小鼠外周血淋巴细胞亚群在细粒棘球蚴包虫致敏反应中的作用

Role of peripheral blood lymphocyte subsets in echinococcosis-induced sensitization in mice

目的建立细粒棘球蚴过敏反应BALB/c小鼠模型,研究和探讨淋巴细胞亚群在细粒棘球蚴致敏反应中的作用。方法从自然感染细粒棘球蚴的绵羊肝脏中提取原头蚴,培养40 d后,以50个微囊/鼠的剂量,通过腹腔注射接种BALB/c小鼠,对照组注射无菌生理盐水。感染6个月之后,每只小鼠按0.1 ml/10 g经腹腔注射羊源细粒棘球蚴粗制囊液致敏。对照组继续注射无菌生理盐水。激发过敏后1 h,根据评分将小鼠分为正常对照组、未致敏组、致敏组,每组各6只。取小鼠内眦静脉血,使用流式细胞仪检测B细胞、NK细胞、CD3^(+)/CD4^(+)/CD8^(+)T细胞,液相多重蛋白定量技术检测白细胞介素-4(interleukin-4,IL-4)、IL-6、IL-13的变化。结果与对照组比较,未致敏组的B细胞百分比显著升高(P<0.001),致敏组的B细胞百分比上升,但差异无统计学意义。与未致敏组比较,致敏组的B细胞百分比显著下降(P<0.01)。与对照组比较,未致敏组和致敏组的NK细胞百分比显著下降(P<0.001和P<0.01)。与未致敏组比较,致敏组的NK细胞百分比显著上升(P<0.05)。与对照组比较,未致敏组的CD3^(+)T细胞和CD4^(+)T细胞百分比显著降低(P<0.05),CD8^(+)T细胞百分比显著升高(P<0.01);致敏组的CD3^(+)T细胞和CD4^(+)T细胞百分比显著升高(P<0.05和P<0.001),CD8^(+)T细胞百分比无显著变化。与未致敏组比较,致敏组的CD3^(+)T细胞和CD4^(+)T细胞百分比显著升高(P<0.05),CD8^(+)T细胞百分比显著降低(P<0.01)。与对照组比较,未致敏组IL-4、IL-6、IL-13含量都显著升高(P<0.001);致敏组IL-4含量升高,但差异无统计学意义(P>0.05),IL-6含量显著升高(P<0.01),IL-13含量下降,但差异无统计学意义(P>0.05)。与未致敏组比较,致敏组IL-4、IL-6、IL-13含量都显著降低(P<0.001)。结论本研究成功建立了细粒棘球蚴过敏反应BALB/c小鼠模型,证明了Th2细胞引导的体液免疫应答在细粒棘球蚴囊液引起的过敏性休克中发挥着重要作用,为建立细粒棘球蚴囊液外溢所致过敏性休克患者的防治策略提供了重要的科学依据。

Objective To establish a BALB/c mouse model of Echinococcus granulosus allergy and investigate the role of lymphocyte subsets in Echinococcus granulosus-induced sensitization.Methods Echinococcus granulosus was isolated from the liver of sheep naturally infected with Echinococcus granulosus and cultured for 40 d.BALB/c mice were intraperitoneally injected with 50 microcapsules and sensitized by intraperitoneal injection of 0.1 ml/10 g of larval Echinococcus granulosus capsule six months after infection.According to the symptom scores 1 h after sensitization,these mice were divided into two groups:non-sensitized group(n=6)and sensitized group(n=6).The mice(n=6)in control group were injected with sterile saline.Blood sample was collected from the angular vein of each mouse.Flow cytometry was used to detect B cells,NK cells and CD3^(+)/CD4^(+)/CD8^(+)T cells.Cytometric bead array was used to measure IL-4,IL-6 and IL-13.Results The percentage of B cells was significantly higher in the non-sensitized group than in the control group(P<0.001),but no significant difference was observed between the sensitized group and the control group.Compared with the non-sensitized group,the percentage of B cells in the sensitized group decreased significantly(P<0.01).Compared with the control group,the percentages of NK cells in the non-sensitized group and the sensitized group decreased significantly(P<0.001 and P<0.01).Compared with the non-sensitized group,the percentage of NK cells in the sensitized group increased significantly(P<0.05).Compared with the control group,the percentage of CD3^(+)and CD4^(+)T cells in the non-sensitized group decreased significantly(P<0.05),while the percentage of CD8^(+)T cells increased significantly(P<0.01).Compared with the control group,the percentage of CD3^(+)and CD4^(+)T cells in the sensitized group increased significantly(P<0.05 and P<0.001),while no significant change in the percentage of CD8^(+)T cells was detected.Compared with the non-sensitized group,the percentage of CD3^(+)and CD4^(+)T cells in the sensitized group increased significantly(P<0.05),while the percentage of CD8^(+)T cells decreased significantly(P<0.01).The levels of IL-4,IL-6 and IL-13 were significantly higher in the non-sensitized group than in the control group(P<0.001).Compared with the control group,the sensitized group showed increased IL-4(P>0.05),significantly increased IL-6(P<0.01)and decreased IL-13(P>0.05).The levels of IL-4,IL-6 and IL-13 in the sensitized group were significantly lower than those in the non-sensitized group(P<0.001).Conclusions The BALB/c mouse model of allergy induced by larval Echinococcus granulosus was successfully established.This study proved that the humoral immune response induced by Th2 cells played an important role in Echinococcus granulosus-induced sensitization,which provides an important scientific basis for establishing a prevention and treatment strategy for patients with anaphylactic shock caused by extravasation of Echinococcus granulosus fluid.