儿童轮状病毒腹泻特异性免疫应答研究

目的 通过对儿童轮状病毒腹泻特异性免疫应答的研究,探索轮状病毒感染的有效预防与治疗方法.方法 收集75例急性期轮状病毒腹泻患儿的血和粪便标本,记录临床表现,收集同期健康儿童45例标本作为对照组.ELISA法检测轮状病毒抗原,逆转录PCR法确定轮状病毒基因型.建立间接ELISA法检测血浆和粪便的轮状病毒特异性抗体.流式细胞仪进行淋巴细胞亚群的检测,荧光定量PCR法进行外周血单个核细胞细胞因子mRNA表达的检测.荧光定量PCR法测外周血单个核细胞5种TLRmRNA表达的变化.结果 急性轮状病毒感染患儿特异性免疫应答的主要特点：（1）轮状病毒G分型以G3为主,占77.3%,P分型以P[8]为主,占82.7%.（2）血浆轮状病毒特异性IgM/IgG/IgA抗体滴度均明显高于对照组,粪便中IgA抗体滴度的增高尤为显著,不仅高于对照组而且高于血浆.病情重的患儿血浆和粪便中的IgA抗体更高.（3）患儿CD19＋细胞百分比（30.8%±7.9%）显著高于对照组（23.1%±7.7）%.CD4＋细胞的比例明显下降.起病3d以内患儿IFN-γmRNA的平均表达水平升高2倍,IL-12p40mRNA的表达水平在疾病全程均比对照组明显升高.结论 急性轮状病毒感染儿童早期出现免疫应答,以特异性体液免疫尤其是黏膜免疫的显著增强为主要特点.

Specific humoral immune response of children with rotavirus diarrhea

Objective To investigate the specific humoral immune response in children with rotavirus diarrher. Methods Blood and stool specimens were collected from 75 children diagnosed as rotavirus diarrhea in acute phase. The rotavirus antigen was detected by ELISA, and the genotype of rotavirus was determined by RT-PCR method. Rotavirus-specific antibody in plasma and stool were detected by indirect ELISA method. The lymphocyte subsets were determined by flow cytometry; the expression of cytokine mRNAs and five TLR mRNAs in peripheral blood mononuclear cells were detected by fluorescence quantitative PCR method. Results In 75 cases, G3 was dominated for G typing, accounting for 77.3%; P[8] was dominant for P typing, accounting for 82.7% and the rest types were G1, G2, P[4] and P[9]. Plasma rotavirus-specific IgM/IgG/IgA antibody titers in children with rotavirus infection were significantly higher than those in controls; IgA antibody titers in fecal were increased significantly, which were higher than the control group and plasma levels of infected children. In children with severe infection their plasma and fecal IgA antibodies titers were markedly higher. The percentage of CD19＋ cells （30.8 ±7.9%） in infected children was significantly higher than that in controls （23.1 ±7.7%）; while the proportion of CD4＋ cells was decreased significantly. The expression of IFN-ymRNA in infected children within 3d after onset was increased by 2-fold, the expression level of IL-12p40mRNA during the entire process of disease was significantly higher than those of controls. Conclusion Children with acute rotavirus infection present early humoral immune response, especially mucosal immune response.