反复自然流产的免疫因素分析探讨

为分析探讨反复自然流产的免疫性因素，应用补体微量淋巴细胞毒实验方法检测女性反复自然流产的封闭抗体，用ELISA法检测女性反复自然流产的抗心磷脂抗体(ACA)、抗精子抗体(ASA)共752例。结果显示，封闭抗体与反复自然流产明显相关，抗心磷脂抗体与反复自然流产有一定关系，抗精子抗体与反复自然流产无明显相关。研究表明，封闭抗体、抗心磷脂抗体是造成反复自然流产的重要免疫因素。     

正常妊娠期及正常生育期妇女体内的封闭抗体

<正> 妊娠中的胚胎类似同种异体移植,但结局却迥然不同。其主要原因可能由于以胎盘滋养叶细胞与母体脱膜间的复杂免疫调节环路。本文报道的正常生理妊娠及正常生育妇女体内的封闭抗体可能在母胎免疫调节中发挥重要作用。 材料和方法 一、研究对象:①正常妊娠组:无流产史的中孕妇女;②正常生育组:1～2年内有足月分娩史的生育期妇女。 二、实验方法:①补体依赖的微量细胞毒试验:参照McIntyre报道的研究方法加以改良,用男方外周血淋巴细胞检测女方血清中的细胞毒抗体,②单向混合淋巴细胞反应封闭试验:参照Takakuwa等研究方法加以改良,将男方外周血淋巴细胞用丝裂霉素处理,分别在12.5％AB型血清中或在12.5％女方血清中与女方外周血淋巴细胞,置于     

主动免疫疗法治疗反复自然流产145例疗效分析

目的探讨研究淋巴细胞主动免疫疗法治疗由于封闭抗体缺乏导致的同种免疫型反复自然流产的临床治疗效果。方法以经临床确诊为封闭抗体缺乏所致的反复自然流产的145例患者为研究组,取其丈夫或无关第三方男性外周静脉血,提取淋巴细胞后注射患者前臂内侧,进行淋巴细胞主动免疫的治疗,并观察患者的受孕和胚胎发育情况,进行疗效评价。结果 145例患者采用主动免疫治疗后,其中再次妊娠成功128例,早孕流产17例,妊娠成功率达88.27%。结论淋巴细胞主动免疫治疗能有效治疗封闭抗体阴性导致的反复性流产,并且封闭抗体阴性导致复发性流产的患者孕前淋巴细胞免疫治疗转阳后,妊娠成功率显著提高,临床效果显著,安全性高,操作便捷,经济性高,患者容易接受。     

配偶淋巴细胞免疫治疗反复自然流产91例分析

目的探讨反复自然流产患者,运用配偶淋巴细胞免疫治疗后,体内封闭抗体水平的变化及其临床疗效.方法对确诊为封闭抗体缺乏的RSA患者91例,用分离的配偶淋巴细胞,皮内注射法免疫治疗3～5疗程,治疗前后运用流式细胞仪测定患者体内封闭抗体水平.结果 91例RSA患者经免疫治疗后,体内封闭抗体的水平显著提高,抗配偶CD3%、抗配偶CD4%、抗配偶CD8%,分别由治疗前的-1.32±0.98、-1.39±0.80、-0.96±0.54上升为0.99±0.74、1.01±0.64、0.75±0.49(P＜0.05);妊娠成功80例,成功率为87.9%(80 / 91).结论配偶淋巴细胞免疫治疗,可以提高患者体内封闭抗体水平,有助于妊娠成功.     

反复自然流产患者血清中封闭抗体的检测

本文根据病史将反复自然流产(RSA)分类为原发性RSA 及继发性RSA,分别以单向混合淋巴细胞反应封闭试验及补体依赖微量细胞毒试验评价封闭抗体与两类流产的关系。结果发现,原发性RSA主要因封闭抗体缺乏所致;而继发性RSA 无封闭抗体缺乏迹象。因此,尽管原发性RSA 与继发性RSA 临床表现类似,但其免疫学病因及发病机理可能不同。     

