寻常型天疱疮患者抗原特异性Th细胞的研究

Antigen.specific helper T cells in patients with pemphigus vulgaris

目的探讨寻常型天疱疮（pemphigusvulgaris，PV）患者抗原特异性Th1和Th2细胞在不同疾病阶段的变化，进一步了解自身反应性T细胞在疾病中的作用。方法收集24例PV患者资料，合成桥粒芯糖蛋白抗原肽段DG3（96～112）.体外用该肽段分别刺激患者的外周血单一核细胞（PBMC），用酶联免疫斑点（EusPOT）方法检测干扰素（IFN）-γ＋Th1细胞和白介素（IL）-4＋Th2细胞的数量，以及记忆性B细胞的数量。用t检验或单因素方差分析（one—wayANOVA）对各组数值进行比较，用Pearson相关系数对Th细胞、记忆性B细胞及抗桥粒芯糖蛋白3（Dsg3）抗体滴度进行相关性分析。结果24例Pv患者男女比例1．67：1，平均年龄（56．59±14．66）岁。5×10^5个PBMC中，PV患者特异性IFN-γ＋Th1细胞绝对数为420．18±350．29，高于健康对照145．12±86．56，差异有统计学意义（P〈0．05）。PV患者特异性IL-4＋Th2的细胞数（366．76±192．44）与健康对照（335．88±164．96）之间差异无统计学意义。PV患者外周血中特异性IL-4＋Th2占总Th2细胞的百分率为37．03％±23．44％，特异性IFN-γ＋Th1细胞为23．62％±16．77％；7例患者进行了治疗前后的自身对照比较。特异性IL-4＋Th2细胞的数量在治疗后（241．68±160．60）较治疗前（452．82±199．29）明显下降，差异有统计学意义（t=2．48，P〈0．05）。特异性卟细胞、记忆性B细胞及抗Dsg3抗体滴度之间无统计学相关性（均P〉0．05o结论抗原肽段DG3（96～112）含有被PV患者特异性Th细胞识别的致病性抗原表位；特异性IFN-γ＋Th1细胞和IL-4＋Th2细胞均在疾病中发挥一定的作用；特异性IL-4＋Th2细胞在疾病活动中可能起到了更重要的致病作用。

Objective To study the changes of antigen-specific T helper type 1 （Thl） cells and Th2 cells in patients with pemphigus vulgaris （PV） at different stages, so as to better understand the roles of autoreactive T cells in PV. Methods The DG3 （96 - 112） peptide was synthesized. Twenty-four patients with PV and 10 health checkup examinees were included in this study. Peripheral blood mononuclear cells （PBMCs） were obtained from the health checkup examinees and all the patients before treatment and seven patients about one month after treatment, and stimulated with the DG3 peptide of 10 mg/L for different durations. Then, enzyme-linked immunospot （ELISPOT） assay was performed to count the number of DG3-specific IFN-γ＋ Thl cells, IL-4＋ Th2 cells and memory B cells. One-way analysis of variance （ANOVA） and t test were done to compare the number of cells between different groups, and Pearson correlation coefficient was used to evaluate the correlation among Th cells, memory B cells and anti-desmoglein 3 （Dsg3） antibody titers. Results In these patients, the male to female ratio was 1.67 ： 1, and the average age was （56.59 ± 14.66） years. Compared with the health check-up examinees, the patients with PV showed a higher absolute number of DG3-specific IFN-γ＋ Th1 cells （420.18 ± 350.29 vs. 145.12 ± 86.56, t = 3.25, P 〈 0.05）, but a similar absolute number of specific IL-4＋ Th2 cells （366.76 ± 192.44 vs. 335.88 ± 164.96, P 〉 0.05） per 5 x 105 PBMCs. The percentage of DG3-specific IL-4＋ Th2 cells in Th2 cells was 37.03% ± 23.44%, and the percentage of IFN-γ＋ Thl cells was 23.62% ± 16.77% in peripheral blood of patients with PV. The number of DG3-specific IL-4＋ Th2 cells per 5 × 10^5 PBMCs significantly decreased from 452.82 ＋ 199.29 before treatment to 241.68 ＋ 160.60 after treatment in seven patients （t = 2.48, P 〈 0.05）. There was no significant correlation between specific Th cells, memory B cells and anti-Dsg3 antibody titers （all P 〉 0.05）. Conclusions The peptide DG3 （96-112） has pathogenic epitopes which could be recognized by specific Th cells of patients with PV. Both antigen-specific IFN-γ＋ Thl cells and IL-4＋ Th2 cells play certain roles in the pathogenesis of pemphigus vulgaris, and IL-4＋ Th2 cells appear to be more important.