白细胞介素-8蛋白在反复自然流产患者绒毛及蜕膜组织中的表达

目的 探讨反复自然流产患者绒毛及蜕膜组织中白细胞介素-8 （IL-8）蛋白水平的表达。 方法 应用免疫组织化学染色和Western blotting 技术,分别对30例反复自然流产患者（流产组）和30例正常妊娠者（对照组）绒毛、蜕膜组织中IL-8的蛋白表达进行定位及半定量分析。 结果 免疫组织化学染色结果显示,在绒毛组织中,流产组和对照组IL-8的蛋白表达主要定位于绒毛上皮细胞的细胞质内;在蜕膜组织中,流产组IL-8蛋白表达于蜕膜细胞的胞质内,对照组蜕膜细胞则为阴性,但两组腺上皮细胞均为阳性反应。Western blotting结果显示,在绒毛组织中,流产组IL-8的蛋白相对含量明显高于对照组（P﹤0.01）;在蜕膜组织中,流产组IL-8的蛋白相对含量高于对照组（P﹤0.05）。 结论 流产组绒毛及蜕膜组织中IL-8蛋白表达增强与反复自然流产有关,IL-8可能参与反复自然流产的病理过程。     

CXCR2在不明原因自然流产患者绒毛和蜕膜组织中的表达

目的研究CXCR2在不明原因自然流产患者绒毛及蜕膜组织中的表达情况。方法应用免疫组织化学染色和图像分析技术,观察CXCR2在不明原因自然流产患者（病例组）和正常人工流产者（对照组）绒毛及蜕膜组织中的表达情况;HE染色后光镜下观察不明原因自然流产患者绒毛与蜕膜组织的形态学变化。结果免疫组化染色结果显示：两组CXCR2蛋白主要表达于绒毛的滋养层、蜕膜组织中的腺体,病例组绒毛滋养层和腺体CXCR2蛋白表达均强于对照组,两组比较,有显著性差异（P〈0.01）。病例组蜕膜细胞CXCR2蛋白呈阳性表达,对照组呈阴性;HE染色可见不明原因自然流产患者绒毛组织的滋养层变薄、滋养层细胞变性甚至坏死、滋养层嗜酸性增强、绒毛中轴纤维化程度增强;蜕膜组织中蜕膜细胞连接松散、蜕膜细胞空泡化、蜕膜组织中有大量炎细胞浸润。结论 CXCR2可能通过与其配体白细胞介素-8结合而参与不明原因自然流产的病理过程。     

白细胞介素-2在反复自然流产患者绒毛及蜕膜组织中的表达

目的:研究白细胞介素-2(IL-2)在反复自然流产患者绒毛及蜕膜组织中的表达水平及意义.方法:应用免疫组织化学和图像分析,分别对30例反复自然流产患者(病例组)和30例正常妊娠者(对照组)绒毛和蜕膜组织中IL-2的表达进行定位和半定量分析;应用显微镜观察反复自然流产患者绒毛和蜕膜组织的形态学变化.结果:免疫组织化学显色显示,IL-2蛋白定位于绒毛组织的滋养层细胞和蜕膜组织的蜕膜细胞细胞质内,且病例组的IL-2蛋白表达高于对照组.H-E染色结果显示,病例组的蜕膜组织结构模糊、细胞变性坏死、部分细胞连接消失,血管内皮有缺损;绒毛组织的滋养层变薄,细胞变性坏死,细胞嗜酸性增强,绒毛中轴纤维化程度增强.结论:绒毛和蜕膜组织中IL-2蛋白的高表达与反复自然流产有关,可能参与反复自然流产的病理过程.     

白细胞介素-6在反复自然流产患者绒毛和蜕膜组织中的表达

目的:研究白细胞介素-6(IL-6)在反复自然流产患者绒毛与蜕膜组织中的表达情况,为防治反复自然流产提供理论依据.方法:应用免疫组织化学显色法和图像分析技术,对IL-6蛋白在反复自然流产患者(病例组)和正常人工流产者(对照组)绒毛和蜕膜组织中的表达进行定位和半定量分析;H-E染色法观察反复自然流产患者绒毛和蜕膜组织的形态学变化.结果:免疫组织化学显色显示,在绒毛组织中,IL-6蛋白定位于滋养层细胞的细胞质内,病例组的IL-6蛋白表达明显低于对照组;在蜕膜组织中,IL-6定位于蜕膜细胞和腺上皮细胞的细胞质内,且病例组蜕膜细胞的表达明显高于对照组,而2组在腺上皮细胞的表达无差异;H-E染色结果显示,病例组绒毛组织的滋养层变薄,细胞变性、坏死,细胞嗜酸性增强,绒毛中轴纤维化程度增强,蜕膜细胞失去细胞间连接,部分蜕膜细胞解体、核消失,细胞嗜酸性增强.结论:绒毛和蜕膜组织中IL-6蛋白表达的变化与反复自然流产有关,IL-6可能参与反复自然流产的病理过程.     

