先兆子痫患者外周血、脐血及蜕膜NK细胞的相关研究

目的：对先兆子痫孕妇外周血、脐血及蜕膜NK细胞进行研究，以探讨先兆子痫的病因。方法：对42例先兆子痫患者及20例正常孕妇外周血、脐血NK细胞分离后记数。采用免疫组织化学法检测胎盘蜕膜NK表型后记数并检测其平均灰度。结果：①轻度及重度先兆子痫患者母血及脐血NK细胞数均显著高于正常孕妇（P〈0．05）；②重度先兆子痫患者蜕膜NK细胞数高于正常孕妇（P〈0．05）；③轻度及重度先兆子痫患者胎盘蜕膜NK细胞CD56组化染色平均灰度值高于正常孕妇（P〈0．05）。结论：先兆子痫患者外周血、脐血及蜕膜的NK细胞数量均高于正常孕妇，且呈激活状态的胎盘蜕膜NK细胞较多，可能与发病有关。     

重度子痫前期蜕膜NK细胞Tim-3的表达及其相关作用

目的 探讨重度子痫前期Tim-3+CD56+CD16-NK细胞的表达及其对滋养细胞侵袭及血管内皮细胞成管能力的影响.方法 分离重度子痫前期患者(sPE组)及正常妊娠孕产妇(Normal组)的胎盘蜕膜组织中单个核细胞,细胞流式检测蜕膜NK细胞(decidual NK cell,dNK)细胞所占比例;免疫磁珠分选CD56+...     

可溶性人类白细胞抗原G与重度子痫前期的相关性研究

目的：在重度子痫前期患者血浆中的含量水平,探讨s HLA-G在早发型及晚发型重度子痫前期患者血浆中表达的差异性。方法：选择南京医科大学附属南京医院2013年07月至2014年04月间收治的6例早发型重度子痫前期患者（早发型组）、18例晚发型重度子痫前期患者（晚发型组）的临床资料,比较早发型组及晚发型组患者的一般临床情况;应用免疫组织化学染色法（enzyme linked immunosorbent assay,ELISA）,检测6例早发型组,18例晚发型组及32例正常足月妊娠者（正常对照组）的血清中s HLA-G的表达,并比较三组s HLA-G含量的差异。结果：1）早发型组与晚发型组的发病孕周,分娩孕周及期待治疗天数差异均有统计学意义（P〈0.05）,而年龄差异无统计学意义（P〉0.05）;2）早发型组与晚发型组的收缩压及舒张压差异均有统计学意义（P〈0.05）;3）早发型组中s HL A-G含量均值为（4.284±1.932）ng/m L,晚发型组中s HL A-G含量均值为（7.036±1.623）ng/m L,正常孕妇组中s HLA-G含量均值为（10.399±3.168）ng/m L。正常对照组s HLA-G含量高于重度子痫前期组（P〈0.01）,且早发型组低于晚发型组（P〈0.05）。     

妊娠早期蜕膜CD56~+NK样细胞杀伤活性的研究

分离早孕妇女蜕膜CD56~+NK样细胞,采用改进的乳酸脱氢酶(LDH)释放试验,比较蜕膜与外周血NK细胞杀伤活性。结果发现:蜕膜NK样细胞杀伤活性低于孕妇外周血及正常育龄妇女外周血NK细胞。提示妊娠早期蜕膜局部NK细胞活性受到抑制,可能有利于滋养细胞侵袭和胎盘形成。     

