sLeX/L-选择素介导的双向黏附在胚胎着床中的作用

目的:探讨寡糖sLeX与L-选择素(L-selectin)作为黏附分子在母-胎之间的识别与黏附的作用。方法:通过免疫荧光方法检测胚胎细胞(JAR细胞)与子宫内膜细胞(RL95-2细胞)的sLeX与L-选择素的表达,通过转染α1,3-岩藻糖基转移酶VII(fucosyltransferase VII,FUT7)过表达载体至JAR细胞和RL95-2细胞,调节细胞表面sLeX的表达,以及通过sLeX/L-选择素抗体封闭细胞,计算不同处理后JAR细胞与RL95-2细胞黏附率。结果:①胚胎细胞与子宫内膜细胞均表达sLeX和L-选择素;②sLeX抗体及L-选择素抗体分别封闭JAR细胞与RL95-2细胞后,JAR细胞与RL95-2细胞的黏附率下降;③FUT7过表达增加sLeX寡糖抗原的合成,促进JAR细胞与RL95-2细胞的黏附率。结论:sLeX/L-选择素组成的黏附系统在胚胎与子宫内膜的识别与黏附过程中发挥重要作用。     

黄芩、白术通过调节岩藻糖基转移酶Ⅳ、Ⅶ的表达促进胚胎体外黏附

目的:探讨黄芩、白术对胚胎体外黏附的影响及其调控的糖分子机制。方法:培养人子宫内膜细胞(RL95-2)和人绒毛膜细胞(JAR),并经米非司酮诱导后建立着床障碍模型。通过黏附实验观察不同浓度黄芩、白术水煎液对胚胎黏附率的影响,采用RT-PCR、Western blotting检测着床相关寡糖合成关键酶的表达变化。结果:黄芩、白术50 mg/L治疗组胚胎黏附率较模型组显著升高(P0.05),着床相关的岩藻糖基转移酶Ⅳ、Ⅶ(FUT4、FUT7)的m RNA及蛋白表达显著增加(P0.001)。结论:中药黄芩、白术可通过上调FUT4、FUT7的表达促进胚胎体外黏附。     

肝素结合表皮生长因子通过诱导人子宫内膜细胞骨桥蛋白的表达促进人滋养层细胞的体外侵袭

目的:探讨肝素结合表皮生长因子(HB-EGF)对人子宫内膜细胞RL95-2与人滋养层细胞Be Wo体外黏附和铺展的影响及其作用机制。方法:利用免疫荧光法观察骨桥蛋白(OPN)在RL95-2细胞中的表达定位,采用RT-PCR和Western blotting检测HB-EGF对RL95-2细胞中OPN表达水平的影响,并通过黏附和铺展实验观察HB-EGF对Be Wo球状体细胞在RL95-2单层细胞上侵袭活性的影响。结果:OPN在RL95-2细胞膜表面高表达,HB-EGF能诱导RL95-2细胞中OPN的表达,并促进Be Wo细胞球在RL95-2细胞上的黏附和铺展。结论:HB-EGF可能通过上调子宫内膜细胞膜OPN的表达而促进滋养层细胞的体外侵袭活性。     

人α1,3-岩藻糖基转移酶VII荧光真核表达载体的构建及鉴定

目的:构建并鉴定绿色荧光蛋白为报告基因的pIRES2-EGFP-FUT7真核表达载体。方法:采用RT-PCR方法获得α1,3-岩藻糖基转移酶VII(FUT7)全长基因,克隆至pMD18-TSimple载体后,将FUT7亚克隆至pIRES2-EGFP表达载体并进行鉴定。通过脂质体转染到子宫内膜RL95-2细胞中,荧光显微镜下观察并经RT-PCR检测FUT7的表达。结果:测序及酶切鉴定证明获得人全长FUT7基因,FUT7基因正确插入pIRES2-EGFP中,在荧光显微镜下观察到了绿色荧光蛋白在RL95-2细胞中的表达,半定量RT-PCR检测结果显示FUT7的表达明显增加。结论:成功构建了绿色荧光蛋白为报告基因的FUT7真核表达载体,并检测到在RL95-2细胞中的表达,为研究FUT7及其合成的糖抗原sLex在胚胎着床中的作用奠定了基础。     