孕酮及其受体、IL-8与原因不明自然流产的相关性研究

目的：探讨孕酮（P）及其受体（PR）、IL-8在原因不明自然流产患者外周血及母胎界面的表达及相关性。方法：ELISA检测30例原因不明自然流产患者（流产组）和30例正常妊娠妇女（对照组）血清P、IL-8水平,免疫组织化学染色法检测两组流产绒毛和蜕膜组织PR、IL-8表达。结果：流产组血清P水平明显低于对照组（P<0.01）,血清IL-8水平明显高于对照组（P<0.01）。两组绒毛组织PR蛋白表达均为阴性;两组蜕膜组织中PR均表达于蜕膜细胞的细胞核,且流产组表达明显低于对照组（P<0.01）。两组绒毛组织中IL-8均表达于绒毛上皮细胞的细胞质,且流产组表达明显高于对照组（P<0.01）;两组蜕膜组织中IL-8均表达于蜕膜细胞的细胞质,且流产组表达高于对照组（P<0.05）。流产组血清P与IL-8呈显著负相关（r=-0.870,P<0.01）,流产组蜕膜组织PR与IL-8呈显著负相关（r=-0.650,P<0.01）。结论：P、PR低表达、IL-8高表达可能与原因不明自然流产发生相关,且P、PR可能通过下调IL-8表达维持正常妊娠。     

Expression of CXCR2 in villous tissues and decidua tissues of patients with spontaneous abortion

目的:研究不明原因自然流产患者绒毛及蜕膜组织中CXCR2在mRNA水平和蛋白质水平的表达情况.方法:应用半定量RT-PCR、免疫组织化学显色和图像分析技术,观察CXCR2在不明原因自然流产患者(病例组)和正常人工流产者(对照组)绒毛及蜕膜组织中的表达情况.结果:CXCR2 mRNA在对照组和病例组的绒毛及蜕膜组织中均有表达,且2组CXCR2mRNA的表达并无差异；两组CXCR2蛋白主要表达于绒毛的滋养层,蜕膜组织中的腺体,病例组绒毛滋养层和腺体CXCR2蛋白表达均显著强于对照组.病例组蜕膜细胞CXCR2蛋白呈阳性表达,对照组呈阴性.结论:CXCR2可能通过与其配体白细胞介素-8结合而参与不明原因自然流产的病理过程.     

白细胞介素-8mRNA与反复自然流产的相关性

目的:探讨反复自然流产患者绒毛、蜕膜和外周血单个核细胞白细胞介素-8(IL-8) mRNA 的表达与反复自然流产的相关性.方法:采用半定量逆转录-聚合酶链反应(RT-PCR)技术,检测 30 例反复自然流产患者(病例组)和 30 例正常妊娠妇女(对照组)绒毛、蜕膜及外周血单个核细胞内 IL-8 mRNA 表达水平的相对含量(%).结果:在绒毛组织中,病例组 IL-8mRNA 的表达明显高于对照组;在蜕膜组织中,病例组 IL-8 mRNA 的相对含量为(68.57±4.73)%,对照组的为0;在外周血单个核细胞中,病例组 IL-8 mRNA 的表达高于对照组.结论:病例组绒毛、蜕膜组织及外周血单个核细胞中 IL-8 mRNA 表达水平显著升高,提示 IL-8 可能与反复自然流产相关.     

