绒毛外滋养细胞浸润能力的调节

细胞滋养细胞起源于胚泡的滋养外胚层，是胎盘的主要细胞。妊娠早期，细胞滋养细胞代表着一个异源细胞群，在胚泡植入子宫壁后，其沿两条途经分化，即分化为绒毛滋养细胞(VCTs)和绒毛外滋养细胞(EVTs)^[1]。。其中EVTs是具有浸润能力的滋养细胞。Aplin^[2]将其分为浸润型和非浸润型两种亚型。定位在蜕膜组织的EVTs称间质滋养细胞。     

细胞过度凋亡是脂多糖抑制细胞滋养细胞侵入能力的重要机制

目的:探讨脂多糖(LPS)对细胞滋养细胞侵入能力的影响及细胞凋亡在此过程中的作用。方法:对体外培养的早孕细胞滋养细胞,给予不同浓度的LPS(0ng/ml、25ng/ml、50ng/ml、100ng/ml、200ng/ml),采用Transwell检测侵入能力的改变,荧光显微镜、电镜观察凋亡细胞的形态学改变,流式细胞仪定量检测细胞凋亡指数,western blot检测Caspase-3、MMP-2和MMP-9的表达。结果:LPS诱导细胞滋养细胞出现过度凋亡,抑制其侵入能力,促进Caspase-3的表达,抑制MMP-2、MMP-9的表达。以上各指标组间差异显著,P值均＜0．01。细胞凋亡指数与其侵入能力以及MMP-2和MMP一9的表达呈负相关,P值均＜0．01。结论:LPS对细胞滋养细胞的侵入能力有显著的抑制作用,细胞过度凋亡是这一作用发生的重要机制。     

子痫前期血清对滋养-内皮细胞共培养体系滋养细胞侵袭力和内皮细胞凋亡的影响

目的:探讨子痫前期血清对滋养-内皮细胞共培养体系滋养细胞侵袭力改变及对内皮细胞凋亡的影响.方法:选择正常足月妊娠剖宫产孕妇10例(正常对照组),子痫前期患者20例(子痫前期组).采用组织块贴壁法培养人孕早期细胞滋养细胞(CTB),胰酶消化法培养正常妊娠人脐静脉内皮细胞(HUVEC),传代后建立滋养细胞与脐静脉内皮细胞共培养模型,并分别加入正常对照组和子痫前期组患者静脉血清培养24小时;Transwell小室检测细胞滋养细胞的侵袭能力;流式细胞学检测人脐静脉内皮细胞凋亡率;逆转录-聚合酶链反应(RT-PCR)检测细胞滋养细胞MMP-2、MMP-9mRNA及人脐静脉内皮细胞自杀相关因子(Fas)mRNA的表达.结果:子痫前期患者血清共培养体系的细胞滋养细胞的侵袭能力低于正常对照组(P<0.01);人脐静脉内皮细胞凋亡率明显低于正常对照组(P<0.05);MMP-2、MMP-9和FasmRNA的表达明显下降(P<0.05).结论:子痫前期血清可能通过降调细胞滋养细胞MMP-2、MMP-9表达而影响滋养-内皮细胞共培养体系滋养细胞的侵袭能力,并且其滋养-内皮细胞共培养体的内皮细胞Fas的表达异常可能与内皮细胞凋亡下降有关.提示滋养细胞MMP-2、MMP-9、Fas/Fasl的表达异常可能参与了子痫前期子宫螺旋动脉重铸障碍的病理过程.     

滋养细胞肿瘤侵袭转移的分子机制研究进展

妊娠滋养细胞肿瘤是来源于胎盘滋养细胞的特殊疾病，其发生、发展是多种因素参与的、极其复杂的病理过程，主要是滋养细胞运动、浸润及迁移能力发生异常。癌基因、抑癌基因、蛋白水解酶、细胞因子等在妊娠滋养细胞肿瘤侵袭转移中起到不同的作用，认识这些因素对妊娠滋养细胞肿瘤侵袭转移的调节作用，可加深对妊娠滋养细胞肿瘤发生发展的认识，有望对其进行早期预测并进行有效治疗。     

