早孕蜕膜及绒毛组织中趋化因子CXC受体3、4及其配体的表达变化和意义

目的 探讨早孕蜕膜及绒毛组织中趋化因子CXC受体(CXCR)3、4及其配体CXCL9、CXCL10和CXCL12的表达变化和意义.方法 体外分离正常早孕蜕膜组织中单个核细胞,免疫磁珠分选试剂盒纯化CD+56自然杀伤(NK)细胞,流式细胞仪分析其纯度和表型;RT-PCR技术榆测早孕蜕膜NK细胞中CXCR3和CXCR4、早孕蜕膜及绒毛组织中CXCL9、CXCL10、CXCL12的表达情况;免疫组化链霉菌抗生物素蛋白-过氧化物酶连接(SP)法检测正常子宫内膜、早孕蜕膜组织中CXCL9和CXCL10的表达及CD+56NK细胞的分布情况,分析CXCL9、CXCL10表达量(灰度值)与CD+56NK细胞数的相关性.结果 分离纯化的早孕蜕膜NK细胞中,98.7%的细胞表型为CD56bright;早孕蜕膜NK细胞中有CXCR3和CXCR4表达;早孕蜕膜组织中有CXCL9、CXCL10表达,早孕绒毛组织中有CXCL12的表达.分泌期子宫内膜中CXC19、CXCL10表达最为56±43、59±47,较增生期子宫内膜的16±18、8±14明显升高,差异有统计学意义(P<0.05),而早孕蜕膜组织中CXCL9、CXCL10表达量为143±35、158±29,较分泌期子宫内膜进一步升高(P<0.05);分泌期子宫内膜中NK细胞数量为(60±20)个,增生期子宫内膜中NK细胞数量为(23±4)个,两者比较,差异有统计学意义(P<0.05),早孕蜕膜中NK细胞数量为(114 ±15)个,较分泌期子宫内膜进一步增多(P<0.05);子宫内膜和蜕膜组织中CXCL9、CXCL10表达量与组织中CD56+细胞数呈正相关关系(rcxL9=0.88,P<0.05;rcxcL10=0.86,P<0.05).结论 早孕蜕膜及绒毛组织中表达的CXCL9、CXCL10及CXCL12可能通过与CD56+NK细胞表面对应的趋化因子受体CXCR3、CXCR4结合而影响早孕期母-胎界面中CD56+NK细胞的聚集,从而对母-胎间免疫平衡起调控作用.     

趋化因子受体4在子宫颈癌中的表达及临床意义

目的探讨趋化因子受体4(chemokine receptor 4,CXCR4)在宫颈癌组织中的表达及其对宫颈癌预后的影响。方法采用免疫细胞ABC法检测60例宫颈癌和10例正常宫颈组织中CXCR4的表达,分析宫颈癌组织中CXCR4阳性表达与各临床病理因素及患者预后的关系。结果 CXCR4在正常宫颈组织中不表达,而在宫颈癌组织中的阳性表达率为78.3%(47/60)。宫颈癌组织CXCR4的阳性表达与盆腔淋巴结转移、淋巴血管间隙浸润、宫颈间质浸润深度有关(P<0.05),与患者年龄、病理类型、病理分级、临床分期、原发肿瘤的大小和阴道残端累及无关(P>0.05)。单因素分析显示,盆腔淋巴结转移、淋巴血管间隙浸润、CXCR4强阳性表达是影响宫颈癌患者总生存时间的危险因素。盆腔淋巴结转移和宫颈癌组织CXCR4强阳性表达是影响宫颈癌患者无瘤生存时间的危险因素。COX回归分析显示,CXCR4强阳性表达是宫颈癌患者总生存和无瘤生存时间的危险因素。结论 CXCR4在宫颈癌组织中高表达,与宫颈癌的侵袭及转移相关。并且其强阳性表达是影响宫颈癌患者预后的重要因素。     