Toll样受体4/髓样分化蛋白88在早期自然流产母-胎界面的表达

目的:探讨Toll样受体4(TLR4)/髓样分化蛋白88(MyD88)与早期自然流产的相关性。方法:选择30例正常妊娠妇女作为对照组,采用免疫组织化学显色对30例早期自然流产患者(流产组)母-胎界面的蜕膜和绒毛组织中TLR4、MyD88蛋白表达进行定位观察,采用RT-PCR技术对其TLR4和MyD88mRNA表达进行半定量分析。结果:TLR4和MyD88蛋白在流产组和对照组的绒毛及蜕膜组织中均有表达,定位表达于绒毛滋养层细胞、蜕膜细胞的细胞质内,阳性反应强弱不等。RT-PCR半定量分析结果显示,在绒毛组织中,流产组TLR4 mRNA、MyD88 mRNA的表达显著高于对照组;在蜕膜组织中,流产组TLR4 mRNA、MyD88 mRNA的表达显著高于对照组。流产组绒毛和蜕膜组织中TLR4与MyD88的表达均呈正相关。结论:TLR4和MyD88可能参与早期自然流产的病理过程,且TLR4可能通过MyD88信号途径参与早期自然流产的发生。     

早期自然流产患者母胎界面E-钙黏素与β-连环素的异常表达

目的探讨早期自然流产患者绒毛和蜕膜组织E-钙黏素（E—cadherin）与β-连环素（β-catenin）的异常表达。方法采用免疫组化sP法和显微图像分析系统，检测20例早期自然流产（自然流产组）及24例正常早期人工流产（正常妊娠组）绒毛和蜕膜组织内E—cadherin、β-catenin表达的平均光度及积分光度。结果E—cadherin、β-catenin．在自然流产组和正常妊娠组绒毛与蜕膜组织中均有表达。自然流产组绒毛细胞滋养细胞和蜕膜细胞表面E—cadherin、β-catenin表达平均光度及积分光度均明显高于正常妊娠组，P〈0．001，差异有统计学意义。结论E—cadherin及β-catenin对妊娠维持起一定的作用。蜕膜及绒毛组织E—cadherin与β-catenin的高表达可能是导致早期自然流产的原因之一。     

β-catenin及DDK1在URSA患者的绒毛滋养层细胞和蜕膜组织中表达

目的分析β-链蛋白(β-catenin)及重组人Dickkopf相关蛋白1(DDK1)在不明复发性流产(unexplained recurrent spontaneous abortion,URSA)患者的绒毛滋养层细胞和蜕膜组织中表达,为URSA的诊断与治疗提供理论依据。方法以2014年2月~2016年2月,医院收治的URSA患者40例,作为研究对象,入选病例组,另选择同期收治的正常妊娠早期女性40例,纳入对照组。人工流产术后,取蜕膜以及绒毛组织,一部分迅速放入无RNA酶的EP管中,液氮下保存,进行实时定量PCR,免疫组化染色镜下检查,另一部分直接采用4%福尔马林溶液固定,进行蛋白提取与Western blot分析。结果病例组的绒毛组织、蜕膜组织β-catenin蛋白光密度与细胞中β-catenin蛋白表达低于对照组,差异有统计学意义(P0.05);病例组绒毛组织、蜕膜组织β-catenin蛋白高表达率低于对照组,病例组细胞滋养细胞膜浅黄色着色率低于对照组,差异有统计学意义(P0.05)。病例组绒毛组织、蜕膜组织中的DKK1 m RNA水平分别是对照组的1.84、2.34倍。结论 URSA患者的绒毛滋养层细胞和蜕膜组织中β-catenin蛋白表达显著下降,DDK1 m RNA水平上升。     

IL-18 is present at the maternal-fetal interface and enhances cytotoxic activity of decidual lymphocytes

