【摘要】目的比较供者及配偶白细胞免疫疗法治疗母-胎免疫识别低下型反复自然流产(RSA)的临床疗效。方法将40例封闭抗体缺乏的RSA患者随机分成两组，分别采用供者白细胞(静脉)与配偶白细胞(皮内加静脉)行免疫治疗。封闭抗体及抗其独特型抗体分析采用单向混合淋巴反应封闭试验、单向混合淋巴细胞反应封闭抑制试验、细胞毒试验及封闭抗体的流式细胞分析。结果经供者或配偶白细胞免疫后，封闭抗体及抗其独特型抗体呈明显升高（P＜0.05, P＜0.01）。再次妊娠成功率为86.2%；新生儿体重为3 300±116 g(±s),与同期正常妊娠组比较，差异无显著性(P＞0.05)。对封闭抗体的动态分析显示，配偶白细胞免疫疗法优于供者白细胞免疫疗法；但再次妊娠成功率及新生儿体重在两者间差异无显著性。结论供者或配偶白细胞免疫，能有效刺激母-胎免疫识别低下型RSA患者产生封闭抗体及抗其独特型抗体，并导致妊娠成功。白细胞免疫疗法对新生儿体重无明显影响。

Comparative Study of the Third Party and Paternal Leukocyte Immunization in Recurrent Spontaneous Abortion of Lowered Maternal-Fetal Immuno-RecognitionLi dajin, Li Chaojing, Zhu Ying, et al. Laboratory of Reproductive Immunology, Institute of Obstetrics and Gynecology, Shanghai Medical University, Shanghai 200011

【Abstract】ObjectiveTo compare curative effect of leukocyte immunization to recurrent spontaneous aborters(RSA) with lowered maternal-fetal immuno-recognition between transfusions of the third party leukocyte-rich erythrocyte concentrates and injections of paternal leukocytes.Methods Forty RSA patients with lack of blocking antibodies were randomly divided into two groups. Blocking antibodies were analysed by one-way mixed lymphocyte reaction blocking test and complement-dependent cytotoxic test, anti-idiotypic antibodies to blocking antibodies by one-way mixed lymphocyte reaction blocking inhibiting test, and flow cytometry evaluation of blocking antibodies were used to investigate effect of blocking antibodies on the CD antigens of T cells. The immunization was achieved by transfusions of the third party leukocytes or injections of the paternal leukocytes for 3 times or more at an interval of 4 weeks.Results It was found in dynamical analyses that levels of the blocking antibodies and their anti-idiotypic antibodies increased significantly after the aborters had been immunized. The successful pregnancy rate was 86.2% following immunization, and body weight of the infants was 3 300±116 g(±s), with no significant difference to 3 279.0±113.8 g of the infants from normal pregnancy. According to dynamics of blocking antibodies and their anti-idiotypic antibodies, the paternal leukocyte immunotherapy appeared superior to the third party one, but the successful pregnancy rate and the body weight of infants were with not different between the two immunotherapy groups.ConclusionThe third party and paternal leukocyte immunization could effectively stimulate the aborters of lowered maternal-fetal immuno-recognition to produce blocking antibodies and their anti-idiotypic antibodies, leading to successful pregnancy. The immunotherapy does not apparently influence the body weight of the delivered infants.

【Key words】Abortion, habitual ImmunotherapyAntibodies, blockingAntibodies, anti-idiotypic

反复自然流产(recurrent spontaneous abortion, RSA)患者中，除少数因生殖内分泌、染色体及生殖器官解剖学异常外，多数患者病因及发病机理不详^［1］。随着生殖免疫学及其技术的发展，发现 RSA 多因母、胎免疫调节异常所致^［2］。根据免疫流行病学分析，RSA可进行以下免疫病因分类：母-胎免疫识别低下型、母-胎免疫识别过度型、母-胎免疫识别紊乱型^［3］。其中，以封闭抗体缺乏为主要特征的母-胎免疫识别低下型是最常见的一种类型。本研究分别选用供者白细胞静脉免疫及配偶白细胞皮内加静脉免疫，对母-胎免疫识别低下型RSA患者进行免疫治疗，观察并评价两种白细胞免疫疗法后治疗封闭抗体及抗其独特型抗体的动态变化及妊娠预后。