趋化因子受体6在子宫内膜异位症在位内膜及异位灶中的表达

目的分析趋化因子受体6(chemokine receptor,CCR6)在子宫内膜异位症(内异症)患者的在位内膜及异位内膜中的表达水平,以探讨其在发病中的作用。方法采用免疫组化技术检测内异症患者在位内膜及异位灶CCR6的表达。结果对照组子宫内膜中CCR6的表达分泌期明显强于增殖期(P0.05);内异症患者在位子宫内膜间质和腺体CCR6的表达比较,差异无统计学意义(P0.05),但其表达强度高于对照组(P0.05),异位灶CCR6的表达明显高于对照组(P0.05),略高于在位子宫内膜,但无统计学意义(P0.05)。结论CCR6的表达具有周期特异性,可能受雌/孕激素调节,参与子宫内膜异位症的发生和进展。     

趋化因子CXCL12及其受体CXCR4在子宫腺肌病组织中的表达及意义题录

目的观察子宫腺肌病(AM)子宫内膜和子宫肌层中CXC型趋化因子配体12(CXCL12)及CXC型趋化因子受体4(CXCR4)的表达、相关性和意义。方法选取2017年10月至2018年12月于首都医科大学附属北京妇产医院, 因AM行子宫切除术的患者38例作为AM组, 选择同期因子宫颈上皮内瘤变Ⅲ级或子宫颈癌行子宫切除术的患者31例作为对照组, 通过免疫组化二步法和实时荧光定量PCR技术检测两组患者子宫内膜和子宫肌层组织中CXCL12、CXCR4 mRNA和蛋白的表达水平, 并分析CXCL12与CXCR4表达的相关性。结果 (1)CXCL12和CXCR4蛋白在AM组子宫内膜组织中的表达水平分别为0.229±0.025、0.226±0.016, 显著高于对照组的0.153±0.018、0.178±0.026, 分别比较, 差异均有统计学意义(P均<0.05);CXCL12和CXCR4蛋白在AM组子宫肌层组织中的表达水平分别为0.222±0.045、0.126±0.058, 显著高于对照组的0.091±0.029、0.099±0.020, 分别比较, 差异均有统计学意义(P均<0.05)。(2)CXCL12和CXCR4 mRNA在AM组子宫内膜组织中的表达水平分别为6.31±0.12、8.49±0.21, 显著高于对照组的1.23±0.10、1.36±0.13, 分别比较, 差异均有统计学意义(P均<0.05);CXCL12和CXCR4 mRNA在AM组子宫肌层组织中的表达水平分别为9.11±0.12、8.45±0.16, 显著高于对照组的1.18±0.08、1.46±0.13, 分别比较, 差异均有统计学意义(P均<0.05)。(3)AM组中CXCL12与CXCR4 mRNA在子宫内膜和子宫肌层组织中的表达呈线性正相关关系(r=0.478、0.542, P均<0.05)。结论 AM子宫内膜和子宫肌层组织中CXCL12、CXCR4的表达增加且呈正相关, 两种因子可能协同作用参与AM的发生、发展。     

趋化因子及其受体在子宫内膜异位症发病中的作用

子宫内膜异位症（EMs）发病机制复杂,近年免疫学概念的引入对深入理解EMs发病机制产生了重要影响。趋化因子及受体在EMs的发生和发展中起重要作用。EMs患者腹腔内多种趋化因子及受体表达水平升高,其异常表达可能招募免疫细胞进入腹腔,促进腹腔炎症反应,并通过分泌细胞因子及促进内膜的黏附、侵袭及新生血管生成等复杂机制促进EMs的形成。     

趋化因子及其受体在子宫内膜异位症发病中的作用

子宫内膜异位症(EMs)发病机制复杂,近年免疫学概念的引入对深入理解EMs发病机制产生了重要影响.趋化因子及受体在EMs的发生和发展中起重要作用.EMs患者腹腔内多种趋化因子及受体表达水平升高,其异常表达可能招募免疫细胞进入腹腔,促进腹腔炎症反应,并通过分泌细胞因子及促进内膜的黏附、侵袭及新生血管生成等复杂机制促进EMs的形成.     

