.01].The IFN-γ in mice blood plasma of T-bet groups in abrogating and reversing trial were(130.2±10.5)and(145.7±16.7)pg/ml respectively,both significantly higher than those of the abrogating and reversing trial normal control groups[(25.0±6.5)and(24.6±5.9)pg/ml respectively],asthmatic model control groups[(20.7±4.5)and(16.5±7.0)ps/ml respectively] and LacZ groups[(17.6±7.0)and(24.2±9.0)pg/ml respectively](all P＜0.01).However,the IL-4 levels in mice blood plasma of T-bet groups were both significantly lower than those of asthmatic model control groups and LacZ groups(all P＜0.01).The airway inflammation of T-bet groups were remarkable milder than those of the model control groups and LacZ groups.Conclusion The asthma management strategy based on T-bet gene modified DCs is feasible with the plausible mechanism that the T-bet gene modified DCs regulate the T cells differentiation and polarization on the antigen presenting level.