RNA干扰抑制EZH2基因表达对子宫内膜癌细胞上皮-间质转化的影响

目的:利用RNA干扰技术靶向沉默EZH2基因,检测其对子宫内膜癌细胞增殖、迁移能力及上皮-间质转化(EMT)的影响。方法:实时荧光定量PCR、Western blot检测EZH2在子宫内膜细胞ESC及子宫内膜癌细胞株ECC-1、RL95-2中的表达差异。利用化学技术合成小分子siRNA转染子宫内膜癌细胞,靶向抑制EZH2基因表达。CCK-8、Transwell小室检测干扰前后子宫内膜癌细胞株ECC-1、RL95-2增殖、迁移能力变化。Western blot检测干扰前后子宫内膜癌细胞株ECC-1、RL95-2中EMT相关蛋白E-cadherin、α-catenin、N-cadherin和Vimentin的表达变化。结果:(1)EZH2在子宫内膜癌细胞株ECC-1、RL95-2中的表达明显高于子宫内膜细胞ESC(P0.01)。(2)RNA干扰抑制EZH2基因表达后,ECC-1、RL95-2细胞增殖、迁移能力降低(P0.01)。(3)RNA干扰抑制EZH2基因表达后,ECC-1、RL95-2细胞中上皮标志物E-cadherin和α-catenin的蛋白表达水平上调(P0.05),间质标志物N-cadherin和Vimentin的蛋白表达水平下调(P0.01)。结论:EZH2在子宫内膜癌细胞中高表达,siRNA靶向沉默EZH2基因可抑制子宫内膜癌细胞的增殖、迁移和上皮-间质转化。     

TRAIL在小鼠胚胎着床过程中子宫内膜的表达

目的:检测TRAIL在小鼠胚胎着床过程中子宫内膜的表达,探讨它在蜕膜细胞凋亡中的作 用。方法:采用RT-PCR及免疫组化技术检测妊娠d 1-8小鼠子宫组织TRAILmRNA及蛋白的表 达情况。结果:妊娠d 1-8的小鼠子宫组织均有TRAIL mRNA的表达,且着床期间的表达较着床前 明显增加(P<0．05)。妊娠d 1-3,小鼠子宫内膜无TRAIL蛋白表达;妊娠d 4,TRAIL表达在小鼠胚 胎定位、黏附点的子宫内膜腔上皮细胞;妊娠d 5-6,TRAIL定位于胚胎着床点附近的蜕膜细胞中; 妊娠d 7-8,TRAIL表达在与子宫蜕膜邻近的胚胎滋养层细胞中。结论:在小鼠胚胎着床过程中, TRAIL诱导子宫内膜腔上皮细胞凋亡可能是胚胎跨越上皮屏障的重要机制之一,且TRAIL诱导的 蜕膜细胞和胚胎滋养层细胞的凋亡在滋养层细胞对子宫内膜的适度侵入过程中起重要作用。     

骨桥蛋白在肝素结合表皮生长因子样生长因子介导的小鼠胚胎对子宫内膜黏附和扩展中的作用研究

目的:探讨肝素结合表皮生长因子样生长因子(HB-EGF)对小鼠胚胎黏附和扩展中的作用机制。方法:建立小鼠胚胎与小鼠子宫内膜细胞共培养模型,分A、B、C、D、E共5组,分别加入不同浓度的HB-EGF(0μg/L、20μg/L、50μg/L、100μg/L、200μg/L),于倒置显微镜下统计各组胚胎黏附率及扩展率;用免疫组织化学法检测小鼠子宫内膜细胞骨桥蛋白(OPN)的表达及定位,Western blotting法测定各浓度HB-EGF对子宫内膜细胞OPN表达水平的影响,并分析其与HB-EGF的关联。结果:OPN在小鼠子宫内膜细胞高表达,主要分布于胞质中,随HB-EGF浓度升高,小鼠子宫内膜细胞中OPN的表达增强,小鼠胚胎对子宫内膜的黏附和扩展率也逐渐增高。结论:HBEGF可能通过上调小鼠子宫内膜细胞OPN的表达,促进小鼠胚胎对子宫内膜的黏附和扩展。     

降钙素是孕激素调节的子宫内膜容受性标志

降钙素是含有 32个氨基酸残基的肽类激素 ,分子量为 350 0。主要生理功能是调节细胞外 Ca2 + 生成。近年来发现子宫内膜降钙素表达与子宫内膜容受性密切相关 ,并受孕激素调节 ,着床结束后 ,表达下降 ,阻断其表达 ,导致着床失败。通过对假孕小鼠子宫降钙素m RNA水平的检测 ,发现子宫内膜降钙素的表达不需要胚胎存在 ,只受母体因素调控。提示 :降钙素在子宫可能是子宫内膜容受性的标志 ,在胚胎着床过程中起重要作用     

