【作者】 刘丽平；

【导师】 廖爱华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2009， 硕士

【摘要】 妊娠期高血压疾病(Hypertensive disorder complicating pregnancy,HDCP)严重威胁母胎健康,是世界范围内导致围产期母胎高发病率和死亡率的主要原因之一。正常妊娠的维持,需要体液免疫与细胞免疫的共同参与。诸多研究表明,在HDCP患者中,存在着不同程度的免疫失衡。有关细胞免疫因素参与HDCP发病机制的研究报道较多,而有关体液免疫因素对其影响的研究较缺乏,有限的研究多聚焦于血清中细胞因子的变化。本研究从胎儿和母体方面了解HDCP患者真实的体液免疫状态,并从抗胎儿免疫角度探讨其在HDCP发病机制中的作用。目的检测HDCP患者及正常足月剖宫产孕妇外周血中抗HLA抗体,比较母体血循环中抗HLA抗体特异性是否与胎儿HLA基因型一致,评价母体抗HLA抗体是否是被胎儿源性抗原致敏,探讨母体抗胎儿源性体液免疫在HDCP中的作用。方法选择46例正常足月剖宫产妇女为对照组,60例HDCP患者为病例组;病例组按病程进展分为妊娠期高血压组、轻度子痫前期组和重度子痫前期组,每组各20例。用Flow PRA法对母体血清中抗HLA抗体进行初筛,对于抗HLA抗体阳性者,用Luminex法检测其抗体特异性;同时用SSP-PCR法检测相应孕妇及胎儿的HLA基因型,比较母体外周血中抗HLA抗体类型是否与其胎儿HLA基因型一致。结果对照组母体外周血抗HLA-Ⅱ类抗体阳性率为6.5%;妊娠期高血压组为20%,轻度子痫前期组为20%,重度子痫前期组为25%。病例组HLA-Ⅱ类抗体阳性率较对照组高,其差异具有统计学意义(P < 0.05)。病例组各组间母体外周血抗HLA-Ⅱ类抗体阳性率随疾病进展加重而升高,但组间差异无统计学意义(P > 0.05)。对照组母体外周血抗HLA-I类抗体阳性率为19.6%,妊娠期高血压组、轻度子痫前期组和重度子痫前期组均为20%,其差异无统计学意义(P > 0.05)。比较母胎HLA基因类型,母体外周血中抗HLA-Ⅱ类抗体类型与其胎儿HLA基因型一致。结论HDCP患者外周血中抗胎儿HLA-Ⅱ类抗体明显增加,且被胎儿源性HLA致敏。目的比较HDCP患者及正常足月剖宫产孕妇胎儿脐血中IL-6阳性单核细胞百分比,了解HDCP患者胎儿单核细胞的激活状态。方法选择46例正常足月剖宫产妇女作为对照组,60例HDCP患者为病例组;病例组按病程进展分为妊娠期高血压组、轻度子痫前期组和重度子痫前期组,每组各20例。采集脐血(EDTA抗凝)经密度梯度离心分离单个核细胞,经双色荧光标记(FITC-CD14和PE-IL-6单克隆抗体)染色、流式细胞技术测定胎儿脐血中IL-6阳性单核细胞百分比。结果对照组胎儿脐血中IL-6阳性单核细胞百分比为0.78%±0.54%;妊娠期高血压组为4.38%±0.91%,轻度子痫前期为5.73%±2.18%,重度子痫前期为6.29%±2.70%。病例组胎儿脐血中IL-6阳性单核细胞百分比较对照组高,其差异有统计学意义(P < 0.01)。病例组各组间胎儿脐血中IL-6阳性单核细胞百分比随疾病进展加重而升高,各组间差异有统计学意义(P < 0.05)。结论HDCP患者脐血中胎儿单核细胞是被激活的,且随其严重程度增强。目的以正常足月剖宫产为对照,比较HDCP妇女外周血中抗胎儿HLA抗体分泌记忆B细胞的频数差异,探讨抗胎儿HLA抗体分泌记忆B细胞在HDCP发病机制中的作用。方法选择46例正常足月剖宫产妇女作为对照组,60例HDCP患者为病例组;病例组按病程进展分为妊娠期高血压组、轻度子痫前期组、重度子痫前期组,每组各20例。取相应胎儿脐血(EDTA抗凝),采用密度梯度离心法分离脐血中单个核细胞,与IFN-γ共培养72h后制备成B细胞溶胞体,作为胎儿源性抗原来源,包被96微孔硝酸纤维素膜板;加入体外培养并经美洲商陆(Pokeweed mitogen, PWM)预刺激的孕妇外周血单个核细胞(Peripheral blood mononuclear cells,PBMC),采用酶联免疫斑点法(Enzyme-linked Immunospot Assay,ELISPOT)检测HLA抗体分泌记忆B细胞。比较各组记忆B细胞频数(特异性ASC/总ASC百分比)的差异。结果正常妊娠足月剖宫产组,抗胎儿HLA抗体分泌记忆B细胞频数为0.37%±0.13%;妊娠期高血压组为0.67%±0.11%,轻度子痫前期组为0.79%±0.21%,重度子痫前期组为0.94%±0.27%。HDCP组抗胎儿HLA抗体分泌记忆B细胞频数高于正常妊娠组,其差异有统计学意义(P < 0.01)。HDCP各组间抗胎儿HLA抗体分泌记忆B细胞频数随疾病进展加重而增高,重度子痫前期与妊娠期高血压组比,其差异有统计学意义(P < 0.05);但轻度子痫前期组与妊娠期高血压组相比,其差异无统计学意义(P > 0.05)。结论HDCP组母体血循环中抗胎儿HLA抗体分泌记忆B细胞频数较正常妊娠组增加,其可能是母体抗胎儿异体免疫增强的原因。更多还原