子痫前期患者血清对滋养-平滑肌细胞共培养体系平滑肌细胞凋亡的影响

目的:探讨子痫前期血清对滋养-平滑肌细胞共培养体系滋养细胞侵袭力改变及对平滑肌细胞凋亡的影响.方法:采用组织块贴壁法培养人孕早期细胞滋养细胞(CTB)和人脐动脉平滑肌细胞(HUASMC),传代后建立滋养细胞与HUASMC共培养模型并分别加入正常和子痫前期患者静脉血清培养24小时并设立相对应的单培养组;用不同的方法检测细胞滋养细胞的侵袭能力、HUASMC活力、HUASMC凋亡率、滋养细胞基质金属蛋白酶MMP-2mRNA、MMP-9mRNA、人脐静脉内皮细胞自杀相关因子(Fas)及HUASMC的Fas配体(Fasl)mRNA的表达;HUASMC的Fas蛋白的表达.结果:子痫前期患者血清能显著抑制共培养体系中的细胞滋养细胞的侵袭能力,上调HUASMC活力,降低HUASMC早期凋亡率,降低细胞滋养细胞MMP-2mRNA、MMP-9mRNA、FasmRNA及HUASMC的Fasl mRNA和Fas蛋白水平的表达.结论:子痫前期血清可能通过降调细胞滋养细胞MMP-2、MMP-9表达而影响滋养-平滑肌细胞共培养体系滋养细胞的侵袭能力,子痫前期血清培养滋养-平滑肌细胞共培养体的Fas/Fasl的表达异常可能与HUASMC凋亡下降有关.提示滋养细胞MMP-2、MMP-9、Fas/Fasl 系统的表达异常可能参与了子痫前期子宫螺旋动脉重铸障碍的病理过程.     

MMPs及TIMPs在滋养细胞疾病中表达的分析

目的 :分析基质金属蛋白酶 (MMPs)及其组织抑制物 (TIMPs)在滋养细胞疾病中表达水平与预后的关系。方法 :采用免疫组化S P法检测 17例正常绒毛、39例葡萄胎 (HM)、4 2例侵蚀性葡萄胎 (IM)和 2 0例绒毛膜癌 (CC)组织中MMP 2、MMP 9及其TIMP 1、TIMP 2的表达情况。结果 :葡萄胎恶变组MMP 2、MMP 9表达较正常绒毛和葡萄胎未恶变组高 (P =0 .0 0 0 ,0 .0 0 0 ;P =0 .0 4 0 ,0 .0 11)。未化疗滋养细胞肿瘤 (包括IM和CC)MMP 2表达强于早孕绒毛和葡萄胎组 (P均 =0 .0 0 0 ) ,而≥ 3疗程化疗后MMP 2、9表达明显低于未化疗和≤ 2疗程化疗组 (P =0 .0 0 0 ,0 .0 35 ;P =0 .0 10 ,0 .0 18) ,TIMP 1在未化疗滋养细胞肿瘤组表达低于早孕绒毛和葡萄胎组 (P =0 .0 0 5 ,P =0 .0 0 0 )。MMP 2在Ⅱ、Ⅲ期的表达明显强于Ⅰ期 (P均 =0 .0 0 0 ) ,WHO预后评分中、高危者明显强于低危者 (P =0 .0 0 2 ,P =0 .0 0 5 )。结论 :MMPs及TIMPs表达水平高低与滋养细胞疾病预后有关 ,深入了解两者在滋养细胞疾病中的生物作用 ,有望为耐药患者的治疗提供新的途径     

miR-18a对人正常滋养细胞中MMP-2、MMP-9表达的影响

目的:研究子痫前期(pre-eclapmpsia,PE)相关微小RNA-18a(miR-18a)对人正常滋养细胞(HTR-8细胞)中基质金属蛋白酶2(MMP-2)、MMP-9的影响。方法:按照HTR-8细胞中miR-18a的表达情况分为3组(每组重复5次):miR-18a过表达组、miR-18a抑制组、阴性对照组;实时定量聚合酶链反应(RT-qPCR)检测转染后HTR-8细胞中miR-18amRNA的表达量;RT-qPCR和蛋白质印迹(Western blotting)检测转染后HTR-8细胞中MMP-2、MMP-9mRNA和蛋白表达量。结果:miR-18a过表达组miR-18amRNA表达量比阴性对照组升高,miR-18a抑制组miR-18amRNA表达量比阴性对照组降低,差异均有统计学意义(均P<0.001)。miR-18a抑制组MMP-2、MMP-9mRNA和蛋白表达量比阴性对照组降低,差异均有统计学意义(均P<0.001)。结论:miR-18a影响人正常滋养细胞中MMP-2、MMP-9在mRNA及蛋白水平的表达,可能通过该途径参与对滋养细胞浸润能力的调控。     