补肾益气方对不明原因反复自然流产患者外周血Foxp3~+ Treg/Th1/Th17细胞的影响

为探讨补肾益气方对反复自然流产患者外周血Treg/Th1/Th17的影响,选取我院自然流产专科门诊30例反复自然流产(recurrent spontaneous abortion,RSA)患者,诊断为正常妊娠后开始服用补肾益气方中药,采用流式细胞仪检测服药前后Treg/Th1/Th17细胞数量,ELISA方法检测外周血TNF-α、IFN-γ、IL-4、IL-10及IL-17水平变化。结果显示中药治疗后RSA患者外周Treg细胞数量上升(5.85±2.76),明显高于服药前(3.26±1.19),P<0.05。Th1和Th17细胞数量下降(8.38±4.38和0.95±0.15),明显低于治疗前(23.59±8.14和1.58±0.71),P<0.05。IL-10和IL-4水平显著上升(P<0.05),TNF-α、IFN-γ和IL-17水平明显下降(P<0.05)。实验表明,补肾益气方中药能够调控Treg/Th1/Th17细胞水平,改变母体细胞因子分泌格局,保护胎儿不被排斥。     

系统性红斑狼疮重症血小板减少患者血清对巨核祖细胞的影响

研究系统性红斑狼疮(SLE)合并重症血小板患者的血清对正常人巨核祖细胞增殖分化的影响。以12例SLE重症血小板减少患者为研究对象,12份正常骨髓单个核细胞,培养中分别加入患者血清或灭活补体的患者血清,免疫化学染色检测巨核细胞克隆形成单位(CFU-MK),流式细胞仪检测CD41+细胞数。结果是正常对照组骨髓CFU-MK集落数为(61.22±29.71)个/片,加入SLE重症血小板减少患者血清后减少为(29.44±23.35)个/片,P<0.05;加灭活补体血清后减少为(22.56±15.21)个/片,与正常对照相比P<0.05;灭活补体与否差异不显著,P>0.05。加入患者血清或灭活补体的血清后,CD41+细胞数由正常(2.30%±1.63%)分别减少为(1.15%±0.85%)和(1.07%±0.76%),与正常对照相比P<0.05;灭活补体与否差异不显著。SLE血小板正常但活动期病人的血清对正常人CFU-MK和CD41+细胞的生成均无显著抑制。提示SLE重症血小板减少患者血清能抑制正常人骨髓巨核祖细胞的增殖分化,这种抑制作用是非补体依赖性的。     

哮喘患者诱导痰中GM-CSF mRNA表达的研究

目的利用非创伤方法获取样本的方法,在细胞和分子水平上探讨哮喘气道炎症的指标.方法3%高渗盐水诱导痰液,利用逆转录-聚合酶链反应技术(RT-PCR)检测哮喘患者治疗前后诱导痰中炎性细胞粒-巨噬细胞集落刺激因子(GM-CSF)信使核糖核酸(mRNA)表达和各种炎性细胞的变化.结果哮喘组15例,痰中嗜酸细胞百分数为(33.4±6.7)%,与慢性支气管炎(慢支炎)组(15例)的(2.1±0.4)%和对照组(10例)的(0.8±0.3)%比较,差异有显著性(P＜0.05);哮喘组诱导痰中的炎性细胞GM-CSFmRNA为0.32±0.05,与慢支炎组的0.19±0.02和对照组0.06±0.03比较,差异有显著性(P＜0.05),慢支炎组与对照组比较,差异有显著性(P＜0.05).哮喘患者(7例)治疗后诱导痰中嗜酸细胞为(32.6±11.9)%,与治疗前(29.6±8.7)%比较,差异无显著性(P＞0.05);诱导痰中炎性细胞GM-CSFmRNA表达为0.42±0.12,与治疗前的0.66±0.26相比,差异有显著性(P＜0.05).结论诱导痰中炎性细胞GM-CSFmRNA表达增高是一较特异的气道炎症指标.     

环孢菌素A对小鼠T细胞活化影响的血清免疫药理学评价

目的　应用血清免疫药理学技术 ,探讨环孢菌素A(CsA)对T细胞体外活化的影响 ,以进一步揭示CsA免疫调节的分子机制 ,为临床用药提供理论依据。方法　分离小鼠淋巴结细胞 ,分别与CsA、5 %CsA血清预孵后 ,加入多克隆刺激剂继续培养共 2 4h。收获细胞 ,进行双色免疫荧光标记 ,以流式细胞术分析T细胞上CD6 9分子的表达情况。结果　在CsA和 5 %CsA血清作用下 ,ConA活化的T细胞CD6 9表达的百分率分别为 (2 4 15± 3 38) %和 (2 3 6 8±6 89) % ,与相应对照组 [分别为 (6 4 6 7± 5 88) %和 (6 4 35± 10 13) % ]相比较均有显著差异 (P <0 0 1) ;在CsA和5 %CsA血清作用下 ,PDB活化的T细胞CD6 9表达的百分率分别为 (78 86± 7 70 ) %和 (80 0 5± 9 91) % ,与相应对照组 [分别为 (84 79± 6 87) %和 (79 6 8± 4 17) % ]相比较均无显著差异 (P >0 0 5 )。结论　CsA(410nmol/L)和含CsA血清均可强烈抑制ConA刺激的T细胞活化 ,而对PDB刺激的T细胞活化无抑制效应。 5 %CsA血清与CsA单纯药物体外实验的结果相一致 ,在一定程度上反映了临床等效剂量下血清免疫药理技术的有效性与可行性     