早孕期人绒毛趋化因子CCL2及其受体CCR2的表达

目的:分析人早孕绒毛组织趋化因子CCL2及其受体CCR2的转录和翻译表达。方法:收集人早孕期胎盘组织30例,分离出绒毛组织,采用半定量RT-PCR、免疫组织化学法分析正常人早孕绒毛组织CCL2/CCR2的表达。结果:人早孕绒毛滋养细胞中等水平转录和翻译CCL2及CCR2。结论:CCL2/CCR2表达于母-胎界面的胎儿面绒毛组织,可能参与滋养细胞功能调控。     

CXCL12/CXCR4在绒毛外滋养细胞中的表达及其意义

目的:研究趋化因子CXCL12及其受体CXCR4在绒毛外滋养细胞(extravillous trophoblasts,EVTs)中的表达及其在妊娠中的作用。方法:取妊娠早期绒毛,培养绒毛外滋养细胞,用免疫细胞化学技术检测其中CXCR4与CXCL12的表达;并通过体外侵袭实验,分析绒毛外滋养细胞在CXCL12作用下的侵袭能力。结果:在EVTs的胞膜和胞浆中均检测到CXCL12和CXCR4的表达;EVTs在CXCL12作用下的侵袭能力在一定范围内与CXCL12浓度呈正相关(r=0.79,P<0.01),当CXCL12浓度达到10ng/ml时最强,达到100ng/ml时则开始下降。最大趋化指数为1.71±0.14。结论:CXCL12/CXCR4受体配体系统参与绒毛外滋养细胞的侵蚀,在妊娠过程中有重要意义。     

子宫颈癌和盆腔淋巴结组织中趋化因子受体CXCR4及其配体CXCL12基因DNA的表达及其意义

宫颈癌转移是一个复杂的、连续的、多阶段的过程,表现出高度的组织特异性.能转移到特定器官的肿瘤细胞,均具有多种机制促进其侵袭组织、刺激血管或淋巴管形成、产生各种细胞因子等作用,为肿瘤转移创造条件.趋化因子在肿瘤转移中发挥着多种作用,包括控制白细胞浸润至肿瘤、调节肿瘤相关的血管生成、激活宿主对肿瘤的特异性免疫应答、以旁分泌方式刺激肿瘤细胞增殖、控制肿瘤细胞运动等.趋化因子的基本功能就是对表达有相应趋化因子受体细胞的定向趋化作用,趋化因子和趋化因子受体的相互作用能诱导靶细胞趋化性迁移及细胞骨架的重排,增强靶细胞与内皮细胞的黏附能力等,在肿瘤的生长和转移过程中发挥着重要的作用.本研究通过对宫颈癌和盆腔淋巴结组织中的趋化因子受体CXCR4及其配体CXCL12基因DNA表达的研究,探寻其临床意义.     

趋化因子CXCL12及其受体CXCR4对卵巢上皮性癌细胞增殖、迁移和侵袭能力的影响

目的 探讨趋化因子CXCL12及其受体CXCR4对卵巢上皮性癌（卵巢癌）细胞增殖、迁移和侵袭能力的影响。方法采用RT—PCR技术和免疫细胞化学染色法检测卵巢癌细胞株CAOV3细胞中CXCL12、CXCR4mRNA和蛋白的表达,以及CXCL12（100ng／m1）作用后CAOV3细胞中整合素B1和血管内皮生长因子C（VEGF—C）mRNA的表达。实验分为6组：对照组（未加药）,实验组1（加100ng／ml的CXCL12）,实验组2（加10ng／ml的CXCL12）,实验组3（加100ng／ml的CXCL12和10斗g／ml的CXCR4抗体）,实验组4（加100ng／ml的CXCL12和1pg／ml的CXCR4拮抗剂——AMD3100）,实验组5（加10μg／ml的CXCR4抗体或卵巢癌腹水）。采用四甲基偶氮唑蓝比色法检测CXCL12对CAOV3细胞增殖的影响。以穿膜小室为模型,采用重组细胞基底膜迁移、侵袭实验检测CXCL12和卵巢癌腹水对CAOV3细胞迁移、侵袭的影响。结果 CAOV3细胞中,CXCR4蛋白呈棕黄色强阳性表达,CXCR4mRNA的表达水平为0．70±0．10,经100ng／ml的CXCL12作用24h时CXCR4mRNA的表达水平（1．24±0．14）显著增加（t=-7．1088,P=0．0021）;CXCL12蛋白和mRNA均无表达。100ng／ml的CXCL12作用3h时整合素131mRNA表达水平即显著增加（作用前后分别为0．53±0．10、1．53±0．16,P〈0．01）,24h时VEGF—CmRNA表达水平显著增加（作用前后分别为0．52±0．09、1．11±0．15,P〈0．01）。对CAOV3细胞的增殖作用,实验组1分别与对照组和实验组2比较均明显增强（3组分别为0．428±0．051、0．325±0．045、0．328±0．039,P〈0．05）;实验组1分别与实验组3和实验组4比较均明显被抑制（后两组分别为0．356±0．031、0．373±0．029,P〈0．05）;而实验组5（0．349±0．038）与对照组比较,差异则无统计学意义（P〉0．05）。对CAOV3细胞的迁移和侵袭作用,实验组1分别与对照组和实验组2比较均明显增强[迁移细胞数分别为（523．3±25．2）、（108．0±7．2）、（211．7±24．7）个,侵袭细胞数分别为（39．3±4．0）、（4．0±1．0）、（15．7±3．1）个;P均〈0．01];实验组1分别与实验组3和实验组4比较均明显被抑制（P〈0．05）;但实验组5[即加入卵巢癌腹水组;迁移细胞数为（706．6±30．6）个,侵袭细胞数为（61．7±7．6）个]显著多于实验组1（P〈0．05）。结论 CXCL12可促进卵巢癌细胞增殖、迁移和侵袭,并上调整合素B1和VEGF—CmRNA的表达,此作用可被CXCR4抗体抑制,表明CXCL12在卵巢癌的生长和转移中发挥一定作用。     

