在变应性鼻炎小鼠模型中经鼻应用爱泼斯坦-巴尔病毒/脂质复合物以消除嗜酸性粒细胞增多症。

Han De-min, Zhou Bing, Wang Tong, Wang Xiang-dong, Fan Er-zhong

Department of Otorhinolaryngology, Head and Neck Surgery, Beijing Tongren Hospital, Capital University of Medical Sciences, Beijing 100730, China.

Chin Med J (Engl). 2006 Jun 20;119(12):991-7.

BACKGROUND

Currently anti-inflammatory therapy with steroids for allergic rhinitis need long-term repeated administration, although it is effective. Gene therapy is being suggested to substitute it. The aim of this study was to investigate nonviral vector mediated exogenous gene expression in COS-7 cells in vitro and the effect of intranasal mouse interleukin (mIL)-12 transgene expression on allergen induced eosinophil infiltration of nasal mucosa in a murine model of allergic rhinitis.

METHODS

In vitro COS-7 cells were infected with Epstein-Barr virus (EBV)/lipoplex. The expression of IL-12 p70 in cell culture supernatant was examined by enzyme-linked immunosorbent assay (ELISA). In mice with ovalbumin (OVA) induced allergic rhinitis, EBV/lipoplex was administered by nasal drops before OVA challenge once a day from day 1 to day 10. The expression of IL-12 mRNA and protein, the change of eosinophil count in nasal mucosa and serum total IgE were measured 24 hours after the last challenge.

RESULTS

EBV/lipoplex could effectively transfect COS-7 cells. The expression of IL-12 p70 in cell culture supernatant was significantly more than in blank control. IL-12 via EBV plasmid vector transduction could be overexpressed in vivo. In pGEG.mIL-12 treated models, the nasal mucosa revealed a high level of widespread mIL-12 transduction by immunohistochemistry and in situ hybridization. Histological evaluation revealed marked suppression of eosinophil infiltration in nasal mucosa. The eosinophil count in allergic rhinitis group [(26.5 +/- 9.8)/high-power field (HPF)] was significantly increased over control group [(0.40 +/- 0.52)/HPF] (F = 56.94, P < 0.01), while the count in IL-12 gene therapy group [(4.60 +/- 2.63)/HPF] was significantly less than that of allergic group (F = 56.9, P < 0.01). Serum total IgE between in gene therapy mice [(88.83 +/- 6.71) ng/ml] and allergic rhinitis mice [(103.1 +/- 5.7) ng/ml] showed a significant difference (F = 1216, P < 0.05).

CONCLUSIONS

Nonviral EBV plasmid vector, pGEG.mIL-12 was able to overexpress exogenous gene both in vitro and in murine nasal mucosa in vivo. IL-12 overexpression via EBV/lipoplex could stem allergen induced eosinophil infiltration in nasal mucosa in murine models of allergic rhinitis, which may suggest a new cytokine immunogenetic therapy for allergic rhinitis.

背景

目前，用于过敏性鼻炎的类固醇抗炎疗法虽有效，但需要长期反复给药。有人提出用基因疗法来替代它。本研究的目的是在体外研究非病毒载体介导的外源性基因在COS-7细胞中的表达，以及在过敏性鼻炎小鼠模型中鼻内小鼠白细胞介素（mIL）-12转基因表达对变应原诱导的鼻黏膜嗜酸性粒细胞浸润的影响。

方法

体外将爱泼斯坦-巴尔病毒（EBV）/脂质体复合物感染COS-7细胞。通过酶联免疫吸附测定（ELISA）检测细胞培养上清液中IL-12 p70的表达。在卵清蛋白（OVA）诱导的过敏性鼻炎小鼠中，从第1天到第10天，每天在OVA激发前经滴鼻给予EBV/脂质体复合物一次。在最后一次激发后24小时，测量IL-12 mRNA和蛋白的表达、鼻黏膜中嗜酸性粒细胞计数的变化以及血清总IgE。

结果

EBV/脂质体复合物能有效转染COS-7细胞。细胞培养上清液中IL-12 p70的表达明显高于空白对照。通过EBV质粒载体转导的IL-12在体内可过度表达。在pGEG.mIL-12处理的模型中，通过免疫组织化学和原位杂交显示鼻黏膜有高水平的广泛mIL-12转导。组织学评估显示鼻黏膜中嗜酸性粒细胞浸润明显受到抑制。过敏性鼻炎组的嗜酸性粒细胞计数[（26.5±9.8）/高倍视野（HPF）]明显高于对照组[（0.40±0.52）/HPF]（F = 56.94，P < 0.01），而IL-12基因治疗组的计数[（4.60±2.63）/HPF]明显低于过敏组（F = 56.9，P < 0.01）。基因治疗小鼠[（88.83±6.）ng/ml]和过敏性鼻炎小鼠[（103.1±5.7）ng/ml]之间的血清总IgE有显著差异（F = 1216，P < 0.05）。