蜕膜基质细胞条件培养液对滋养细胞浸润功能的影响

目的:探讨蜕膜基质细胞条件培养液(DSCM)对滋养细胞浸润力的影响。方法:体外培养人早孕正常蜕膜基质细胞,收集DSCM处理早孕滋养细胞系(B6Tert)。应用浸润实验分析B6Tert细胞浸润力的改变,采用RT-PCR与明胶酶谱技术检测B6Tert细胞中基质金属蛋白酶(MMPs)的表达变化。结果:DSCM浓度较低时(占细胞培养液5%),可促进B6Tert细胞的浸润力与该细胞MMP-2mRNA及pro-MMP-2的表达;反之,高浓度的DSCM(20%)则抑制滋养细胞的浸润力与MMP-2的表达,差异有统计学意义(P<0.05)。DSCM不影响B6Tert细胞中MMP-9的表达。结论:早孕DSCM可能通过调节滋养细胞中MMP-2的表达影响滋养细胞的浸润能力。     

PTEN在子痫前期胎盘滋养细胞浸润不足中的作用及机制研究

目的:检测子痫前期(PE)患者胎盘组织中PTEN表达,探讨PTEN对滋养细胞迁移功能的调控及其可能机制。方法:收集重度PE患者和正常孕妇的胎盘组织各20例,实时定量PCR和Western blot法检测胎盘组织中PTEN表达;将PTEN过表达质粒(pcDNA3.1-HA-PTEN)或特异性siRNA(siPTEN)瞬时转染至HTR-8/SVneo细胞,同时转染pcDNA3.1或siCTL作为对照。采用划痕实验评估细胞迁移能力,Western blot法检测p-Akt(Thr308)、p-Akt(Ser473)、Akt、MMP-2和MMP-9表达。结果:重度PE患者胎盘组织中PTEN mRNA和蛋白表达明显高于正常孕妇,差异均有统计学意义(P0.01)。与转染pcDNA3.1组比较,pcDNA3.1-HA-PTEN瞬时转染HTR-8/SVneo细胞48h后,细胞迁移率明显降低[(26.42±6.68)%vs(52.92±6.71)%,P0.01],p-Akt(Thr308)和p-Akt(Ser473)表达显著下调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达下降;与转染siCTL组比较,siPTEN瞬时转染HTR-8/SVneo细胞48h后,细胞迁移率则明显升高[(50.39±6.84)%vs(30.04±5.40)%,P0.01],p-Akt(Thr308)和p-Akt(Ser473)的表达明显上调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达升高。结论:重度PE患者胎盘组织中PTEN表达明显升高,PTEN可通过下调Akt活性降低MMP-2和MMP-9表达,抑制滋养细胞的迁移。提示PTEN在PE的发病中发挥了一定的作用。     

PTEN在子痫前期胎盘滋养细胞浸润不足中的作用及机制研究26

目的:检测子痫前期(PE)患者胎盘组织中PTEN表达,探讨PTEN对滋养细胞迁移功能的调控及其可能机制.方法:收集重度PE患者和正常孕妇的胎盘组织各20例,实时定量PCR和Western blot法检测胎盘组织中PTEN表达;将PTEN过表达质粒(pcDNA3.1-HA-PTEN)或特异性siRNA(siPTEN)瞬时转染至HTR-8/SVneo细胞,同时转染pcDNA3.1或siCTL作为对照.采用划痕实验评估细胞迁移能力,Western blot法检测p-Akt(Thr308)、p-Akt(Ser473)、Akt、MMP-2和MMP-9表达.结果:重度PE患者胎盘组织中PTEN mRNA和蛋白表达明显高于正常孕妇,差异均有统计学意义(P<0.01).与转染pcDNA3.1组比较,pcDNA3.1-HA-PTEN瞬时转染HTR-8/SVneo细胞48 h后,细胞迁移率明显降低[(26.42±6.68)%vs(52.92±6.71)%,P<0.01],p-Akt(Thr308)和p-Akt(Ser473)表达显著下调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达下降;与转染siCTL组比较,siPTEN瞬时转染HTR-8/SVneo细胞48 h后,细胞迁移率则明显升高[(50.39±6.84)%vs(30.04±5.40)%,P<0.01],p-Akt(Thr308)和p-Akt(Ser473)的表达明显上调,Akt表达无明显改变,MMP-2和MMP-9蛋白表达升高.结论:重度PE患者胎盘组织中PTEN表达明显升高,PTEN可通过下调Akt活性降低MMP-2和MMP-9表达,抑制滋养细胞的迁移.提示PTEN在PE的发病中发挥了一定的作用.     

Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion

Human trophoblast research relies on a combination of in vitro models, including isolated primary cultures, explant cultures, and trophoblast cell lines. In the present study, we have utilized the rotating wall vessel (RWV) bioreactor to generate a three-dimensional (3-D) model of human placentation for the study of cytotrophoblast (CTB) invasion. The RWV supported the growth of the human CTB cell line SGHPL-4 and allowed for the formation of complex, multilayered 3-D aggregates that were morphologically, phenotypically, and functionally distinct from SGHPL-4 monolayers. The cells cultured three-dimensionally differentiated into an aggressively invasive cell population characterized by the upregulation of matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9 and urokinase-type plasminogen activator (uPA) secretion and activation. Microarray analysis of the 3-D and 2-D cultured cells revealed increased expression in the 3-D cells of various genes that are known mediators of invasion, including MT1-MMP, PECAM-1 and L-selectin, as well as genes not previously associated with CTB differentiation such as MMP-13 and MT5-MMP. These results were verified by quantitative real-time PCR. These findings suggest that when cultured in 3-D, SGHPL-4 cells closely mimic differentiating in utero CTBs, providing a novel approach for the in vitro study of the molecular mechanisms that regulate CTB differentiation and invasion.     

细胞外基质金属蛋白酶诱导因子在子痫前期及子痫患者胎盘组织中表达的研究

目的探讨细胞外基质金属蛋白酶诱导因子(EMMPRIN)在子痫前期及子痫患者胎盘组织中的表达及其与子痫前期及子痫发病的关系。方法采用免疫组化链霉菌抗生物素蛋白-过氧化酶连接法和RT-PCR技术检测不同孕周的44例子痫前期(重度)孕妇(子痫前期组)、38例子痫孕妇(子痫组)和49例正常孕妇(正常妊娠组)胎盘组织中EMMPRIN蛋白及mRNA的表达。结果(1)EMMPRIN蛋白阳性表达：子痫前期组EMMPRIN蛋白表达中度阳性率为18%(8/44),强阳性率为9%(4/44);子痫组EMMPRIN蛋白中度阳性率为21%(8/38),强阳性率为13%(5/38),两组分别比较,差异均无统计学意义(P＞0．05)。正常妊娠组EMMPRIN蛋白表达中度阳性率为12%(6/49),强阳性率为82%(40/49),与子痫前期组及子痫组比较,差异均有统计学意义(P＜0．001)。(2) EMMPRIN mRNA表达：子痫前期组孕晚期(37～40周)EMMPRIN mRNA为0．342±0．002,子痫组为0．344±0．023,两组比较,差异均无统计学意义(P＞0．05)。正常妊娠组孕晚期(37～40周) EMMPRIN mRNA为0．872±0．094,分别与子痫前期组及子痫组比较,差异均有统计学意义(P＜0．001)。结论子痫前期及子痫患者胎盘组织中EMMPRIN表达水平下降是子痫前期及子痫发病的重要原因;EMMPRIN可作为子痫前期及子痫发病的一个预测指标。     