Th17细胞相关因子在子痫前期和正常妊娠孕妇外周血和胎盘中表达量的对比

目的通过对子痫前期及正常妊娠的孕妇外周血及胎盘组织中Th17细胞相关因子IL-17、IL-21、IL-22、HLA-G及RORc表达量的检测以及miRNA-21相对表达量的比较,探讨Th17细胞相关因子以及miRNA-21在子痫前期发病中的作用。方法选取2012年3月至2014年5月镇江妇幼保健院的40例孕妇为研究对象,其中子痫前期孕妇20例,正常孕妇20例,采用流式细胞仪分别检测子痫前期组和正常对照组外周血中Th17细胞的水平,采用实时定量PCR法检测外周血和胎盘组织中Th17细胞相关细胞因子IL-17、IL-21、IL-22、HLA-G、RORc的表达量和相对表达量,采用实时定量PCR法检测胎盘组织中miRNA-21的相对表达量。结果初步证实子痫前期患者和正常妊娠女性外周血Th17细胞相关细胞因子IL-17、IL-21、IL-22、HLA-G、RORc水平之间存在差异,子痫前期患者和正常妊娠女性胎盘组织中Th17细胞相关细胞因子IL-17、IL-21、IL-22、RORc、HLA-G水平存在差异。(1)子痫前期患者的IL-17、IL-21、IL-22的相对表达量明显高于正常对照组,差异有统计学意义(P0.05)。(2)子痫前期组的HLA-G、RORc的相对表达量明显低于正常对照组,差异有统计学意义(P0.05)。(3)子痫前期组的miRNA-21的相对表达量明显高于正常对照组,差异有统计学意义(P0.05)。(4)各项指标之间及各检测指标与临床指标间存在相关性。结论 Th17细胞相关细胞因子IL-17、IL-21、IL-22、HLA-G、RORc水平在子痫前期的发病过程中可能发挥重要作用,通过对相关指标的监测可能预测子痫前期的发生和监测病情变化,阻断这一途径就有可能有效预防和治疗子痫前期。     

CD4+CD25+Foxp3+调节性T细胞在子痫前期患者外周血与蜕膜中水平变化研究

目的本文检测CD4+CD25+Foxp3+调节性T细胞在子痫前期患者外周血及胎盘附着处蜕膜中的表达,探讨其在子痫前期免疫耐受失衡中的作用。方法选择子痫前期患者20例,正常晚期妊娠患者20例。采用流式细胞仪检测外周血CD4+CD25+Foxp3+的表达;免疫组织化学法检测蜕膜CD4+CD25+调节性T细胞的特异性转录因子Foxp3的表达。结果 1.子痫前期组外周血CD4+CD25+Foxp3+T细胞表达率(1.70±0.23%)明显低于正常晚期妊娠对照组(3.55±0.47%)(P<0.05)。2.蜕膜中Foxp3在子痫前期组的表达阳性率为(16.67%)明显低于正常对照组(66.67%)(P<0.05)。结论子痫前期患者外周血和蜕膜组织中的CD4+CD25+Foxp3+调节性T细胞均低于正常孕妇,提示其数量的减少使其免疫抑制功能减弱,母胎免疫耐受失衡,导致子痫前期的发生。     

基质金属蛋白酶-9和血管内皮生长因子在子痫前期患者胎盘组织中的表达

目的 检测子痫前期患者胎盘组织中RECK、基质金属蛋白酶-9（MMP-9）、血管内皮生长因子（VEGF）的基因表达,探讨它们与胎盘滋养细胞浸润过程的调控关系。 方法 采用RT-PCR、Western blotting、免疫组织化学检测120例妊娠足月剖宫产（正常妊娠、轻度子痫前期、中度子痫前期、重度子痫前期各30例）胎盘组织中RECK、MMP-9、VEGF的基因表达。结果 3组子痫前期患者胎盘组织中MMP-9及VEGF的mRNA表达水平均显著低于正常妊娠组（P<0.05）;重度子痫前期组中RECK mRNA的表达显著高于正常妊娠组（P<0.05）;3组子痫前期患者胎盘组织中MMP-9及VEGF蛋白表达均显著低于正常妊娠组（P<0.05）,中度和重度子痫前期组中RECK蛋白表达显著高于正常妊娠组（P<0.05）。结论 子痫前期患者胎盘中RECK与MMP-9、VEGF之间存在负相关性,它们可能参与了胎盘滋养细胞浅浸润过程的调控。     