白血病抑制因子及其受体与妊娠

白血病抑制因子 ( LIF)是一种多生物学活性的细胞因子。近年来的研究表明 ,LIF存在于子宫内膜、妊娠期蜕膜及胎盘上。它通过与其受体 ( LIFR)结合 ,从而调节胚胎的着床及妊娠的维持。LIF不仅与其他细胞因子之间相互调节 ,且它还受到激素内分泌的严格调控。进一步研究还发现 ,原因不明不孕症妇女子宫内膜 LIF的表达较正常妇女减弱甚至缺失 ,这提示 LIF的异常表达可能是原因不明不孕症患者着床失败的原因之一 ,也为不孕症的治疗及节育技术提出了一条新途径     

小鼠囊胚在子宫内膜体外共培养模型上着床的超微结构观察

目的:进一步证明已建立的体外着床模型的可行性。方法:将妊娠d4的小鼠囊胚培养在已建立的体外子宫内膜共培养模型上,电镜观察胚胎植入时囊胚与子宫内膜上皮细胞相互关系的超微结构。结果:小鼠囊胚能正常脱透明带、黏附和扩展。扫描电镜见黏附在子宫内膜上的囊胚呈椭圆形或扁平形;胚胎表面具微绒毛;在胚胎与子宫内膜细胞接触点可见胚胎滋养细胞黏附于胞饮突上。透射电镜观察显示,胚胎滋养细胞通过微绒毛黏附于上皮细胞上,有些滋养细胞通过微绒毛开始植入,局部滋养细胞与上皮细胞形成紧密连接。胚胎滋养细胞与上皮细胞间形成许多镶嵌连接。超微结构显示粗面内质网、线粒体、溶酶体和核糖体非常丰富。结论:小鼠囊胚在子宫内膜体外模型上生长发育良好,该胚胎与子宫内膜共培养系统是研究胚胎着床机理的理想体外模型。     

米非司酮对小鼠着床期子宫内膜活化的β-连环蛋白(active β-catenin)表达的影响

目的:探讨活化的β-连环蛋白(active β-catenin)在胚胎着床过程中的作用。方法:将孕第2日(d2)昆明小鼠随机分成3组,每组30只,A组:孕d2单次背部皮下注射0.1ml米非司酮(1.2mg/ml);B组(溶剂对照):以等量1,3-丙二醇代替米非司酮;C组:不作任何处理。用免疫组织化学、间接免疫荧光、Western blotting方法定性、定量、定位检测3组小鼠在妊娠d3￣7活化的β-连环蛋白的动态表达。结果:B、C组小鼠子宫内膜中活化的β-连环蛋白于孕d3在腺上皮和腔上皮、基质细胞质和细胞膜表达;妊娠d4在腔上皮细胞质和细胞膜强表达,并达高峰;妊娠d5￣7在腺上皮、血管内皮、腔上皮下基质细胞膜和细胞质表达,且B、C组间在妊娠d3￣7各时间点的表达无显著性差异(P>0.05);A组小鼠妊娠d3￣7活化的β-连环蛋白的表达较B、C组显著降低(P<0.01),在妊娠d4表达量未见高峰,且表达部位局限于基质细胞,腔上皮无表达。结论:米非司酮可能通过抑制孕激素与其受体结合而下调活化的β-连环蛋白表达,进而抑制子宫内膜黏附性,影响子宫内膜容受性的建立而阻碍胚胎顺利着床。     

FUT8 drives the proliferation and invasion of trophoblastic cells via IGF-1/IGF-1R signaling pathway

