A unifying concept of trophoblastic differentiation and malignancy defined by biomarker expression

Several trophoblast phenotypes, including cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast, emerge during gestation. To clarify the lineage relationship between these subtypes, we profiled p63 localization in developing and term placental tissue, as well as in trophoblastic tumors, using antibodies specific to full-length (TAp63) and one against all p63 isoforms (TAp63 and DeltaNp63). Localization of p63 was compared with that of biomarkers of proliferation and trophoblastic differentiation, including mib-1, inhibin, and MelCAM. In early gestation, p63 was localized principally to villous cytotrophoblast after contact with the villous mesenchyme, absent in the trophoblast columns, and early implantation trophoblast. In the maturing placenta, intraplacental perivillous fibrin correlated with the emergence of a p63-positive "transitional" (vacuolated) extravillous trophoblast from cytotrophoblast, which differentiated further into a "mature" p63-negative extravillous trophoblast. The same lineage pathway emerged from entrapped villi on the chorionic membrane. Virtually all p63 immunopositivity was attributed to dominant-negative p63. The immunophenotypic patterns seen in the immature and mature placenta permit the resolution of all trophoblastic phenotypes within 3 lineage pathways of cytotrophoblast differentiation, including cytotrophoblast-to-trophoblast column/implantation site, cytotrophoblast-to-syncytiotrophoblast, and cytotrophoblast-to-mature extravillous trophoblast. In the latter pathway, a transitional (vacuolated) p63-positive extravillous trophoblast emerges from and links cytotrophoblast to mature extravillous trophoblast in intraplacental fibrin, chorionic membrane, and basal plate. The placental trophoblast is thus resolved within this continuum of differentiation. Terms such as transitional and mature extravillous trophoblast are proposed to reflect the differentiation phases of this unique epithelium. p63 staining patterns in trophoblastic tumors reflect timing of neoplastic transformation during trophoblastic differentiation.     

Distribution of human endogenous retrovirus type W receptor in normal human villous placenta

BACKGROUND: The fusion of trophoblast cells into the villous syncytiotrophoblast is crucial for appropriate placental function and fetal development. Fusion occurs following the interaction of syncytin-1, an envelope protein of the endogenous retrovirus HERV-W, and the RD114/mammalian type D retrovirus receptor (RDR/ASCT2) on adjacent cell membranes. This process must be tightly regulated in order to maintain the proliferative pool of cytotrophoblast cells as well as the function of the syncytia. AIM: We sought to investigate whether syncytial fusion of placental cytotrophoblast cells may be regulated via modulation of RDR/ASCT2 expression. METHODS: Expression of RDR/ASCT2 in term and first trimester villous placenta was assessed along with a number of molecular markers using immunofluorescent staining. In a complementary approach, Western blotting was used to investigate RDR/ASCT2 expression in a panel of choriocarcinoma cell lines before and after stimulation of fusion. RESULTS: Villous placental RDR/ASCT2 expression was found to be restricted to the cytotrophoblast compartment, being largely absent in the syncytiotrophoblast. Local variations in RDR/ASCT2 expression were not associated with the proliferative status of cytotrophoblast cells. RDR/ASCT2 expression was also shown to be down-regulated in BeWo choriocarcinoma cells after stimulation of syncytial fusion. CONCLUSION: This first report of the localisation and distribution of RDR/ASCT2 in human placental villi suggests that the fusion of placental trophoblast cells is not regulated by local or temporal variations of RDR/ASCT2 expression in villous cytotrophoblast cells.     

