毒蕈碱3型受体胞外区第二环多肽诱导NOD-scid小鼠体内IL-17和IFN-γ的分泌

Immunization with 2nd extracellular loop peptide of muscarinic acetylcholine 3 receptor induces the secretion of IL-17 and IFN-γ in NOD-scid mice

目的:观察用毒蕈碱3型受体(M3R)胞外区第二环多肽免疫NOD-scid小鼠(nonobese diabetic mice CB17.Prkdcscid/J,非肥胖糖尿病/重症联合免疫缺陷小鼠)后,小鼠体内IL-17和IFN-γ分泌水平的变化。方法:200μg的M3R胞外区第二环多肽和不完全弗氏佐剂(IFA)以1∶1的比例配制而成后,皮下免疫NOD-scid小鼠。免疫后1、7、14、21 d尾部采血检测血清中的抗M3R第二环多肽抗体,血清中IL-17和IFN-γ含量的变化,同时测定每星期每只小鼠的饮水量。21 d给予小鼠安乐死,取小鼠脾脏细胞与抗M3R第二环多肽共同培养,观察细胞上清中IL-17和IFN-γ含量的变化。同时取小鼠泪腺做免疫荧光观察IL-17和IFN-γ的变化。结果:用M3R胞外区第二环多肽免疫NOD-scid小鼠14 d后,小鼠体内的抗M3R第二环多肽抗体显著高于对照肽组和PBS组(P<0.05),血清中IL-17和IFN-γ含量在第14天和第7天显著升高,具有统计学意义(P<0.01)。脾细胞多肽共培养后细胞上清中的IL-17的含量维持在一定的水平,而对照肽组和PBS组则显著下降(P<0.01)。组织免疫荧光显示泪腺中的IL-17和IFN-γ的含量也显著提高。每只小鼠的饮水量3组没有显著差别(P>0.05)。结论:用M3R胞外区第二环多肽免疫NOD-scid小鼠后成功诱导出抗M3R第二环多肽抗体,并且促进了NOD-scid小鼠体内IL-17和IFN-γ的分泌。

AIM： To evaluate whether or not the changes in the secretions of IL-17 and IFN-γ can be induced by the immunization with 2nd extracellular loop peptide of muscarinic acetylcholine 3 receptor （M3R） in NOD-scid （ nonobese diabetic-severe combined immunodeficiency） mice. METHODS： We synthesized the 2nd extracellular loop peptide of M3R and immunized NOD-scid mice subcutaneously with the 1：1 mixture of the peptide and the incomplete Freund＇s adjuvant （IFA）. At day 1, 7, 14, 21 after immunization, tail blood samples were taken to determine the antibody titer and evaluate the secretions of IL-17 and IFN-γ）, in sera. Meanwhile, we recorded the fluid intake amount per mouse every week. At day 21, all of the NOD-scid mice were killed to measure the concentrations of IL-17 and IFN-γ in cell supernatants. Immunofluorescence staining of lacri- mal glands was performed to observe the changes in the secretions of IL-17 and IFN-γ. RESULTS： Compared with the control group, the sera titers of anti-2nd extracellular loop peptide antibodies were significantly higher in 2nd extracellular loop peptide immunized NOD-scid mice at day 14 （P 〈 0.05）. The concentrations of IL-17 and IFN-γ increased significantly in sera of the 2nd extracellular loop peptide immunized NOD-scid mice at day 7 and 14 （P〈0.01）. The concentration of IL-17 maintained at a certain level in the supematants of spleen cells co-cultured with 2nd extracellular loop peptide, while it decreased significantly in the control groups （ P 〈 0.01 ）. Immunofluorescence staining demon- strated that the production of IL-17 and IFN-γ, increased in the lacrimal glands of NOD-scid mice immunized with the 2nd extracellular loop peptide. However, no changes in fluid intake was observed in NOD-scid mice immunized with the 2nd extracellular loop peptide （ P 〉 0.05 ）. CONCLUSION： Immunization with 2nd extracellular loop peptide of M3R can induce the production of anti-2nd extracellular loop peptide antibodies and the secretions of IL-17 and IFN-γ in NOD- scid mice.