T淋巴细胞亚群及其细胞因子在子痫前期患者的表达及意义

目的通过检测T淋巴细胞亚群及其所分泌的细胞因子IL-10、TNF-α在子痫前期患者外周血及胎盘中的变化规律及其相关性,探讨其与子痫前期发病的关系。方法取60例孕妇(正常晚孕组20例;子痫前期组40例,其中轻度20例,重度20例)外周血,分别检测T淋巴细胞亚群比例及其细胞因子IL-10、TNF-α的水平;分娩后检测胎盘组织中的IL-10、TNF-α的表达。结果①轻度子痫前期组胎盘IL-10、TNF-α的表达强度与对照组相比均无显著差异(P>0.05),而重度子痫前期组IL-10表达强度显著降低(P<0.05),且与子痫前期病情呈负相关(rs=-0.342P=0.009);TNF-α表达强度显著升高(P<0.05),且表达强度与子痫前期病情呈正相关(rs=0.349P=0.007)。②子痫前期组IL-10外周血中含量明显低于对照组(P<0.05),其中重度子痫前期组IL-10外周血中的含量比轻度子痫前期者降低(P<0.05),差异有显著性;而TNF-α的含量明显升高(P<0.05)。③子痫前期组血清CD4 、CD8 的细胞百分比与对照组相比无显著差异,但CD4 /CD8 比例上升明显,两者比较有显著性差异(P<0.05)。子痫前期组中重度子痫前期者血清CD4 /CD8 比例比轻度子痫前期者血清CD4 /CD8 比例上升,且差异有显著性(P<0.05)。结论T淋巴细胞亚群及其所分泌的细胞因子的变化可能与子痫前期的发病中起着重要作用,且与病情密切相关。     

不明原因自然流产与蜕膜NK细胞表面活化性受体表达的相关性研究

探讨不明原因自然流产患者蜕膜NK细胞杀伤活性与其细胞表面活化性受体NKp46、NKp44、NKp30和NKG2D表达的相关性。选取21例早孕不明原因自然流产患者为病例组,25例正常早孕人流妇女为对照组,收集两组的蜕膜组织,Ficoll密度梯度离心分离淋巴细胞,MACS磁珠分选CD3-CD56+NK细胞。以K562细胞为靶细胞,用细胞染色及流式细胞技术检测两组蜕膜NK细胞杀伤活性,用流式细胞技术检测两组蜕膜CD56brightCD16-NK和CD56dimCD16+NK细胞上活化性受体NKp46、NKp44、NKp30和NKG2D的表达,并与NK细胞杀伤活性进行相关性分析。结果:(1)早孕蜕膜NK细胞具有杀伤活性;(2)病例组蜕膜NK细胞的杀伤活性较正常对照组显著增强(P=0.014);(3)病例组蜕膜CD56brightCD16-NK细胞中NKp44的表达比正常对照组显著升高(P=0.021);病例组蜕膜CD56dimCD16+NK细胞中NKp46和NKp44的表达比正常对照组显著升高(分别P=0.026,P=0.041);其余活化性受体的表达两组未见明显差异;(4)蜕膜NK细胞杀伤功能与蜕膜CD56brightCD16-NK细胞中NKp44的表达呈显著正相关(r=0.677,P<0.05),和蜕膜CD56dimCD16+NK细胞中NKp46的表达呈显著正相关(r=0.634,P<0.05)。蜕膜NK细胞活化性受体NKp46和NKp44表达增加,从而使蜕膜NK细胞的杀伤功能增强可能在不明原因自然流产的发病中起重要作用。     

重度子痫前期患者胎盘中内源性一氧化碳的检测

目的检测重度子痫前期患者胎盘内源性一氧化碳（CO）的水平。方法采用改良的双波长分光光度法，建立回归方程，以此测定18例健康晚孕妇女与19例重度子痫前期患者胎盘匀浆中内源性CO的浓度，并进行分析。结果建立的回归方程为：Y=263、3X-0．178，相关系数为0．966。重度子痫前期患者胎盘内源性CO的水平较正常晚孕组下降（P〈0．05）。结论胎盘内源性一氧化碳可能在妊娠期高血压疾病特别在重度子痫前期的发生中，参与了其病理生理过程，发挥着重要的生理作用。     