乙型肝炎患者外周血单个核细胞凋亡的检测及意义

目的研究激活诱导细胞死亡 (AICD)现象在乙型肝炎慢性化和重型化机制中的意义。方法分离 2 0例慢性 /慢性重型乙型肝炎病人与 10例健康献血员外周血单个核细胞 (PBMC) ,在 PHA的刺激下培养 72 h后收集细胞 ,经流式细胞仪检测凋亡。结果 PBMC凋亡率乙型肝炎组明显高于正常对照组 [(2 5 .48± 14.0 7) % vs(11.45± 5 .2 7) % ,P<0 .0 1];慢性乙型肝炎组 [(30 .5 7± 13.43) % ]明显高于正常对照组 [(11.45± 5 .2 7) % ,P<0 .0 1]和慢性重型乙型肝炎组 [(13.5 9± 6 .44 ) % ,P<0 .0 1];PBMC凋亡率乙型肝炎 HBe Ag(+ )组明显高于正常对照组 [(2 9.5 0± 12 .5 4) % vs(11.45± 5 .2 7) % ,P<0 .0 1]。结论 AICD可能是形成 HBV慢性感染免疫耐受的一个重要机制。     

Proportion of CD56+3+ T cells in decidual and peripheral lymphocytes of normal pregnancy and spontaneous abortion with and without history of recurrent abortion

PROBLEM: The present study investigated the proportion of CD56+3+ T cells in maternal peripheral and decidual lymphocytes in normal pregnancy and spontaneous abortion with and without history of recurrent spontaneous abortion (RSA). METHOD OF STUDY: Maternal peripheral blood and decidua were taken from normal pregnancies and missed abortions with and without RSA. Decidual lymphocytes were prepared from decidual tissue and analyzed by flow cytometry. RESULTS: In normal pregnancy, the percentages of CD56+3+ T cells in decidual lymphocytes did not differ from those in the peripheral blood. However, the proportion of CD56+3+ T cells in decidual CD3+ T cells increased higher than that in the peripheral CD3+ T cells. The percentages of decidual CD56+3+ T cells in missed abortions with and without RSA were lower than those in normal pregnancies. CONCLUSION: CD56+3+ T cells may play a role in the maintenance of pregnancy. The phenomenon, where the proportion of CD56+3+ T cells in decidual lymphocytes decreases, may be due to an immunologic event leading to missed abortion.     

Cytokine production by maternal lymphocytes during normal human pregnancy and in unexplained recurrent spontaneous abortion

It has been proposed that successful pregnancy is a T helper 2-type phenomenon, and that T helper (Th)1-type reactivity is deleterious to pregnancy. The objective of this study was to compare the concentrations of Th1 and Th2 cytokines produced by peripheral blood mononuclear cells from women undergoing unexplained recurrent spontaneous abortion (RSA) with those produced during normal pregnancy at a similar gestational stage. The control group consisted of 24 women with a history of successful pregnancies and the abortion group comprised of 23 women with a history of unexplained RSA. Blood from the control group was obtained at the end of the first trimester as gestational age controls for the abortion group from whom blood was collected at the time of abortion. Phytohaemagglutinin-stimulated peripheral blood cell culture supernatants were analysed for concentrations of cytokines. Significantly higher concentrations of Th2 cytokines were produced by the first trimester normal group than by the RSA group, while significantly higher concentrations of Th1 cytokines were produced by the abortion group as compared to first trimester normal pregnancy, indicating a distinct Th2-bias in normal pregnancy and a Th1-bias in unexplained RSA.     