人胎盘滋养层细胞的分离培养及IgG FcγRⅢ在滋养层细胞的表达

目的：体外分离培养人胎盘滋养层细胞，观察其生物学特性，研究IgGFcγRⅢ（CD16）在人胎盘组织中的定位及体外培养的滋养层细胞是否存在CD16，从而为HCV母婴传播机制的研究提供细胞学实验基础。方法：采用胰蛋白酶消化法消化人足月胎盘组织，以35%、45%两个Percoll密度梯度进行分离纯化，用ABC免疫组化染色法对足月分娩后的胎盘组织石蜡包埋切片及体外分离培养的胎盘滋养层细胞进行染色。结果：胎盘组织中滋养层细胞角蛋白染色阳性，血管内皮细胞及基质成分波形蛋白染色阳性，经该法分离纯化的细胞角蛋白染色阳性者（滋养层细胞（占90%以上，胎盘组织切片的滋养层细胞及体外分离培养的滋养层细胞抗CD16染色阳性，阳性信号定位于胞质，未见胞核着色，结论：改进后的分离方法可能得到较高纯度的滋养层细胞，胎盘组织的滋养层细胞和体外分离培养的滋养层细胞均有CD16表达。     

Rho激酶抑制剂Y-27632对细胞滋养细胞侵袭能力的研究

目的 观察Rho激酶特异抑制剂Y-27632对体外培养的细胞滋养细胞侵袭能力的影响。方法2006年3月至8月在中国医科大学附属第二医院收集妊娠6～8周行人工流产术的健康妇女绒毛组织，通过免疫组化法检测细胞滋养细胞中RhoA和ROCKⅡ蛋白的表达；利用原代细胞培养技术，采用MTT、Transwell体外浸润实验和细胞运动实验检测Rho激酶抑制荆Y-27632对细胞滋养细胞生长和浸润运动能力的影响。结果在细胞滋养细胞有RhoA和ROCKⅡ蛋白的表达，原代培养的细胞滋养细胞经Y-27632处理后，侵袭及运动能力明显下降。结论Rho／Rho激酶信号转导系统对细胞滋养细胞侵袭有重要作用．可能参与由胎盘植入异常引起的妊娠相关疾病。     

内毒素调节细胞滋养细胞侵入能力的机制

目的:探讨内毒素(lipopolysacchraride,LPS)对细胞滋养细胞侵入能力的影响。方法:留取正常早孕绒毛,分离细胞滋养细胞,用无血清培养基培养。对照组:培养基中不添加内毒素。实验组:培养基中加入不同浓度的内毒素,其终浓度分别为25、50、100、200ng/m l。Transwell小室检测细胞滋养细胞的侵入能力;采用激光共聚焦-免疫荧光技术检测基质金属蛋白酶-2、9(MMP-2、9)蛋白的表达;RT-PCR检测MMP-2、MMP-9 mR-NA表达水平。结果:内毒素降低细胞滋养细胞侵入Transwell小室的能力,在浓度为0、25、50、100、200ng/m l的LPS作用24h后,细胞滋养细胞侵至滤膜下表面的细胞为145.6±20.7、139.6±18.8、123.1±17、76.5±18、47.9±16,差异有统计学意义(P<0.01);内毒素显著抑制细胞滋养细胞MMP-2、MMP-9蛋白和mRNA的表达。结论:内毒素能够抑制细胞滋养细胞的侵入能力,可能是通过抑制基质金属蛋白酶表达来实现。     

MMP-2和MMP-9在早孕胎盘滋养细胞中的表达及意义

目的探讨MMP-2和MMP-9在早孕胎盘滋养细胞中的表达及意义。方法本研究通过免疫组织化学技术检测早孕早期和早孕晚期绒毛组织中MMP-2和MMP-9蛋白的表达。结果MMP-2和MMP-9蛋白主要定位在绒毛细胞滋养细胞和合体细胞滋养细胞胞浆内，绒毛间质细胞内亦有少量阳性染色，且旱孕早期绒毛滋养细胞中MMP-2表达量高于早孕晚期（P〈0．05），而MMP-9表达量在早孕晚期高于早期（P〈0．05）。结论MMP-2和MMP-9在调节滋养细胞浸润过程中起重要作用，且在早孕不同时期，二者表达不同，为深入探讨MMPs在滋养细胞浸润机制中的作用提供理论基础。     