流式细胞仪分离人早孕绒毛滋养细胞亚型及鉴定

目的用流式细胞仪分离早期绒毛滋养细胞各细胞亚型，以便对各类型滋养细胞功能进行进一步研究。方法用胰蛋白酶消化法得到早期绒毛滋养细胞，用流式细胞仪分离纯化各滋养细胞亚型。所得的细胞亚型用免疫细胞化学、光镜、透射电镜检测以及Western印迹方法鉴定。结果使用流式细胞仪成功的分离了绒毛外滋养细胞与绒毛滋养细胞，纯度超过98％。结论此方法可快速准确大量获得滋养细胞亚型。     

Diagnostic value of elevated CXCR4 and CXCL12 in neonatal sepsis

Objective: Neonatal sepsis remains a major cause of morbidity and mortality in newborns. The chemokine CXCL12 and its receptor CXCR4 are now known to play an important role in inflammatory states. However, it is unclear how chemokines respond to late-onset neonatal sepsis.

Methods: Patients were classified into the groups of septic and non-septic ones. Samples of venous blood were obtained from all septic and non-septic newborns at the beginning and within 48–72?h after initiation of treatment. Serum levels of CXCR4 and CXCL12 were measured.

Results: Concentrations of IL-6, CXCR4 and CXCL12 at the time of diagnosis were significantly higher in the septic neonates compared with the non-septic ones. Additionally, there were statistically significant differences in septic neonates between the first and the second levels of IL-6, CXCR4, CXCL12 and I/T ratio. ROC curve analyses revealed that IL-6, CXCR4, CXCL12 and I/T ratio resulted in significant AUC with respect to early identification of septic neonates. Univariate logistic regression analysis showed that increased IL-6, CXCR4 and CXCL12 were strong predictors of neonatal LOS.

Conclusions: Serum CXCR4 and CXCL12 levels increase in septic neonates and that both chemokines decrease within 48–72?h of treatment. Serum concentrations of both chemokines represent promising novel biomarkers for neonatal sepsis.     

