类天疱疮和天疱疮患者肿瘤坏死因子α及可溶性肿瘤坏死因子Ⅰ型受体的测定
类天疱疮和天疱疮患者肿瘤坏死因子α及可溶性肿瘤坏死因子ⅰ型受体的测定
中华皮肤科杂志 1999年第6期第32卷 论著
作者：陈向东 翁孟武 陈洪 苏玮 侯爱琴
单位：陈向东 200011上海第二医科大学附属第九医院皮肤科；翁孟武 苏玮 侯爱琴 上海医科大学附属华山医院皮肤科；陈洪 上海第二医科大学微生物教研室
关键词：类天疱疮；大疱性天疱疮肿瘤坏死因子受体；肿瘤坏死因子
【摘要】目的 探讨肿瘤坏死因子α(tnfα)及其可溶性ⅰ型受体(stnf-rⅰ)在类天疱疮、天疱疮患者疱液和血清中的变化及其临床意义。方法 应用elisa方法对比研究31例bp、9例pv患者血液和疱液中tnfα和stnf-rⅰ的含量。并以15例正常人吸疱液和30例正常人血清作对照。结果bp、pv患者活动期疱液和血液中stnf-rⅰ水平均高于对照，且疱液中浓度更高；bp缓解初期血中stnf-rⅰ水平高于活动期；bp患者血液和疱液中均未测到或有极少量tnfα，pv患者血液中仅测到低浓度的tnfα，而疱液中tnfα浓度明显高于对照。结论 细胞因子stnf-rⅰ和tnfα可能参与了bp和pv的发病，stnf-rⅰ在bp中可能对机体起保护作用，有望成为病情好转和治疗有效的新的观察指标。
levels of tumour necrosis factor α and its soluble receptor type ⅰ in sera and blister fluids of patients with autoimmune bullous dermatoses
chen xiangdong,weng mengwu,chen hong,et al.
department of dermatology,huashan hospital
of shanghai medical university,shanghai 200040
【 abstract】 objective to determine the relationship between tumour necrosis factor α (tnfα ) and its soluble receptor type ⅰ (stnf-rⅰ) and their significance in autoimmune bullous dermatoses.methods the levels of tnfα and stnf-rⅰ in both sera and blister fluids of 31 cases with bullous pemphigoid(bp) and 9 cases with pemphigus vulgaris(pv)were investigated by elisa.fifteen suction blister fluids and 30 sera from normal individuals were taken as the control.results at active stage， the levels of stnf-rⅰ were higher in both the sera and blister fluids from patients with bp and pv than those from the controls,and were much higher in blister fluids than in sera.the serum level of stnf-rⅰ was even higher at early remission stage than at active stage of bp.there were traces or undetectable amount of tnfα in both sera and blister fluids of bp cases. tnfα level was much low in sera of pv cases, but higher in pv patients′ blister fluids than in control group′s.conclusion stnf-rⅰ and tnfα might be related to the pathogenesis of the two diseases, and the former might exert a protective effect on the inflammatory reaction and might serve as a new indicator for evaluation of the severity of bp and the effectiveness of therapy. the effects of stnf-rⅰ and tnfα in bp and pv might be different. tnfα might be much important for blister formation in pv.
【 key words】 pemphigoid,bullous pemphigus tumor necrosis factor receptors,tumor necrosis factor
肿瘤坏死因子α是一种多功能的细胞因子，它不仅对肿瘤有直接的细胞毒作用，还具有多种免疫调节活性。与之相对应的可溶性肿瘤坏死因子ⅰ型受体在调节tnfα的生物学活性中起了很大的作用。我们对40例自身免疫性大疱病患者的血液和疱液中tnfα及stnf-rⅰ进行测定。
材料和方法
病例：40例患者均选自本院门诊或住院病例，其中类天疱疮31例、天疱疮9例。男23例，女17例，平均年龄岁。所有病例经临床、病理、dif、iif及1mol/lnacl盐裂后免疫荧光证实，并排除其它感染情况。
血清和疱液：所有患者同时抽取未经治疗的活动期(指患者不断出现水疱或红斑)血液及24h内新发之水疱疱液。11例bp患者在病情控制后即抽取缓解期(指治疗后无新发水疱，无明显炎症约1周)血液。离心后分别取血清及上清液，分装，－30℃保存。另外，30例正常人血清及15例正常人的吸疱液作为对照。
(三)elisa方法测定血清、疱液中tnfα、stnf-rⅰ：①从试剂盒中取出所需已包被相应单抗的板条。②加入标准品稀释液100μl至空白孔和零孔，将待测血清或疱液和已稀释的不同浓度的标准品各100μl加入相应孔中，用封板胶封住反应孔，37℃孵育90min，用清洗液洗板。③除零孔外，每孔加稀释好的1∶600生物素标记二抗100μl，封住板孔，37℃孵育60min后洗板。④除零孔外，每孔加稀释好的酶联物100μl，封住板孔，37℃孵育30min后洗板。⑤每孔加底物a、b各50μl，避光，37℃孵育15min。⑥每孔加终止液50μl，混匀。⑦即刻在450nm波长的酶标仪下读数。⑧制作标准曲线并根据标准曲线查找各标本相应浓度。
(四)统计学方法：检测数据用spss统计软件进行统计学处理。变量用秩和检验或t检验。
结果
31例bp、9例pv患者活动期血清、疱液中tnfα、stnf-rⅰ的检测结果见表1。bp、pv患者活动期疱液和血液中stnfri水平均高于对照，且疱液中浓度更高。bp患者活动期血液和疱液中tnfα的含量明显低于对照。pv患者血液中仅测到极低浓度的tnfα，而疱液中tnfα浓度明显高于对照。
11例bp患者活动期和缓解初期血清中tnfα、stnf-rⅰ的比较见表2。bp缓解初期血清中stnfrⅰ水平较活动期高。tnfα水平则明显低于对照。另外，我们还对2例疗效不佳一直未愈的bp患者发病初期、病程4周和8周时血中stnf-rⅰ水平进行动态观察，检测结果分别为2150、1800、1600pg/ml和1275、1175、1050pg/ml，均显示stnf-rⅰ在4周和8周时反较发病初期逐渐降低。
表1 类天疱疮与天疱疮患者活动期血清和疱液肿瘤坏死
因子α及可溶性肿瘤坏死因子ⅰ型受体的测定结果(pg/ml)
组别例数肿瘤坏死因子α可溶性肿瘤坏死因子ⅰ型受体
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