5一羟色胺对人胎盘滋养细胞L-型钙通道的影响

目的探讨5一羟色胺对胎盘滋养细胞钙通道的影响。方法将体外培养的胎盘滋养细胞分为对照组和低（1．01xmol／L）、高（10．0μmol／L）浓度5一羟色胺组。采用全细胞膜片钳实验方法,在待测胎盘滋养细胞外液中分别加入Nimodpine（10μmol／L）、Flunarizne（10μmol／L）和替曲朵辛（Trxl01．Lmol／L）,进行电流检测;同样的实验条件和方法,在待测胎盘滋养细胞外液中分别加入5-TH（1μmol／L和101xmol／L）,测试电流值;在细胞外液中分别加入5-TH（μmol／L）和Nimodpine（10μmol／L）、5-TH（10μmol／L）和Nimodpine（10μmol／L）后再检测电流。结果胎盘滋养细胞L-型钙通道开放在一60--50mV之间,最大峰值电流在一40～30mV为82．31±6．46（pA）。加入Nimodpine（10μmol／L）。可阻断此电流大部分成份。Flunarizne（10Ixmol／L）和’兀X（10肛m0ⅣL）不能抑制该电流成份。加入5-TH（1μmol/L和10μmol／L）峰值电流明分别为104．77±10．84（pA）和117．16±9．67（pA）,与对照组相比有显著差异（P〈0．05）;加入5-TH（1μmo]／L）和Nimodpine（101xmol／L）、5一TH（10μmol／L）和Nimodpine（10μmol／L）后;峰值电流分别为85．35±6．54（pA）、89．42±7．62（pA）,与对照组相比无显著差异（P〉0．05）。结论5-TH可激动胎盘滋养细胞L-型钙通道,促使钙电流值增加。     

The biological function of hyperglycosylated hCG

ObjectiveHyperglycosylated human chorionic gonadotropin (hCG) is a variant of hCG made by cytotrophoblast cell. Here we examine the role of hyperglycosylated hCG in placenta growth and invasion.MethodsJEG-3 choriocarcinoma cells and term cytotrophoblast monolayer culture were prepared. The effect of supplemental hyperglycosylated hCG and hCG was investigated. Growth of these cells was examined by increase in cell number. Invasion was investigated using Matrigel basement membrane cells. The proportion of cell invading Matrigel was determined.ResultsTerm cytotrophoblast cell and JEG-3 choriocarcinoma cells grew to 5 427±834 cells (109%) and 7 114±553 cells (142%). With the supplementation of hyperglycosylated hCG, they grew significantly wider to 7 633±177 cells (142%) and 10 315±1 477 cells (206%). With the supplementation of hCG they diminished to 4 227±769 cells (78%) and 5 620±657 cells (79%). Term cytotrophoblast cell and JEG-3 choriocarcinoma cells penetrated Matigel membranes to (40.0±10.0)% and (46.0±9.8)%. Hyperglycosylated hCG significantly enhanced penetration to (76.0±13.0)% and (84.0±6.6)%. hCG diminished penetration to (32.0±9.1)% and (32.0±4.5)%.ConclusionsHyperglycosylated hCG enhances both cytotrophoblast growth and cytotrophoblast cell invasion. hCG minimally suppresses growth and invasion.     

氧化低密度脂蛋白及其受体对滋养细胞凋亡的影响

目的:探讨氧化低密度脂蛋白(oxLDL)及血凝集素样氧化低密度脂蛋白受体1(LOX-1)对滋养细胞凋亡的影响。方法:采用蛋白印迹法检测滋养细胞LOX-1蛋白的表达;RT-PCR技术检测LOX-1mRNA及caspase-3mRNA的表达;流式细胞仪检测滋养细胞凋亡指数。结果:不同浓度oxLDL(0.01,0.05,0.10mg/ml)培养下细胞滋养细胞中LOX-1蛋白、mRNA及caspase-3mRNA表达水平均明显高于对照组(P<0.01),各组间比较差异均有统计学意义(P<0.01)。拮抗剂组LOX-1蛋白、mRNA及caspase-3mRNA表达水平均显著低于同浓度oxLDL刺激组,差异亦有统计学意义(P<0.01)。结论:oxLDL可通过上调滋养细胞中LOX-1表达引起滋养细胞的过度凋亡。