The cytotrophoblastic shell and complications of pregnancy

Many complications of pregnancy have their pathophysiological roots in the early stages of placentation. Impaired trophoblast invasion and deficient remodelling of the maternal spiral arteries are a common feature. While malperfusion of the placenta may underpin cases of fetal growth restriction and early-onset pre-eclampsia, the mechanistic links to spontaneous miscarriage, pre-term labour and premature rupture of the membranes are less obvious. Here, we speculate that formation of a well-developed cytotrophoblastic shell at the maternal-fetal interface is crucial for pregnancy success. Initially, extravillous trophoblast cells differentiate from the outer layer of the shell in contact with the endometrium. Impaired development may thus contribute to reduced invasion and deficient remodelling. In addition, the extent of the shell influences the timing and spatial configuration of onset of the maternal arterial circulation. A thin and fragmentary shell results in premature and disorganised onset, leading to spontaneous miscarriage. In less severe cases it may predispose to haemorrhage at the interface and formation of intrauterine haematomas. If pregnancy continues, these haematomas may act as a source of oxidative stress, promoting senescence and weakening of the membranes, and stimulating inflammation in the uterine wall and premature contractions. Formation of the shell is dependent on proliferation of cytotrophoblast progenitor cells during the first weeks after implantation, when the developing placenta is supported by histotrophic nutrition from endometrial glands. Hence, we propose the fitness of the endometrium prior to conception, and the peri-conceptional dialogue between the endometrium and the trophoblast is critical for avoidance of later complications of pregnancy.     

β_1-整合素与纤维粘连蛋白联合促进卵巢癌细胞侵袭转移

目的:探讨β1-整合素及纤维粘连蛋白对卵巢癌细胞粘附侵袭能力的影响和作用机制。方法:流式细胞仪间接免疫荧光染色检测人卵巢癌SKOV3-S1细胞β1-整合素表达。用人工基底膜粘附实验和体外侵袭实验观察经β1-整合素抗体处理的SKOV3-S1细胞粘附侵袭能力的变化。逆转录PCR检测β1-整合素处理及与纤维粘连蛋白结合后SKOV3-S1细胞基质金属蛋白酶基因表达的变化,同时用酶谱法分析细胞上清MMPs活性变化。结果:SKOV3细胞高侵袭克隆SKOV3-S1均表达β1-整合素,β1-整合素抗体对其粘附及侵袭人工基底膜能力的抑制率分别为69·8%和65·7%。纤维粘连蛋白能诱导癌细胞MMP2基因表达并分泌蛋白酶,β1-整合素抗体能减弱这种诱导作用。结论:β1-整合素与纤粘维连蛋白结合能诱导卵巢癌细胞粘附并表达MMP2基因,分泌蛋白酶降解ECM,从而促进肿瘤侵袭转移。     

A simple histochemical method for the identification of cytotrophoblasts in tissue sections

A simple method for the demonstration of placental cytotrophoblast cells is described, utilising the affinity of the lectin from Bandeiraea simplicifolia-II (BSA-II) for intracellular amylase-sensitive glycogen and a protocol using biotinylated BSA-II followed by an avidin-peroxidase revealing system. In early pregnancy, cytotrophoblast cells in chorionic and anchoring villi are deeply stained and with ongoing differentiation the staining gradually decreases in intensity, suggesting that this lectin can be a useful marker for these cells.     

低分子肝素对不同氧浓度下早孕绒毛滋养层细胞侵袭力的影响作用

目的:研究低分子肝素(LMWH)对不同氧浓度下早孕绒毛滋养层细胞侵袭力的影响。方法:取妊娠8~10周正常绒毛组织,酶消化法分离人早孕绒毛滋养层细胞,分别置2%和20%O_2分压的细胞培养箱培养。用不同浓度LMWH(0、0.1、5、10IU/ml)处理滋养细胞,观察滋养层细胞侵袭力的改变,检测细胞中HIF-1α、MMP-2、MMP-9、TIMP-2、TIMP-3表达。结果:低氧浓度下,早孕绒毛滋养层细胞的侵袭能力随着LMWH浓度(0,0.1,5IU/ml)升高而增强,但高浓度的LMWH(10IU/ml)对其有抑制作用;随着LMWH浓度增加,HIF-1α和MMP-2表达呈先上升后下降的趋势;TIMP-2、TIMP-3表达改变的趋势与MMP-2相同。正常氧浓度下的滋养层细胞,其侵袭能力未见显著增强,各细胞因子的表达无上调。结论:低氧环境下,一定浓度的LMWH可提高早孕绒毛滋养层细胞的侵袭能力,其发生机制可能是通过诱导、增强滋养层细胞HIF-1α和MMP-2基因表达。     