SDF-1/CXCR4对卵巢上皮性癌细胞增殖和侵袭的影响

目的:研究SDF-1/CXCR4在卵巢癌细胞生物学活性中的作用。方法:用RT-PCR方法检测卵巢癌细胞株SW626及Anglne中的SDF-1和CXCR4的表达。细胞经外源性SDF-1或者抗CXCR4单克隆抗体干预后,用MTT法检测细胞增殖,PI测细胞凋亡,Annexin-V/PI法检测细胞早期凋亡,Transwell小室检测细胞的迁移侵袭活性。结果:两株卵巢癌细胞株中,SW626细胞既表达SDF-1又表达CXCR4,Anglne细胞则两者均不表达。在SW626细胞中,SDF-1作用于无血清状态下培养的细胞(吸光度A值为0.911±0.01),与对照组(吸光度A值为0.506±0.01)相比,差异有统计学意义(P<0.01),而加入抗CXCR4单克隆抗体作用后(10μg/ml A值为0.725±0.01,20μg/ml A值为0.650±0.02),与SDF-1组相比,差异均有统计学意义(分别为P<0.05和P<0.01);抗CXCR4单克隆抗体作用于无血清状态下培养的细胞(10μg/ml A值为0.655±0.11和20μg/ml A值为0.520±0.04),与对照组(A值为0.724±0.03)相比,差异均有统计学意义(P<0.05)。加入外源性SDF-1后,SW626细胞在无血清状态下的早期凋亡数为15.6%,相对于对照组,差异有统计学意义(P<0.01)。抗CXCR4单克隆抗体可增加SW626细胞中的PI阳性细胞数(P<0.01)。并且,与对照组相比,SDF-1促进SW626细胞的迁移和侵袭能力增强(P<0.01),而此活性可被抗CXCR4单克隆抗体阻断。结论:SDF-1/CXCR4可能通过促进细胞增殖、迁移、侵袭及抑制其凋亡,使卵巢癌细胞获得进展。     

Leptin induces tube formation in first-trimester extravillous trophoblast cells

Objectives

To study the roles of leptin on tube formation (as a measure of cellular angiogenesis) and expression of associated genes in first-trimester human extravillous trophoblast cells.

Study design

The effects of leptin on tube formation and fatty acid uptake in first trimester extravillous placental trophoblast cells, HTR8/SVneo, were investigated. We also investigated the effects of leptin on the expression of genes involved in angiogenesis and lipid metabolism in these cells.

Results

Leptin at 25 ng/ml maximally stimulated tube formation in the first trimester placental trophoblast cells, HTR8/SVneo, by increasing tube length as well as numbers (10,100 ± 150 pixels) compared with those of control cells (2900 ± 50 pixels) p > 0.05. Leptin-induced tube formation was not inhibited by the selective inhibitor of VEGF, indicating that its action was independent of VEGF. Leptin, however, significantly increased the expression of genes those are involved in angiogenesis pathways such as PECAM1, JAG1, CDH5, IL8, NRP1, SPHK1, S1PR1, CXCL 1 and 6, FGF1, EFNA3 and AKT1, as determined by PCR array. Leptin did not, however, stimulate expression of the primary angiogenic factors known in placenta such as VEGF or ANGPTL4, as determined by both qRTPCR and PCR array assays. Leptin increased 7-fold expression of FABP4, which is known to be involved in VEGF-mediated angiogenesis in endothelial cells. In addition, leptin treatment resulted a 48% increase in the uptake of docosahexaenoic acid, 22:6n-3 (DHA) which also stimulates tube formation in these cells.

Conclusions

Leptin may play an important role in early placentation by stimulating several genes involved in angiogenic signalling pathway and fatty acid metabolism.     

CXCR7 and syndecan-4 are potential receptors for CXCL12 in human cytotrophoblasts

The placenta forms the interface between the mother and the fetus. During placental development cytotrophoblasts differentiate to form the syncytium or to invade the decidual wall to breach maternal vessels and establish the blood flow in the intervillous space. This process is still not well understood but it is proposed that chemokines and their receptors are involved in guiding cytotrophoblasts to the decidua and maternal vessels as well as attracting immunocompetent cells to the implantation site. CXCL12 is a chemokine expressed by cytotrophoblasts and is involved in cytotrophoblast invasion, differentiation and survival. One of its receptors, CXCR4, has been detected on cytotrophoblasts. Recent data show that CXCR7 and syndecan-4 might partially mediate CXCL12 function in other cell types. In this study, we examined CXCR7 and syndecan-4 expression at the maternal-fetal interface via immmunolocalization in placental tissue sections and in isolated cytotrophoblasts. We further used immunoblot analyses to confirm the data. We were able to show that cytotrophoblasts express both receptors and that upregulation occurs during the differentiation process of cytotrophoblasts towards the invasive phenotype. On a functional level CXCR7 seems not to be involved in JAR cell chemotaxis, suggesting a different function of this receptor. In conclusion, we propose that CXCL12 binds to CXCR4, but also to CXCR7 and syndecan-4. These three receptors could mediate different functions of CXCL12, such as cell migration, directed invasion, proliferation and survival. The latter molecules might also be involved in the development of placental pathologies, such as preeclampsia or choriocarcinoma growth.     