子痫前期中IGF—Ⅰ、Ⅱ水平与FGR的相关性研究

目的探讨子痫前期中胰岛素样生长因子Ⅰ、Ⅱ（IGF—Ⅰ、Ⅱ）水平与新生儿出生体重的相关性。方法选择正常晚期孕妇（对照组）32例,轻度子痫前期孕妇20例、重度子痫前期孕妇20例（研究组）,分别于分娩前抽取孕妇静脉血,分娩时留取新生儿脐静脉血和羊水,同时记录新生儿出生体重,应用酶联免疫吸附试验（ELISA）,检测血清及羊水中IGF—Ⅰ、Ⅱ水平,并对检测结果进行分析。结果（1）子痫前期孕妇血及羊水中IGF—Ⅰ、Ⅱ水平与新生儿体重呈正相关。（2）新生儿脐血中IGF—Ⅰ水平与新生儿体重呈正相关,IGF—Ⅱ水平与新生儿体重呈负相关。（3）正常对照组32例均为正常出生体重新生儿,子痫前期孕妇中胎儿生长受限（FGR）总发病率为27．5％,其中轻、重度子痫前期孕妇,FGR发病率分别为5％和50％（P〈0．01）,差异有统计学意义。结论子痫前期状态下IGF—Ⅰ、Ⅱ水平变化与FGR密切相关,重度子痫前期孕妇中FGR发病率较高。     

Increased prevalence of IL-17-producing peripheral blood lymphocytes in pre-eclampsia

Citation
Toldi G, Rigó J Jr, Stenczer B, Vásárhelyi B, Molvarec A. Increased prevalence of IL‐17‐producing peripheral blood lymphocytes in pre‐eclampsia. Am J Reprod Immunol 2011; 66: 223–229 Problem Systemic inflammation is a dominant component in the pathogenesis of pre‐eclampsia. Besides the imbalance of Th1 and Th2 cells, alterations of the prevalence of Th17 and regulatory T cells have also been suggested to contribute to inflammation. We aimed to describe the prevalence of these four CD4 lymphocyte subtypes in pre‐eclampsia and normal pregnancy, along with that of IL‐17‐producing CD8 and NK cells. Method of study Twenty pre‐eclamptic and 22 normal pregnant women were enrolled in this study. Using flow cytometry, we determined the prevalence of IL‐17‐producing cells among the CD4, CD8 and NK cell subsets. Furthermore, we measured the prevalence of CD4+ Tregs, and Th1/Th2 cells were characterized using cell surface chemokine receptor markers. Results We demonstrated that there is a shift not only in the Th1/Th2 but also in the Th17/Treg balance favouring skewness towards a pro‐inflammatory status in pre‐eclampsia. The proportion of CD8 and NK cells that express IL‐17 was also higher in pre‐eclampsia. Conclusion The prevalence of IL‐17‐producing CD4, CD8 and NK cells is elevated in pre‐eclampsia, indicating that both the innate and adaptive arms of the immune system are involved in the development of the exaggerated maternal systemic inflammation observed in this pregnancy‐specific disorder.     

Leukocyte subpopulation in ascites of women with pre-eclampsia

Problem Pre‐eclampsia is associated with excessive maternal systemic inflammatory response. The immune cells in ascites of pre‐eclampsia have not been studied well. In order to investigate the inflammatory response in ascites of pre‐eclampsia, we studied leukocytes subpopulation in ascites. Method of study Maternal peripheral blood samples and ascites were taken from 10 cases of pre‐eclampsia. Antibody‐labeled cells were analyzed by flow cytometry. Results The leukocytes subpopulation in ascites taken from pre‐eclampsia was different from that in the peripheral blood. The largest population of leukocytes in ascites was CD14+ monocyte/macrophage. The percentages of macrophage, CD56+ cells, CD56 + 16?3? NK cell (immunoregulatory NK cell), and CD56 + 3+ T cell (CD56+ T cell including NK T cell) increased in ascites compared with those in the peripheral blood. The median percentages of CD56 + 16?3? NK cells were 22.2%. The half of the CD3+ T cells originated from the increased CD56 + 3+ T cells. Conclusion We demonstrated the accumulation of the macrophage and the CD56+ cells into the ascites taken from pre‐eclampsia patients. This finding may relate to excessive inflammatory response in pre‐eclampsia.     

