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目的观察携带T-bet基因的重组腺相关病毒(rAAV-T-bet)转染对呼吸道合胞病毒(RSV)感染模型小鼠呼吸道炎症的影响。方法健康6～8周龄Balb/c小鼠40只。按随机数字表法分为4组:正常对照组(A组)、RSV感染模型组(B组)、模型/对照病毒rAAV载体介导的增强型绿色荧光蛋白(rAAV-eGFP)干预组(C组)及模型/rAAV-T-bet干预组(D组),每组10只。将B组、C组和D组小鼠用滴度为1.26×1010PFU.L-1的RSV液滴鼻,每只100μL;A组用等量人喉癌上皮细胞(Hep-2细胞)培养上清液代替RSV液。于RSV接种第5天,再次麻醉小鼠,A组和B组经鼻滴入293细胞培养上清液;C组经鼻滴入293细胞包装的rAAV-eGFP对照病毒液,D组经鼻滴入293细胞包装的rAAV-T-bet。7 d后处死小鼠,检测各组小鼠支气管肺泡灌洗液(BALF)中细胞成分及IL-4和γ干扰素(IFN-γ)水平,并观察其肺组织病理学变化。结果 1.B组小鼠BALF中细胞总数和淋巴细胞计数百分比分别为(43.91±10.41)×109L-1、(65.15±1.88)%,A组分别为(14.52±3.57)×109L-1、(25.79±1.07)%,A组与B组比较差异均有统计学意义(Pa<0.05);D组小鼠BALF中细胞总数和淋巴细胞计数百分比分别为(24.84±4.35)×109L-1、(29.48±11.97)%,与B组、C组比较差异均有统计学意义(Pa<0.05)。2.B组小鼠BALF中IL-4和IFN-γ水平分别为(74.32±10.06)ng.L-1、(89.36±20.38)ng.L-1,A组分别为(6.01±1.02)ng.L-1、(177.32±18.16)ng.L-1,A组与B组比较差异均有统计学意义(Pa<0.05);D组分别为(49.14±7.59)ng.L-1、(145.28±43.52)ng.L-1,与B组、C组比较差异均有统计学意义(Pa<0.05)。3.A组小鼠呼吸道无明显炎症改变;B组和C组小鼠小支气管、血管黏膜下和周围肺组织有明显炎性细胞浸润,血管壁明显水肿;D组小鼠呼吸道炎症明显减轻。结论 T-bet基因转染能改善RSV感染模型小鼠呼吸道内细胞因子IL-4和IFN-γ异常表达,同时对淋巴细胞在肺内聚集、肺组织炎症有一定抑制作用。
Objective To investigate the effect of rAAV-T-bet transfection with T-bet gene on respiratory inflammation in respiratory syncytial virus (RSV) -induced model mice. Methods Forty Balb / c mice aged 6-8 weeks were enrolled. The mice were randomly divided into 4 groups according to random number table: normal control group (group A), RSV infection model group (group B), model / control virus rAAV vector mediated enhanced green fluorescent protein (rAAV-eGFP) intervention group Group) and model / rAAV-T-bet intervention group (group D), with 10 rats in each group. Groups B, C and D were intranasally administrated with RSV (1.26 × 1010 PFU.L-1), each 100 μL. Group A was treated with an equal number of human laryngeal squamous cell carcinoma (Hep-2) Serum instead of RSV solution. On the fifth day after RSV inoculation, the mice were anesthetized again, and the culture supernatants of 293 cells were instilled into the groups A and B respectively. In group C, the rAAV-eGFP control virus solution packaged in 293 cells was dripped nasally Into 293 cells packed rAAV-T-bet. Seven days later, the mice were sacrificed and the cellular components and the levels of IL-4 and IFN-γ in the bronchoalveolar lavage fluid (BALF) of the mice in each group were measured. The pathological changes of the lung tissue were observed. Results The percentage of total cell number and lymphocyte count in BALF in group B were (43.91 ± 10.41) × 109L-1, (65.15 ± 1.88)%, and group A were (14.52 ± 3.57) × 109L-1, 25.79 ± 1.07)%, the difference between group A and group B was statistically significant (Pa <0.05). The percentage of total cell number and lymphocyte count in BALF in group D were (24.84 ± 4.35) × 109L-1, 29.48 ± 11.97)% respectively. There were significant differences between group B and group C (P <0.05). The levels of IL-4 and IFN-γ in BALF of group B were (74.32 ± 10.06) ng.L-1 and (89.36 ± 20.38) ng.L-1, respectively, and those in group A were (6.01 ± 1.02) ng (P <0.05). The difference between the two groups was (49.14 ± 7.59) ng.L-1 and (177.32 ± 18.16) ng.L- 145.28 ± 43.52) ng.L-1. There was significant difference between group B and group C (P <0.05). The respiratory tract of group A had no obvious inflammatory changes. In group B and group C, the bronchial, submucosal and surrounding lung tissue had obvious inflammatory cell infiltration and obvious edema on the blood vessel wall; the respiratory tract inflammation of group D was significantly reduced. Conclusion T-bet gene transfection can improve the abnormal expression of IL-4 and IFN-γ in the respiratory tract of RSV-infected mice, and inhibit the lymphocyte accumulation in the lung and inflammation of lung tissue.