屋尘螨变应原重组体免疫治疗的实验研究

Study on immunotherapy by house dust mite allergen recombinants

目的:建立屋尘螨哮喘动物模型,比较分别用屋尘螨粗浸液、重组Derp2(rDerp2)进行免疫治疗的疗效,为变态反应性疾病的免疫治疗提供新的思路。方法:近交系清洁级BALB/c小鼠,腹膜内注射(i.p.)屋尘螨变应原粗浸液与Al(OH)3生理盐水的混悬液致敏小鼠,建立哮喘模型;治疗组在致敏后分别给予i.p.屋尘螨粗浸液、rDerp2溶液进行免疫治疗后,鼻腔滴入(i.n.)屋尘螨粗浸液予以激发;阴性对照组从致敏,阳性对照组在致敏后,均同步生理盐水处理。用ELISA方法检测各组实验动物血清中IgE、IgG1、IgG2a的水平,以及支气管肺泡灌洗液(BALF)与脾细胞培养后上清液(SCCS)中细胞因子IL-4、IFN-γ水平,计数BALF中细胞总数与分类细胞数,并制备肺组织病理切片以观察其病理改变。结果:添加铝佐剂的屋尘螨变应原致敏的小鼠在特异性变应原的连续激发下,肺组织产生明显的炎症反应,表现为BALF中细胞总数增加以及炎症细胞所占比例及绝对计数升高,且以分泌Th2型细胞因子如IL-4为主;血清IgE、IgG1水平升高;肺组织病理切片观察到支气管、细支气管上皮下和小血管周围有明显的炎性细胞浸润,以嗜酸性粒细胞为主,气道上皮结构紊乱,有部分脱落,上皮下充血水肿明显,肺泡间质增厚,也可见到炎性细胞浸润。给予相应免疫治疗后,肺组织炎症反应明显减轻,表现为BALF中细胞总数减少,炎症细胞数减少,且其和SCCS中Th1型细胞因子如IFN-γ占主导;血清IgE、IgG1水平降低,IgG2a水平升高;肺组织病理切片观察到细胞浸润程度明显减轻,气道结果基本恢复正常。结论:本实验研究成功地建立了小鼠哮喘模型。分别用屋尘螨粗浸液及rDerp2进行免疫治疗均显示良好疗效,但前者比后者疗效好。

Objective：To build murine asthma model with Dermatophagoides pteronyssinus. To compare the curative effects of the immunotherapy regimens with either Dermatophagoides pteronyssinus allergen extracts or a recombinant Derp2 （rDerp2）. To offer a novel thread for immunotherapy of allergic diseases. Methods：The BALB/c mice were divided into four groups randomly. They were： negative control, positive control, allergen extracts and rDerp2. Mice were sensitized by i.p. injection of mixture of allergen extracts and AI（OH） 3 except the negative control, and were i. p. given a treatment with either allergen extracts or rDerp2 respectively, and then were i. n. given allergen extracts as a challenge. Negative control was treated by saline always. Assays for IgE, IgG1, IgG2a in serum, IL-4, IFN-γ in BALF and SCCS were performed by ELISA. Total cell number and assorted cell number in BALF samples were determined. The lungs kept in 10% formalin were made into slices stained by HE in order to observe the pathologic changes in lungs. Resuits ： Compared to the negative control, in the positive control the total cell number and the number of inflammatory cell in BALF were significantly increased, BALF eosinophil counts were significantly increased, nentrophils and eosinophils were the predominant BALF inflammatory; and the Th2 cytokine, e.g. IL-4, was the main cytokine in BALF and SCCS. Compared with the positive control, the allergic responses in the mice given relevant immunotherapy were obviously alleviated, and the predominant cytokine in BALF and SCCS was Thl pattern, e.g. IFN-γ. Conclusion：We established successfully the murine asthma model. Immunotherapy with Dermatophagoides pteronyssinus allergen extracts had better curative effects than immunotherapy with recombinant Derp2.