复发性自然流产患者外周血自然杀伤细胞免疫表型检测

目的 检测复发性自然流产(RSA)患者外周血自然杀伤(NK)细胞的免疫表型。方法 应用流式细胞术检测27例RSA妊娠患者、41例RSA非妊娠患者、32例正常妊娠者和25例正常非妊娠者外周血CD56dimCD 16+、CD56brightCD16+/-、CD69+、HLA-DR+ NK细胞表达并进行比较。结果RSA妊娠组、RSA非妊娠组、正常妊娠组和正常非妊娠组外周血CD56dimCD16+ NK细胞占总NK细胞的比例分别为(88.69.±5.86)％、(79.25±9.31)％、(79.24±10.09)％、(75.49±11.96)％;CD56brightCD16+/- NK细胞的比例分别为(8.18±5.54)％、( 12.20±6.49)％、( 13.13±8.65)％、( 11.53±6.23)％;CD69+ NK细胞的比例分别为(3.42±2.13)％、(2.36±1.72)％、(2.68±1.81)％、(2.08±1.73)％;HLA-DR+ NK细胞的比例分别为(15.26±9.32)％、(9.96±7.18)％、( 10.57±8.05)％、(9.64±6.12)％。与RSA非妊娠组、正常妊娠组及正常非妊娠组比较,RSA妊娠组CD56dimCD16+、CD69+、HLA-DR+ NK细胞显著升高（均P＜0.05）。与RSA非妊娠组及正常妊娠组比较,RSA妊娠组CD56brightCD16+/- NK细胞明显降低（均P＜0.05）。结论 RSA妊娠患者外周血中NK细胞CD56dimCD16+、CD69+和HLA-DR+表达增高,而CD56brightCD16+/-表达下降,可能在RSA的发生发展中起着一定的作用。     

解整合素金属蛋白酶19/ADAM19在子痫前期胎盘组织中的表达及其对滋养细胞分泌MMPs的调节

目的通过对子痫前期及正常孕妇胎盘组织中解整合素金属蛋白酶19（ADAM19）的检测及ADAM19对绒毛滋养细胞分泌MMP2,9的调节,探讨ADAM19与子痫前期发病的关系。方法应用免疫组织化学（免疫组化）链霉素抗生物素-过氧化物酶法、免疫印迹技术和RT-PCR技术检测24例正常胎盘组织及46例子痫前期（其中26例为重度子痫前期,20例为轻度子痫前期）胎盘组织中ADAM19蛋白及mRNA的表达;用明胶酶谱方法检测抗ADAM19抗体作用后滋养细胞MMP2,9的分泌变化。结果胎盘组织中,ADAM19主要分布在多种滋养层细胞中,包括细胞滋养层细胞、合体滋养层细胞一和些绒毛间质结缔组织细胞、毛细血管中,其阳性信号不仅定位于细胞膜上,在细胞质中也有分布。正常胎盘中ADAM19的蛋白表达量为0.372±0.016,重度和轻度子痫前期组ADAM19蛋白定量分别为0.766±0.029和0.693±0.041,轻、重度子痫前期组分别与正常组比较,差异均有统计学意义（P〈0.01）,轻度子痫前期与重度子痫前期比较,差异无统计学意义（P〉0.05）。ADAM19mRNA在正常组胎盘中的量为0.205±0.084,在重度和轻度子痫前期组中分别为0.481±0.057和0.454±0.033,轻、重度子痫前期组分别与正常组比较,差异均有统计学意义（P〈0.01）,轻度子痫前期与重度子痫前期比较,差异无统计学意义（P〉0.05）。在体外培养的滋养层细胞中,100μg/L浓度的抗ADAM19抗体在作用12h即可以抑制滋养细胞MMP2,MMP9的分泌,其作用呈浓度依赖。结论子痫前期胎盘组织中ADAM19过表达可能与子痫前期的发生和发展有关;在子痫前期的发生中,ADAM19可能是在妊娠早期抑制滋养细胞的浸润而导致胎盘浅着床的。     

A role for mannose-binding lectin, a component of the innate immune system in pre-eclampsia