Pre-conceptional natural killer cell activity and percentage as predictors of biochemical pregnancy and spontaneous abortion with normal chromosome karyotype

PROBLEM: The aim of the present study was to determine whether pre-conceptional natural killer (NK) cell activity and percentage are predictive of subsequent spontaneous abortion in women with recurrent spontaneous abortion (RSA). METHOD OF STUDY: Pre-conceptional NK cell activity and percentage in peripheral blood of women who had a history of two or more RSA was prospectively assessed. The 51Cr release assay and flow cytometric analysis were performed. A total of 113 RSA women were recruited, and 85 conceived later. RESULTS: Pre-conceptional NK cell activity/percentage values in women whose next pregnancies ended in biochemical pregnancy and spontaneous abortion with normal fetal karyotype (n = 17, median 47%/17.1%), but spontaneous abortion with abnormal karyotype (n = 9, 27%/15.7%), were higher than those in live births (n = 59, 33%/13.1%). High values of pre-conceptional NK cell activity (> 46%; relative risk 3.6, 95%CI 1.6-8.0) and percentage (> 16.4%; 4.9, 1.7-13.8) were found to predict biochemical pregnancy and spontaneous abortion with normal karyotype in the next pregnancy. CONCLUSION: Pre-conceptional NK cell abnormalities were predictive of spontaneous abortion with normal fetal karyotype.     

Immunophenotype of peripheral T lymphocytes, NK cells and expression of CD69 activation marker in patients with recurrent spontaneous abortions, during the mid-luteal phase

PROBLEM: Determination of subpopulations of T lymphocytes, natural killer (NK) and activation status, in peripheral blood during the mid-luteal phase from patients with unexplained recurrent spontaneous abortion (RSA). METHOD OF STUDY: Peripheral blood samples from non-pregnant women with RSA and normal multiparous were taken and evaluated for subpopulations of T lymphocytes: CD4, CD8, ('naive-like' and 'memory-like'), TCR receptor (alphabeta and gammadelta), activation status by CD69(+surface or intracellular)/CD3(+), and NK cells (CD16(+)/CD56(dim)/CD3(-), CD16(+)/CD56 (bright)/CD3(-), CD69(+surface or intracellular)/CD56(+)/CD3(-) cells). RESULTS: The evaluation of T lymphocytes only showed an increase in the expression of CD69 (surface and intracellular) in the RSA group. Additionally, we observed an increase in the total NK cells, CD56(+) NK cells percentages, CD56(dim) NK cells and CD69 NK cells in RSA group. CONCLUSION: These observations support the concept that immunological activation of T lymphocytes and NK cells could be involved in peripheral blood during the mid-luteal phase in patients with unexplained RSA.     

Secretion of Interleukin-2 from Unstimulated Peripheral Blood Mononuclear Cells Is a Possible Pathogenic Mechanism in Recurrent Abortion

PROBLEM: The objective of this study was to evaluate the activated status of helper T cells in women with recurrent abortion. METHOD OF STUDY: The spontaneous secretion of interleukin (IL)-2 from peripheral blood mononuclear cells (PBMCs) obtained from 12 women with primary partner-specific recurrent abortion and 7 normal females and 10 normal males was measured. RESULTS: IL-2 concentrations in medium conditioned with PBMCs from the recurrent abortion group were 1.44 ± 0.32 (mean ± standard deviation) U/ml, whereas those from the normal female and male groups were below the minimum detectable concentration. CONCLUSION: The fact that PBMCs from recurrent aborters spontaneously secrete IL-2 suggests that helper T cells are activated in recurrent aborters.     

Increased natural killer cell cytotoxicity and IL-2 production in recurrent spontaneous abortion

PROBLEM: To determine whether natural killer (NK) cells cytotoxicity in peripheral blood is altered in patients with a history of recurrent spontaneous abortion (RSA); also, if there is any correlation between cytokine production and NK cytotoxicity. METHOD OF STUDY: In this case-control study, 21 patients with RSA within 24 hr of the last abortion (group I), and 32 pregnants with no history of abortion (group II) were surveyed. NK cell cytotoxicity was evaluated by flow cytometry, and IL-2, IL-10, transforming growth factor beta1 were measured in cell culture supernatant by ELISA method. RESULTS: Group I showed higher NK cytotoxicity than group II at all of effector to target (E:T) ratios (P < or = 0.045).The correlation between production of IL-2 and NK cytotoxicity was positively significant (R = 0.350, P = 0.001). Group I had significantly higher levels of IL-2 than group II (P = 0.001). In group II, the production of IL-10 by peripheral blood mononuclear cells was higher than group I (P = 0.002). CONCLUSION: Increased NK cell cytotoxicity and high level of IL-2 may be considered as a risk factor for RSA.     

Decidual natural killer cells in recurrent spontaneous abortion with normal chromosomal content.