Expression and localization of CXCL16 and CXCR6 in ovarian endometriotic tissues

Purpose

Inflammatory mediators, including chemokines, may play crucial roles in the development of endometriosis. Therefore, we investigated the expression and localization of CXCL16 and its receptor, CXCR6, in ovarian endometriotic tissues. We also examined whether CXCL16 induces IL-8 production in endometriotic stromal cells.

Methods

We performed immunohistochemical and Western blotting analyses of in vivo and in vitro samples. IL-8 production was assayed using an ELISA.

Results

Both CXCL16 and CXCR6 were expressed by endometriotic epithelial cells and stromal cells, but not normal ovarian stroma. A Western blotting analysis using primary cultured endometriotic stromal cells showed a constant expression of CXCL16 and CXCR6 in the proliferative phase, secretory phase and during gonadotropin-releasing hormone agonist therapy. CXCL16 induced IL-8 production in several endometriotic stromal cells in vitro.

Conclusions

CXCL16 and CXCR6 might be involved in the pathophysiology of endometriosis through regulation of the inflammatory response.     

siRNA靶向沉默CXCR4基因对人卵巢癌SW626细胞增殖、迁移及侵袭的影响

目的:探讨小分子干扰RNA(siRNA)靶向沉默趋化因子受体4(CXCR4)基因对卵巢癌SW626细胞增殖、迁移及侵袭的影响,为卵巢癌的基因治疗提供新的理论依据。方法:构建CXCR4干扰RNA质粒载体,重组扩增后转染CXCR4阳性的人卵巢癌细胞株SW626,以RT-PCR和Western blot检测CXCR4基因及蛋白表达的变化,MTT检测CXCR4干扰质粒对卵巢癌细胞增殖的影响,Transwell检测CXCR4干扰质粒对卵巢癌细胞迁移、侵袭能力的影响。结果:转染48h后,人卵巢癌细胞株SW626的转染效率为80%～90%,抑制了卵巢癌细胞CXCR4基因及蛋白的表达(P<0.05),部分抑制了癌细胞的增殖(P<0.01),但此作用并不随时间变化而有明显变化;癌细胞的迁移及侵袭率均明显下降,差异有显著统计学意义(P<0.01)。结论:CXCR4 siRNA可通过下调CXCR4的表达抑制人卵巢癌细胞的增殖、迁移及侵袭。     

CXCL12-CXCR4生物轴对宫颈癌Hela细胞株的影响

目的:探讨CXCR4在宫颈癌细胞株Hela细胞中的表达情况,及CXCL12-CXCR4生物轴对Hela的增殖、迁移和侵袭能力的影响,并研究CXCR4的特异性拮抗剂AMD3100对CXCR4的特异性阻断效果。方法:将宫颈癌Hela细胞株细胞进行体外培养。免疫组化检测Hela细胞中CXCR4的表达情况。采用MTT法研究不同浓度CXCL12对Hela细胞增殖的影响;Transwell法进行实验,判断在无血清培养液的生长条件下,不同浓度的CXCL12和CXCR4拮抗剂AMD3100对Hela细胞迁移、侵袭的影响。结果:CXCR4在Hela细胞的细胞膜、细胞质中有表达。1组Hela细胞的增殖数与2组及3组两两比较,差异有统计学意义(P0.05),4组Hela细胞的增殖数较2组、3组低,差异有统计学意义(P0.05)。与A组Hela细胞的迁移数和侵袭数相比,B组明显更高(P0.05);而C组Hela细胞的迁移数和侵袭数明显高于A组和B组;D组Hela细胞的迁移数和侵袭数均低于A、B、C3组,差异有统计学意义(P0.05)。结论:CXCR4在Hela细胞的细胞膜、细胞质中均表达。CXCL12-CXCR4生物轴可促进宫颈癌Hela细胞株的迁移及侵袭,而CXCR4拮抗剂AMD3100能抑制这一过程,CXCL12-CXCR4生物轴在宫颈癌Hela细胞生长和转移过程中可能起着重要作用。