资料及方法

一、研究对象

40例RSA患者来自1990年1月至1996年1月就诊于上海医科大学妇产科医院生殖免疫专科门诊的患者。经生殖免疫学系统分析属母-胎免疫识别低下型，选择标准参见文献［4］。将40例RSA随机分为两组，供者组与配偶组各20例。比较新生儿体重的对照组，为在本院同期无妊娠并发症及妊娠合并症足月分娩的新生儿。

二、免疫方法

供者组：采用供者白细胞静脉免疫疗法，输注红细胞血型一致的红白细胞混悬液4个单位。配偶组：采用配偶白细胞皮内加静脉免疫疗法, 白细胞总数平均为2.74×10⁷/次。两组均间隔4周免疫1次, 免疫3次为1疗程, 末次免疫后2周复查封闭抗体。封闭效率(MLR-BE)仍未升高者加强免疫1次, 直至封闭抗体明显升高。两组患者封闭抗体水平在免疫前经统计学检验差异无显著性。

在发现患者封闭抗体升高后，安排受孕；并于妊娠35天加强免疫1次，此后每间隔4周加强免疫1次，直至孕16周。妊娠16周后一般不需加强免疫。

三、观察方法

1. MLR-BE: 以单向混合淋巴细胞反应封闭试验检测MLR-BE，方法参见文献［3］并对其进行改进：以丝裂霉素处理的男方外周血淋巴细胞作为刺激细胞(1×10⁶/ml，0.1 ml); 以患者外周血淋巴细胞作为反应细胞(1×10⁶/ml，0.1 ml), 将刺激细胞与等量患者血清于37℃水浴1小时后, 去上清。以未经患者血清处理的刺激细胞为对照, 在含有25%正常AB型血清中, 与反应细胞于5% CO₂、37℃孵育5天后，加³H-TdR 0.6 μCi/孔, 继续培养18小时后,收样分别测定各孔放射性核素每分钟闪烁记数(min^-1)值。按下式计算MLR-BE：

MLR-BE=(1-(A)/(B))×100%

A: 刺激细胞与患者血清作用后, 加反应细胞在25%AB型血清中培养的min^-1值

B: 刺激细胞加反应细胞在25%AB型血清中培养的min^-1值

2. 抗封闭抗体的独特型抗体: 以单向混合淋巴细胞反应封闭抑制试验检测抗封闭抗体的独特型抗体。在MLR-BE 实验方法基础上, 增加在25%患者血清中混合淋巴细胞培养，以分析患者血清中可能存在的抗封闭抗体的独特型抗体对MLR-BE的作用。按下式计算抗封闭抗体的独特型抗体:

抗封闭抗体的独特型抗体 = (C-A)/(B)×100%

C: 刺激细胞与反应细胞在患者血清中培养的min^-1值

3. 细胞毒指数(CI): 参见文献［3］: 取微量细胞毒试验板, 分别设定加补体的不加待检血清及加待检血清不加补体的阴性对照,及加抗人淋巴细胞血清(ALS)并加补体的阳性对照; 以男方外周血淋巴细胞作为指示细胞,按标准细胞毒试验法检测患者血清中作用于男方淋巴细胞膜表面抗原的细胞毒抗体。按下式计算CI:

CI=(样品平均死细胞率-阴性对照平均死细胞率)/

(100%- 阴性对照平均死细胞率)×100%

4. 封闭抗体的流式细胞分析: 参见文献［5］。以男方外周血淋巴细胞为抗原靶标，以正常人AB型血清为对照, 分析患者血清中封闭抗体对CD₃、CD₄及CD₈抗原的作用。按下式分别计算抗CD抗原的封闭效率(抗CD-BE):

抗CD-BE＝加AB型血清CD比例-

加患者血清CD比例(%)

四、统计学分析方法

计量资料均数采用t检验，应用SPSS/PC+统计学软件处理系统。 计数资料行χ²检验。

结果

一、免疫后两组封闭抗体的动态变化

母胎同种免疫识别低下型 RSA经供者或配偶白细胞免疫后，反映封闭抗体的各项指标均显示不同程度的上升趋势，特别是MLR-BE、MLR-BE抗独特型抗体、抗CD₃-BE及抗CD₄-BE在免疫后显著升高。其封闭抗体动态变化情况见表1。

二、供者及配偶白细胞免疫疗效的比较

经供者白细胞静脉免疫及配偶白细胞皮内加静脉免疫治疗后，反映封闭抗体的各项指标均呈现明显升高趋势。根据封闭抗体动态变化分析，配<