【Abstract】 Hypertensive disorder complicating pregnancy (HDCP) is still a serious threaten to maternal-fetal health, which is contributed to the high maternal-fetal perinatal morbidity worldwide. A lot of studies have shown that immune imbalance occurs in HDCP. To maintain normal pregnancy, the coordination of cell-mediated immunity and humoral immunity is needed. Many reports indicated that cell-mediated immunity involved in the pathogenesis of HDCP. And most of them focused on the effects of serum cytokine profiles. However, there is lack of data about the effects of humoral immunity on the pathogenesis of HDCP. In the present study, the actual status of humoral immunity in HDCP was explored in view of both mother and fetus. It aimed to explore the effects of anti-fetal immunity on the pathogenesis of HDCP. Objective To detect the anti-HLA antibodies in peripheral blood of women with HDCP and normal term pregnancy, and compare if the antibody specificity was consistent with the HLA genotype of fetus. To evaluate if the HLA antibodies were activated by fetal-derived antigens, illustrating the possible role of maternal anti-fetal immunity in the pathogenesis of HDCP.Methods 60 patients with HDCP were divided into 3 groups, including gestational hypertension group, light pre-eclampsia group and severe pre-eclampsia group. 46 normal term pregnant women were selected as control group. Flow PRA was applied to screen the HLA antibodies in serum. For those women with positive results, the antibody specificity was further determinated by Luminex assay. Meanwhile, the HLA phenotypes of the mother and fetus were detected by SSP-PCR. It was compared if the HLA antibody specificity was in consistent with the HLA genotypes.Results The positive rate of anti-HLA classⅡantibody in maternal peripheral blood of normal term pregnancy was 6.5%, while it was 20% in gestational hypertension group, 20% in pre-eclampsia group and 25% in severe pre-eclampsia group. The difference was statistically significant (P < 0.05). The positive rate of anti-HLA class I in maternal peripheral blood of normal term pregnancy was 19.6%, while it was 20% in gestational hypertension group, pre-eclampsia group and severe pre-eclampsia group, respectively. There was no significant difference (P > 0.05). The fetal HLA genotypes were in good consistence with the specificity of anti-HLA class II antibody in maternal peripheral blood.Conclusion The positive rate of the anti-HLA classⅡantibodies in HDCP groups was increased, which was activated by the fetal-derived antigens. Objective To compare the percentage of IL-6 positive monocytes in fetal cord blood of normal term pregnant women and patients with HDCP, finding out the activation status of fetal monocytes.Methods 60 patients with HDCP were divided into 3 groups, including gestational hypertension group, light pre-eclampsia group and severe pre-eclampsia group. 46 normal term pregnant women were selected as control group. The cord mononuclear cells (CBMC) were isolated by density gradient centrifugation and stained with FITC-CD14 and PE-IL-6 monoclonal antibodies. The percentage of IL-6 positive monocytes in cord blood was determined by flow cytometry.Results The percentage of IL-6 positive monocytes in cord blood of normal group was 0.78%±0.54%; while it was 4.38%±0.91% in the gestational hypertension group, 5.73%±2.18% in light pre-eclampsia and 6.29%±2.70% in severe pre-eclampsia. The difference was statistically significant (P < 0.01). Moreover, the percentage of IL-6 positive monocytes was increased along with the progression of the disease (P < 0.05).Conclusion The fetal monocytes were activated in patients with HDCP and increased with the progression of the disease. Objective To explore the role of anti-fetal HLA antibody-secreting memory B cells in HDCP.Methods 60 patients with HDCP were divided into 3 groups, including gestational hypertension group, light pre-eclampsia group and severe pre-eclampsia group. 46 normal term pregnant women were selected as control group. The CBMC were isolated by density gradient centrifugation. The B cell lysate was obtained by stimulating the CBMC with IFN-γfor 72 h, which was applied as the fetal antigens. The PBMC were stimulated with PWM and cultured in vitro, which owned the ability to produce HLA antibodies. The frequency of anti-HLA antibody secreting memory B cells were detected by ELISPOT assay.Results In control group, the frequency of anti-fetal-HLA secreting memory B cell was 0.37%±0.13%; while it was 0.67%±0.11% in gestational hypertension group, 0.79%±0.21% in light pre-eclampsia group and 0.94%±0.27% in severe pre-eclampsia group. The difference was statistically significant (P < 0.01). The difference between gestational hypertension group and severe pre-eclampsia group was statistically significant (P < 0.05). However, there was no statistically significant between gestational hypertension group and light pre-eclampsia group (P > 0.05).Conclusion The frequency of anti-fetal HLA antibody secreting B cells was increased in HDCP, which might be the cause of enhanced humoral immunity.更多还原