IntroductionTrophoblast proliferation and invasion are essential for embryo implantation and placentation. Protein glycosylation is one of the most common and vital post-translational modifications, regulates protein physical and biochemical properties. FUT8 is the only known fucosyltransferase responsible for catalyzing α1,6-fucosylation in mammals, and α1,6-fucosylated glycoproteins are found to participate in various physiopathological processes. However, whether FUT8/α1,6-fucosylation modulates the functions of trophoblastic cells remains elusive.MethodsFUT8 in human placenta villi during 6-8 gestational weeks and trophoblastic cells were detected by Western blot and immunofluorescent staining. α1,6-fucosylation in tissues or cells were measured by Lectin LCA (Lens culinaris) fluorescent staining and Lectin blot. FUT8 expression was down-regulated by siRNA transfection in JAR and JEG-3 cells, and cell viability, motility and invasiveness ability were detected by the functional experiments. α1,6-fucosylation of insulin-like growth factor receptor (IGF-1R) was examined by immunoprecipitation, and the amount of phosphorylated IGF-1R was detected in FUT8 down-regulated JAR cells.ResultsHuman placenta villi and trophoblastic cells expressed FUT8/α1,6-fucosylation. Knockdown FUT8 by siRNA transfection suppressed the proliferation, epithelial-mesenchymal transition, migration and invasion of JAR and JEG-3 cells. Furthermore, we found that FUT8 modified the α1,6-fucosylation of IGF-1R, and regulated IGF-1 dependent activation of IGF-1R, MAPK and PI3K/Akt signaling pathways in JAR cells.ConclusionsOur results implicate a critical role for FUT8 in maintaining the normal functions of trophoblastic cells, suggesting manipulating FUT8 may be an effective approach in pregnancy.     

早孕绒毛组织中HOXA7、9、10基因的表达及米非司酮对其的影响

目的:探讨米非司酮抗早孕的作用机理。方法:因非意愿妊娠要求终止妊娠的正常早孕妇女60例,分为3组,分别为常规负压吸宫对照组、负压吸引术前服用米非司酮100mg组和150mg,每组20例。运用RT-PCR及免疫组织化学方法检测各组绒毛组织中HOXA7、9、10mRNA和蛋白的表达。结果:①正常早孕绒毛组织在mRNA和蛋白水平均可见HOXA7、9、10基因的表达,表达主要在滋养细胞胞质,间质少量表达。②不同剂量的米非司酮均可以降低绒毛组织中HOXA7、9、10mRNA及蛋白的表达量,但对不同基因的降表达程度有所不同。150mg米非司酮对早孕绒毛组织中HOXA7、10基因的降表达作用明显大于100mg米非司酮(P<0.01)。服用不同剂量米非司酮后的早孕绒毛组织中HOXA7、9、10蛋白的表达位置仍以滋养细胞胞质为主,间质少量表达。结论:HOXA7、9、10基因表达于正常早孕绒毛组织中,服用米非司酮后可降低其表达,呈一定的剂量依赖性,但未改变蛋白的表达位置。米非司酮的抗早孕作用可能是通过对绒毛组织中HOXA7、9、10基因的调控来达到的,且以150mg的米非司酮作用明显。     

IMP1 promotes choriocarcinoma cell migration and invasion through the novel effectors RSK2 and PPME1

Objective

Oncofetal protein insulin-like growth factor II mRNA-binding protein 1 (IMP1) regulates cellular proliferation and migration. Expression of IMP1 is limited to a few adult human tissues. However, it commonly expresses in a variety of cancers. Our objective was to study the regulatory mechanism of IMP1 on the cellular functions of choriocarcinoma (CC) JAR cells.

Methods

IMP1 protein levels were measured in CC tissues via immunohistochemistry. Specific siRNAs were used to down-regulate gene expressions. The abilities of migration and invasion were estimated by wound-healing and Matrigel chamber assays. The profile of IMP1-binding genes was investigated with an Agilent microarray. RT-qPCR, RNA immunoprecipitation, and IMP1 rescue experiments were performed to confirm the association between IMP1 and its binding genes. Gene expression was further analyzed by using RT-PCR and Western blotting.

Results

Strong IMP1 expressions were frequently detected in CC tissues. Knockdown of IMP1 expression in JAR cells inhibited cell migration and invasion, but did not affect cellular proliferation and morphology. Microarray and RNA-immunoprecipitation results revealed several candidate genes regulated by IMP1. Among them, ribosomal protein S6 kinase (RSK2) and protein phosphatase methylesterase 1 (PPME1) were confirmed to be down-regulated in IMP1-depleted JAR cells. Re-expression of IMP1 into the cells restored the expressions of RSK2 and PPME1. Furthermore, the depletion of RSK2 or PPME1 decreased the migration and invasion of JAR cells.