PROBLEM: The maternal local immune responses in unexplained recurrent spontaneous abortion (RSA) are not yet well known. Maternal peripheral and decidual natural killer (NK) cells were evaluated in RSA with normal chromosomal content. METHOD OF STUDY: Maternal peripheral blood, villous trophoblast, and decidua were taken from 15 normal pregnancies and 9 RSA patients with normal chromosomes. The NK cells in decidual lymphocytes were evaluated by flow cytometry using monoclonal antibodies for CD56, CD16, and CD3. RESULTS: The percentages of CD56+ CD16- CD3- cells in decidual lymphocytes in RSA were lower than in normal pregnancies (P < 0.002). The CD56+CD16+/CD56+CD16- cells ratio in RSA was higher than in normal pregnancies (P < 0.02). CONCLUSION: The lower percentages of CD56+CD16-CD3- cells in RSA cases may show an inappropriate accumulation of NK cells in the decidua, and this finding may be a factor involved in RSA.     

活动性巨细胞病毒感染与反复自然流产的关系探讨

目的探讨活动性人巨细胞病毒（human cytomegalovirus,HCMV）感染与反复自然流产（recurrent spontaneous abortion,RSA）的关系.方法采集反复自然流产孕妇和正常产前体检孕妇外周血,分离外周血单个核细胞（PBMCs）和血浆,分别用免疫荧光法和实时定量PCR检测HCMV pp65抗原和HCMV DNA,并比较2种方法的一致性.结果 65例RSA患者HCMV pp65抗原有20例阳性,阳性率30.8%,50例正常体检孕妇 HCMV pp65抗原有4例阳性,阳性率8.0%,2组孕妇HCMV活动性感染率有显著性差异（χ^2=8.87,P＜0.01）.孕妇HCMV pp65抗原阳性率升高,孕妇流产几率增加（χ^2=7.53,P＜0.01）. 免疫荧光法和实时定量PCR有较好的一致性（92.3%）.结论反复自然流产孕妇 HCMV活动性感染率显著高于正常孕妇,HCMV pp65抗原检测也许可作为RSA早期诊断指标之一.     

Leukocyte activation in the decidua of chromosomally normal and abnormal fetuses from women with recurrent abortion

As part of our continuing programme to investigate immunological causes of unexplained recurrent pregnancy losses, we studied subpopulations of white blood cells and their activation status in decidua of women with a history of recurrent spontaneous abortion (RSA). We differentiated specifically between normal karyotyped male fetuses and abnormal karyotyped fetuses with trisomy 16 because trisomy 16 is not compatible with life and is thus a non-controversial cause of spontaneous miscarriage. Leukocytes were counted in paraffin-embedded decidua after immunohistological staining for CD45 (LCA), CD3, CD56, CD68, CD69 and CD25. Numbers of activated versus non-activated T lymphocytes, NK cells and macrophages were compared in decidua from women with: (i) unexplained RSA who had a normal male karyotype (n = 17) miscarriage; (ii) unexplained RSA who had a trisomy 16 (n = 21) miscarriage; and (iii) normal gestationally age-matched first trimester pregnancies following elective termination procedures (n = 20). Significantly more activated leukocytes were detected in the decidua of women with unexplained RSA who had a normal male karyotype compared to the other groups (P < 0.0001). In addition, numbers of cells comprising the major leukocyte subpopulation, CD56+ NK cells, appeared reduced in the decidua of women with unexplained RSA compared to decidua from women having elective terminations. Increased numbers of activated leukocytes in the decidua of women with a history of unexplained recurrent pregnancy loss who had a normal karyotyped pregnancy provide evidence that cellular immunity may be involved in unexplained recurrent pregnancy loss.     

原因不明复发性流产中CD4+ Notch1+T细胞与IL-10的相关性分析

探讨原因不明复发性流产（URSA）中CD4＋Notch1＋T细胞（Notch1＋占CD4＋T细胞的比例）与白介素-10（IL-10）表达水平的相关性。以雌性CBA/J×雄性Balb/c为正常妊娠模型,以雌性CBA/J×雄性DBA/2J为自然流产模型,采用流式细胞术检测6例未孕CBA/J雌性小鼠、6例正常妊娠模型孕14天CBA/J雌性小鼠和6例自然流产模型孕14天CBA/J雌性小鼠脾细胞中CD4＋Notch1＋T细胞,同时运用ELISA法检测其血清中IL-10的表达水平。相比于未孕组,正常妊娠模型中CD4＋Notch1＋T细胞比例减少,而自然流产模型中CD4＋Notch1＋T细胞比例增加,正常妊娠模型与自然流产模型中CD4＋Notch1＋T细胞比例差异有统计学意义（P〈0.05）。CD4＋Notch1＋T细胞比例与IL-10负相关（r=-0.568,P〈0.05）。结论：CD4＋Notch1＋T细胞可能参与原因不明复发性流产发病机制,拮抗Notch1＋表达有可能成为治疗URSA新途径。