雌激素受体亚型α对子宫内膜癌细胞HEC-1B侵袭能力调控的研究

目的：特异性下调子宫内膜癌细胞中的ERα基因，探讨E胁亚型表达在子宫内膜癌侵袭中的作用。方法：将ERa的小干扰RNA（siRNA-small interfering RNA）转染子宫内膜癌细胞HEC-1B，通过RT-PCR和Western blot证实ERα基因的有效阻断。通过transwell小室法检测下调ERa基因表达前后HEC-1B细胞的侵袭能力；应用RT-PCR检测转染前后细胞MMP-2、MMP-9、TIMP-1和TIMP-2 mRNA表达水平的变化；Western blot及明胶酶谱分别检测细胞分泌TIMP-1、TIMP-2、MMP-2和MMP-9蛋白的水平。结果：（1）将ERα-siRNA转染HEC-1B细胞后，转染效率大于90％，ERα mRNA及蛋白的表达水平均明显下调（分别为72％，67％）；（2）下调ERα基因表达后，肿瘤细胞的侵袭能力下降（P〈0．05）；在mRNA水平和蛋白水平均可检测到ERα-siRNA组细胞的MMP-2、MMP-9表达下降（P〈0．05），TIMP-1、TIMP-2表达增加（P〈0．05）。结论：使用ERα-siRNA能够有效地阻断ERa基因表达；子宫内膜癌细胞中，17β-雌二醇对MMPs／TIMPs具有调节作用，这种作用可通过ERα介导；ERα表达水平影响子宫内膜癌细胞的侵袭能力。     

The relation of increased uterine artery blood flow resistance and impaired trophoblast invasion in pre-eclamptic pregnancies

Objective: To assess the association between histopathologically confirmed vascular abnormalities developed during pre-eclampsia and abnormal arterial blood flows recorded during Doppler sonographies. Materials and methods: From pregnant women who attended our clinic between 01/03/2002 and 01/07/2002, a detailed medical history was obtained and routine biochemical blood tests, fetal ultrasonography and UA Doppler scans were performed. In addition, from pre-eclamptic and normal pregnant women who underwent cesarean sections, placental bed biopsies were taken. Thirty two pre-eclamptic [12 mild, 20 severe cases according to American College of Obstetricians and Gynecologogists (ACOG) criteria] cases and as a control group 20 normal pregnancies were included in the study. In our study trophoblast invasion into decidual spiral arteries was observed in 75% of mild (9/12), and 55% of severe (11/20) pre-eclampsias. In the control group all the cases demonstrated trophoblast invasion in decidual spiral arteries. Trophoblast invasion in myometrial spiral arteries was noted in 50% (6/12) of mild and 25% (5/20) of severe pre-eclamptic pregnancies. It was seen in 16 cases out of 20 (80%) pregnancies. In the control group, decidual spiral artery invasion manifests significant differences (P<0.01) among groups studied. Invasion in decidual spiral arteries was seen in all normal pregnancies of the control group. There is not any significant difference between mild and severely pre-eclamptic groups (P>0.05). Conclusion: Doppler ultrasonography is not only a non-invasive method for evaluating fetal status in pre-eclamptic pregnancies, but it also correlates with partial trophoblastic invasion in spiral arteries, which contributes to the pathophysiologic mechanisms involved in pre-eclampsia.     

脂多糖经线粒体途径诱导细胞滋养细胞凋亡

目的:探讨脂多糖(Lipopolysaccharide,LPS)诱导细胞滋养细胞凋亡的机制。方法:从正常早孕绒毛分离细胞滋养细胞,用无血清培养基培养。在培养基中加入不同浓度的LPS,使其终浓度分别为0、25、50、100、200ng/ml。用光镜和透射电镜观察细胞滋养细胞的形态学变化;用Western blot检测Bax、Bcl-2和Caspase-8的表达。结果:LPS作用后24h,光镜和电镜下见细胞滋养细胞出现凋亡的形态学变化;Western blot检测结果表明,LPS抑制Bcl-2表达,促进Bax和Caspase-8表达。结论:LPS能够经线粒体途径诱导细胞滋养细胞凋亡。