Conclusion

Our results suggest that IMP1 plays an essential role in the regulation of migration and invasion of human CC cells, possibly through the novel effectors RSK2 and PPME1.     

米非司酮治疗子宫内膜增生症前后ER和AR的表达及意义

目的:探讨米非司酮抑制子宫内膜增生的机制。方法:37例子宫内膜增生症(不伴非典型增生)患者,连续口服米非司酮(剂量10mg/d)治疗3个月,刮取治疗前、后子宫内膜组织,采用免疫组织化学二步法分别检测其ER和AR的表达进行自身对比;另取10例正常增殖期子宫内膜标本作为正常对照组进行比较。结果:子宫内膜增生症治疗前ER表达、AR表达均显著高于正常对照组(P<0.05),ER在腺体和间质细胞中均有表达,AR主要表达于间质细胞,腺体中几乎无表达。米非司酮治疗后腺体和间质ER表达比治疗前均下降(P<0.05),而间质和腺体中AR表达均比治疗前增加(P<0.05)。结论:米非司酮可通过下调ER和上调AR,从而抑制子宫内膜增生。     

1α,25-二羟基维生素D_3对绒毛膜癌细胞株JAR的影响及其机制

目的:研究生物制剂1α,25-二羟基维生素D3(calcitriol)对绒毛膜癌细胞株JAR的影响,探讨其作用机制。方法:采用四甲基偶氮唑蓝(MTT)比色法检测不同浓度(10-8、10-7、10-6mol/L)calcitriol在不同的时间点(1天、3天、6天)对JAR细胞增殖的影响;流式细胞仪检测calcitriol对JAR细胞周期的影响;RT-PCR检测维生素D受体(VDR)mRNA水平的表达;Western blot检测VDR蛋白表达。结果:MTT比色法检测10-6mol/Lcalcitriol作用JAR细胞3天,10-7、10-6mol/L calcitriol作用6天抑制率分别为(22.74±12.08)%(P<0.05),(29.64±8.66)%(P<0.01),(49.74±17.42)%(P<0.01),呈明显的时间-剂量效应关系。calcitriol作用JAR细胞的半数抑制浓度(IC50)组与对照组比较,细胞周期G1期细胞比例从39.78%增至59.81%(P<0.01),S期细胞数百分比从54.6%减至33.08%(P<0.01)。calcitriol上调JAR细胞的VDR mRNA及蛋白表达水平。结论:calcitriol能明显抑制绒毛膜癌细胞株JAR增殖,并诱导JAR分化,其作用机制与calcitriol上调VDR mRNA,进而增加VDR蛋白的表达有关。     

米非司酮联合宫瘤泄IFI池II=I疗子宫肌瘤临床观察

目的：观察米非司酮和宫瘤消对子宫肌瘤患者的疗效。方法：治疗组：口服米非司酮＋宫瘤消胶囊,观察组：口服米非司酮,治疗三个月。结果：米非司酮联合宫瘤消治疗效果明显高于单用米非司酮,其差异显著意义（P〈0．01）。结论：米非司酮联合宫瘤消是保守治疗子宫肌瘤可行方法。     

Human trophoblast cells express the immunomodulator progesterone-induced blocking factor

Progesterone-induced blocking factor (PIBF) is an immunomoduatory factor with anti-abortive properties. In this study, we present evidence that PIBF is synthesized in the human placenta and determine its cellular source. Expression of PIBF was analysed with polyclonal rabbit anti-human PIBF antibodies against recombinant N-terminal 48kDa PIBF in first trimester and term placental tissues and in the choriocarcinoma cell line JAR by means of immunohistochemistry, confocal laser scanning microscopy of double immunofluorescence labelling, and Western blotting; RT-PCR was performed for analysis of PIBF mRNA in isolated trophoblast cells. PIBF protein is present in human first trimester and term placenta. Double immunofluorescence labelling localised PIBF to the extravillous cytotrophoblast. PIBF is also expressed heterogeneously by syncytiotrophoblast and part of the villous cytotrophoblast. Full-length PIBF mRNA encoded by exons 1-18 is present in isolated first trimester and term villous trophoblast and in the choriocarcinoma cell line JAR. The corresponding 90kDa protein is expressed by JAR cells, first trimester and term villous trophoblast cells. In addition, these cells express PIBF proteins of 50 and 34kDa. Trophoblast is a source of PIBF; its tissue distribution suggests a role both in systemic and local (decidual) immunoregulation.