PROBLEM: Perforin expressing uterine natural killer (uNK) cells are under complex cytokine influence. The aim of the study was to investigate the presence and role of interleukin (IL)-18 on NK cytolytic potential at maternal-fetal (M-F) interface. METHOD OF STUDY: Peripheral blood cells and decidual tissue were obtained from elective pregnancy termination of normal human 6-10-week-old pregnancies. Perforin expression and cytolytic activity of peripheral blood (PBL) and decidual lymphocytes (DL) were analyzed by flow cytometry. IL-18 positive decidual adherent cells (DAC) were detected by the same method. Interleukin-18 and IL-18 receptor (IL-18R) expression on the trophoblastic cells was detected by immunohistology using biotinylated anti-IL-18 and IL-18R monoclonal antibodies. RESULTS: The IL-18 added in a dose of 10 ng/mL up-regulates perforin expression and cytolytic activity of DL. Simultaneous stimulation with IL-18 and IL-12 enhanced DL cytolytic activity, while IL-18 combined with IL-10 or IL-15 did not show this effect. Cytolytic activity of PBL was up-regulated by IL-18 as well, and this effect was enhanced by the addition of IL-12 and IL-15. Interleukin-18 did not affect perforin-protein expression in cultured PBL. Approximately 20% of DAC were IL-18 positive and these cells were mostly human leukocyte antigen (HLA)-DR negative. IL-18R positive cells were found on syncytiotrophoblast cell layer, interstitial tissue cells of villi and fetal blood cells. There was no detectable IL-18 staining on trophoblast cell layer on villi, but strong staining of fetal blood cells in villous vessels. CONCLUSION: These are first results showing IL-18R expression, but not IL-18 expression on villous trophoblastic cells, as well as enhancement of perforin expression and NK cytolytic potential of DL under the influence of IL-18. IL-18 in concert with other cytokines and hormones could play an important role in the regulation of cytolytic potential of first trimester pregnancy decidual and peripheral blood NK cells.     

母胎界面树突状细胞CCL17和CCL22表达在母胎免疫耐受中的作用

目的: 检测非孕期子宫内膜和正常妊娠早期及复发性自然流产患者蜕膜中树突状细胞(DC)CCL17和CCL22的表达差异,探讨母胎界面DC在CD4⁺CD25⁺调节性T细胞(Treg)的募集及母胎免疫耐受微环境形成中的作用。方法: 正常早孕组人工流产时、复发性流产组清宫时取其蜕膜,正常未孕组行子宫切除时取其内膜组织。分离蜕膜或子宫内膜单个核细胞,体外诱导培养DC,用real-time PCR法分析3组DC CCL17和CCL22 mRNA的表达水平,ELISA法检测3组DC培养上清液中CCL17和CCL22蛋白的表达。结果: 正常早孕组蜕膜DC CCL17和CCL22 mRNA的表达分别为3.04±0.40和1.83±0.24,均高于正常未孕组(0.85±0.24和0.31±0.08,P<0.01)和复发性流产组(1.65±0.14和0.96±0.09,P<0.01)。正常早孕组蜕膜DC能够持续旺盛分泌趋化因子CCL17和CCL22,在培养的12~96 h内CCL17和CCL22的分泌量逐渐增多。同一时点早孕组DC分泌的CCL17和CCL22均明显高于未孕组和复发性流产组DC分泌的CCL17和CCL22(P<0.01)。结论: 正常妊娠后蜕膜DC表达CCL17和CCL22增强,DC可能通过高表达CCL17和CCL22而增强对CD4⁺CD25⁺Treg的趋化作用,从而在母胎界面的免疫耐受中发挥重要作用,蜕膜DC表达CCL17和CCL22下降可能与复发性自然流产的发病有关。     

Sperm with damaged membranes are profoundly at risk to have caspases 8, 9 and 3 activated

PROBLEM:  It is almost dogma that IL-2 is not expressed at the M–F interface during normal pregnancy. However, recent results by ours and others clearly showed that IL-15-Th1 type cytokine which shares many similarities with IL-2 is expressed at the interface. IL-15 can affect cytolytic activity of maternal decidual lymphocytes which heavily infiltrate maternal decidua during the first trimester pregnancy. These cells are in a direct contact with trophoblastic cells. IL-18 is a recently discovered Th1 type cytokine with many interesting functions. The aim is to examine IL-18 distribution at the interface and its potential in up-regulating peripheral blood (PB) and decidual lymphocytes (DL) cytotoxicity. Th1 activated lymphocytes are LAK cells and they can kill by both Perforin and Fas pathways in non MHC restricted manner.
METHODS:  PBL and DL were obtained from elective pregnancy termination of pregnancy. IL-18 and IL-18R expression was detected by flow cytometry and immunohistology and cytolytic potential by cytotoxicity against K-562 (NK sensitive) and P815 (NK resistant) cell lines.
RESULTS:  IL-18 positive cells were found in the suspension of Decidual adherent cells and IL-18 R expressions at the trophoblastic cells of villi. Both IL-15 and IL-18 are increasing cytolytic potential of PBL and DL against NK sensitive cell line. Decidual lymphocytes are activated cells with the potential of killing NK resistant but LAK sensitive lines and this is mediated by both perforin and Fas pathways.
CONCLUSIONS:  Physiological and pathophysio- logical role(s) of cytolytic pathways and Th1 cytokines (IL-15 and IL-18) at the interface will be discussed.     

