摘要
目的观察并比较C57BL/6小鼠初次和加强免疫Ⅱ型胶原后的特异性细胞及体液免疫应答,探讨特异性免疫应答在胶原诱导关节炎发病中的意义。方法用鸡Ⅱ型胶原(CⅡ)在C57BL/6小鼠尾部皮内注射,诱导胶原性关节炎(CIA)。分别取初次免疫后19d及加强免疫后7、28dCIA小鼠脾脏淋巴细胞,CⅡ体外刺激扩增后,采用5-溴脱氧尿嘧啶(BrdU)掺入法和流式细胞术分别测定其扩增情况和表型;并通过流式细胞术测定外周血中细胞内细胞因子干扰素(IFN)-γ、白细胞介素(IL)-4的水平分析Th1和Th2亚群的变化;同时用酶联免疫吸附试验(ELISA)测定不同时期CIA模型血清中抗CⅡ抗体的表达。结果①免疫后不同时间,CIA模型组外周血中CD4+IFN-γ+细胞百分率明显高于对照组(P<0.01),但CIA模型组内不同时间CD4+IFN-γ+细胞百分率差异无统计学意义;②特异性增生实验显示,CIA模型组CD4+T细胞中BrdU+细胞率均高于对照组,但加强免疫后BrdU+细胞率明显低于初次免疫(72±6)%,并且有逐渐降低的趋势;初次免疫后CD4+IFN-γ+细胞百分率为(13±4)%,加强免疫后阳性细胞百分率也下降;③ELISA法测定不同时间血清中CIA模型组抗CⅡ抗体水平,28d吸光度(A)值达到最高,其后逐渐减低,到35d左右(即加强免疫后14d)出现较低的抗体水平。
Objective To compare the primary (19 days) and secondary (7 days, and 28 days) CⅡ-specific cellular and antibody responses in C57BL/6 mice. Methods C57BL/6 mice were immunized with emulsified chicken type Ⅱ collagen (CⅡ) in complete Freund′s adjuvant by intradermal injection to induce the collagen-induced arthritis (CIA) mice model. The lymphocytes were obtained from spleen of CIA mice killed at 19 days after initial immunization and 7 days, 28 days after booster immunization, and the CⅡ reactive T cells were stimulated by CⅡ in vitro. The proliferation response and phenotype were analyzed by BrdU incorporation and fluorescence-activated cell sorter (FACS); intracellular cytokines (IFN-γ, IL-4) and surface antigen(CD4) of T cells in the peripheral blood of CIA mice were assayed by FACS; anti-CⅡ antibody in serum was assayed by enzyme-linked immunoadsorbent assay (ELISA) at different timepoints after immunization (7, 14, 21, 28, 35, 42, and 49 days). Results ① The representation of the Th1 cytokine IFN-γ in the peripheral blood of CIA mice was significantly higher than control groups (P<0.01); but a similar representation was observed in the peripheral blood of CIA mice at different timepoints after immun-ization; ② In vitro, the frequency of BrdU+ cells in CD4+ T cells stimulated by CⅡ was significantly higher than that of control group (P<0.01). But CD4+ T cells at 7 days, and 28 days after booster immunization had significantly lower proliferative responses against CⅡ than those of at the initial immunization [the frequency of BrdU+ cells was(72±6)%; and the percentage of IFN-γ-positive cells was (13±4)%]; subsequently there was gradually decreased proliferative responses against CⅡ following secondary boosting immunization; ③ At 28 days of initial immunization, the level of anti-CⅡ antibody in serum reached its peak, and then gradually decreased; at 35 days of initial immunization there was significantly lower level of anti-CⅡ antibody. Conclusion A low specific T cell and B cell responses after secondary immunization in B6 mice leads to the resistance to CIA, and provides a new research clue for CIA and RA pathogenesis and therapy.
出处
《中华风湿病学杂志》
CAS
CSCD
2005年第4期215-218,共4页
Chinese Journal of Rheumatology
基金
国家"863计划"基金资助项目(2001AA215061)