Problem Mannose‐binding lectin (MBL) is a pattern‐recognition receptor that activates complement and modulates inflammation. Homozygosity for the most common allele of the MBL2 gene that is associated with high MBL serum concentrations is more prevalent among patients with pre‐eclampsia. The objective of this study was to determine maternal plasma MBL concentrations in normal pregnant women and patients with pre‐eclampsia. Method of study This cross‐sectional study included normal pregnant women (n = 187) and patients with pre‐eclampsia (n = 99). Maternal plasma MBL concentrations were determined by ELISA. Results Women with pre‐eclampsia had a higher median maternal plasma MBL concentration than normal pregnant women. MBL concentration distribution curves were three‐modal, the subintervals in normal pregnancy were low (<143.7), intermediate (143.7–1898.9) and high (>1898.9 ng/mL). The proportion of normal pregnant women was larger in the low subinterval, while the proportion of patients with pre‐eclampsia was larger in the high subinterval (P = 0.02). Normal pregnant women in the high subinterval had a larger rate of placental underperfusion than those in the low and intermediate subintervals (P = 0.02). Conclusions The median maternal plasma MBL concentration is elevated in patients with pre‐eclampsia and a larger proportion of these patients are in the high subinterval than normal pregnant women, suggesting that this component of the innate immune system is involved in the mechanisms of disease in pre‐eclampsia.     

ORIGINAL ARTICLE: Role of Inflammatory Cytokines and eNOS Gene Polymorphism in Pathophysiology of Pre‐Eclampsia

Citation Singh A, Sharma D, Raghunandan C, Bhattacharjee J. Role of inflammatory cytokines and eNOS gene polymorphism in pathophysiology of pre‐eclampsia. Am J Reprod Immunol 2010; 63: 244–251 Problem Pre‐eclampsia involves endothelial vascular dysfunction. The aim of this study was to test the hypothesis that (i) endothelial nitric oxide (NO) synthase Glu298Asp gene polymorphism limits constitutive NO production causing endothelial dysfunction and (ii) inflammatory cytokines impairs endothelium dependent relaxation in pre‐eclampsia. Method of study This cross‐sectional study included 50 women with pre‐eclampsia and 50 healthy pregnant women. Their blood samples were analyzed for NO, inflammatory cytokines and endothelial NO synthase (eNOS) gene polymorphism. Result Decreased NO levels whereas increased tumor necrosis factor‐α, interleukin (IL)‐6 and interleukin‐2 were found in pre‐eclampsia (P < 0.001). No significant differences were found in genotype/allele distribution between two groups. Significant negative correlation was observed between NO and IL‐6 in pre‐eclamptic group (P = 0.001). Conclusion An IL‐6‐mediated endothelium dependent NO‐cyclic guanine monophosphate‐mediated relaxation pathway may be inhibited in systemic vessels in pre‐eclampsia. As observed in this study Glu298Asp eNOS gene polymorphism did not showed significant association with pre‐eclampsia.     

Role of Endothelin and Inflammatory Cytokines in Pre‐eclampsia – A Pilot North Indian Study

Citation Sharma D, Singh A, Trivedi SS, Bhattacharjee J. Role of endothelin and inflammatory cytokines in pre‐eclampsia – a pilot north Indian study. Am J Reprod Immunol 2011; 65: 428–432 Problem Pre‐eclampsia is new onset hypertension during pregnancy with proteinuria. The initiating event in pre‐eclampsia is postulated to involve reduced placental perfusion, which leads to widespread dysfunction of the maternal vascular endothelium. Cytokines also appear to contribute to the development of the pathological condition. The aim of this study was to evaluate the role of cytokines in pre‐eclampsia and to study the relationship between endothelin‐1 and cytokines with the severity of the disease. Method of study This cross‐sectional study included 300 women with pre‐eclampsia and 200 healthy pregnant women. Their blood samples were analyzed for endothelin‐1 and inflammatory cytokines. Results Increased endothelin‐1 and cytokines [tumor necrosis factor‐α, interleukin‐2 (IL‐2) and γ‐interferon (IFN‐γ)] levels were found in pre‐eclampsia (P < 0.001). Significant positive correlation was seen between endothelin‐1 and cytokine level (IL‐2 and IFNγ) in the pre‐eclamptic group (P = 0.001). Conclusion We conclude that pre‐eclampsia is associated with increased levels of both endothelin‐1 and circulating inflammatory cytokines, which points toward the role of endothelial and inflammatory components.