E003对RSA模型小鼠蜕膜组织超微结构的影响

目的　观察瑞香素（daphnetin）即E003处理后RSA模型小鼠蜕膜组织形态结构的改变。 方法　建立反复自然流产（recurrent spontaneous abortion,RSA）模型小鼠后,实验分正常妊娠组、RSA模型组、E003低剂量组和E003高剂量组共4组,E003连续灌胃处理14 d后,取蜕膜组织。光镜下观察蜕膜组织血管和细胞形态,电镜下观察蜕膜组织超微结构。 结果　解剖子宫计算胚胎吸收率,E003处理组（低剂量组和高剂量组）低于RSA模型组,差异具有统计学意义,P<0.05;E003高剂量组胚胎吸收率与正常妊娠组相比无统计学差异,P>0.05。光镜下可见RSA模型组蜕膜细胞碎裂,数量较正常妊娠组减少;E003低剂量组和高剂量组蜕膜细胞数量均较模型组增多,排列较整齐。电镜下RSA模型组较正常妊娠组蜕膜细胞体积减小,细胞间隙增大,血管壁不完整,血管内皮细胞结构不完整,细胞核呈梭形;E003低剂量组与模型组比较,蜕膜细胞体积增大,细胞间隙增大,血管壁较完整,血管内皮细胞呈长梭形,细胞结构较完整,细胞间隙稍增宽;E003高剂量组蜕膜细胞组织及血管壁接近正常妊娠组。 结论　RSA模型小鼠存在母胎界面血管生成障碍,E003可修复RSA模型小鼠蜕膜组织中破坏的细胞结构,改善血管生成和细胞器形态,对RSA模型小鼠具有一定的治疗作用。     

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.     

Deficiency of decidual IL-10 in first trimester missed abortion: a lack of correlation with the decidual immune cell profile

PROBLEM: To determine if first trimester missed abortion decidua is characterized by an altered immune cell profile and/or a modified interleukin (IL)-10 and interferon (IFN)-gamma production pattern compared with decidua from elective termination. METHOD OF STUDY: Flow cytometry and immunohistochemistry techniques were used to determine the decidual immune cell phenotypic profile and production pattern of IL-10 and IFN-gamma in cases of elective termination (n = 14) and missed abortion (n = 12). RESULTS: Both groups had a similar proportion of CD56+ CD16-, CD56+ CD16+, CD19+, CD3+, CD4+, CD8+, alphabeta T cells and gammadelta T cells. The majority of alphabeta and gammadelta positive T cells in both groups coexpressed the natural killer (NK) cell marker CD56, but lacked cell surface expression of CD3. Diminished decidual IL-10 staining was noted in 7/10 missed abortion cases compared with none of the elective termination cases (n = 12) (P = 0.007). A uniform decidual IFN-gamma staining pattern was observed in both groups. CONCLUSION: Decreased IL-10 production coupled with a sustained IFN-gamma presence noted in missed abortion compared with elective termination cases suggest that these cytokines may be important determinants in pregnancy outcome. In contrast, differences in the proportion of immune cells between both groups may not be a critical factor in early pregnancy loss. In normal pregnancy, decidual alphabeta and gammadelta positive T cells with reduced CD3 on their cell surface may be intrinsically restricted in T-cell receptor (TCR)-mediated activation.     

Immunohistochemical localization of androgen receptor in the human endometrium, decidua, placenta and pathological conditions of the endometrium.

The immunohistochemical localization of the androgen receptor in the human endometrium at various stages of the menstrual cycle and post-menopausal period, in decidua and placenta of early pregnancy, and in several pathological conditions of the endometrium has been investigated. At any phase of the menstrual cycle, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity. There was little staining in myometrium. Even after menopause, endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak. In decidua and placenta of early pregnancy, decidual and trophoblastic cells showed positive staining and there was no staining in the stromal cells of placenta. The expression of the androgen receptor was also detected in adenomyosis, endometriosis and endometrial carcinoma. Although the proliferation and differentiation of endometrium are mediated mainly by oestrogen and progesterone receptors, the androgen receptor may play some role in modulating these changes. These results suggest that it may be involved in both physiological and pathological changes of the endometrium.