Hyaluronan up-regulates growth and invasion of trophoblasts in an autocrine manner via PI3K/AKT and MAPK/ERK1/2 pathways in early human pregnancy

Introduction

As one of the key molecules in the extracellular matrix in human conceptus, hyaluronan (HA) has been receiving particular attention. Here, we have investigated the expression and regulation of different molecular weight HA on the biological behaviors of primary human trophoblasts during the first trimester of pregnancy.

Methods

The expression of HA and HA synthetase (HAS) by human first trimester trophoblasts was analyzed in placentae from normal pregnancy or miscarriage by immunochemistry and real-time RT-PCR, respectively. ELISA was used to measure the secretion of HA by primary trophoblasts. The effects of HA on the proliferation, apoptosis and invasiveness of trophoblasts were examined. We also investigated the signaling pathways involved in HA activation in human trophoblasts.

Results

The higher HAS2 expression and HA secretion were observed in normal villi than that of miscarriage, and the primary trophoblasts secreted HA continuously. High molecular weight HA (HMW-HA) and medium molecular weight HA (MMW-HA) promoted proliferation and invasiveness while inhibited apoptosis of trophoblasts. However, low molecular weight HA (LMW-HA) had no obvious effect on the growth or invasiveness of human trophoblasts. In addition, HMW-HA showed more efficiently than MMW-HA on the growth while MMW-HA displayed a more obvious effect on the invasiveness of trophoblasts than HMW-HA. HMW-HA activated PI3K/AKT and MAPK/ERK1/2 signaling pathways in trophoblasts. Blocking PI3K/AKT or MAPK/ERK1/2 signaling inhibited the HA-upregulated growth and invasiveness of human trophoblasts.

Conclusion

Our results suggest that higher level and greater molecular mass of HA can promote trophoblast growth and invasion in an autocrine manner, which was beneficial to placentation and maintenance of human early pregnancy.     

TLR3配体polyI：C经TRIF／NF—KB途径抑制Bewo细胞中HBV复制

摘要：目的探讨T0Ⅱ样受体3（TLR3）配体聚肌苷酸胞苷酸（polyI：C）抑制人滋养层细胞Bewo中乙型肝炎病毒（HBV）复制的作用机制。方法首先将2斗g1．3倍HBV全基因重组质粒pcDNA3．1（＋）-HBVl．3转染Bewo细胞,12h后,以TLR3配体polyI：C处理3d。然后以polyi：C处理Bewo细胞,观察IFN-B、TNF_d表达的动力学。最后,观察核因子-KB（NF-KB）拈抗剂PDTC对polyI：C诱导Bewo细胞产生细胞因子的作用。采用微粒子酶免疫分析法（MEIA）和荧光定量PCR法分别检测HBsAg、HBeAg和HBVDNA水平,并以ELISA和RT-PCR分别检测IFN-B、TNF-Ⅸ水平及TIR结构域的转接蛋白（TRIF）、髓样分化蛋白88（MyD88）表达。结果与对照组比较,polyIC可显著抑制Bewo细胞中HBV复制,差异有统计学意义（P〈O．01）,且polyI：C可显著诱导Bewo细胞产生IFN-p和TNF_d（P〈O．05）,呈时间和剂量依赖性。PDTC可抑制polyhC诱导细胞产生TNF_Ot,显著低于对照组（P〈0．01）,但对IFN-B无显著作用（乃0D5）。与对照组比较,polyI：c可诱导HBV重组质粒转染的Bewo细胞表达TRIFmRNA（P〈O．01）。结论TLR3配体poyZ：C可能通过rrRIF依赖性途径诱导Bewo细胞产生IFN-B和TNF-d,且TNF-a产生还涉及NF-KB途径,并最终抑制Bewo细胞中HBV复制。     

超声断层显像技术及经阴经腹超声检查结合血HCG对异位妊娠诊断的价值探讨

目的 探讨经阴经腹彩色多普勒超声(彩超)及超声断层显像技术(TUI)结合血人绒毛促性腺激素(HCG)检测诊断异位妊娠(EP)的特征性依据,提高EP诊断的准确性.方法 对188例疑似早期EP者测定血HCG,进行二维经阴经腹彩超及TUI检查,测定子宫内膜厚度及血流分布,测量子宫动脉及滋养动脉血流频谱,并进行比较.结果 188例经阴经腹彩超诊断EP患者中,经手术病例证实EP 183例,超声诊断符合率为97.3%.其超声声像图特征为:(1)附件区妊娠囊内可见胎儿或者卵黄囊,伴或不伴胎心搏动,183例患者中有17例(9.0%)有胎心搏动;(2)Donut征(输卵管环),本组患者中98例(52.1%)有Donut征;(3)附件区内可探及混合性包块,有时伴有少量积液,本组60例(31.9%)患者可探及混合性包块;(4)8例(4.3%)患者仅表现为盆腔积液.血HCG平均为(3 320±2 342)mU/ml.结论 TUI及经阴经腹超声检查能准确迅速地取得EP的证据,结合血HCG检测是早期诊断EP的较好手段;TUI能提供多断面显示,可减少小病灶的漏诊.     

合体细胞滋养层细胞外囊泡阻止母体恶性肿瘤侵袭及转移至胎儿相关机制研究

目的探讨合体细胞滋养层细胞外囊泡(STB-EV)阻止母体恶性肿瘤侵袭、转移至胎儿的相关机制。 方法选择2015年6月,于四川大学华西第二医院进行剖宫产术分娩的1例妊娠合并宫颈癌(CCP)患者被肿瘤侵犯的胎盘组织为研究对象。宫颈癌细胞系SiHa细胞和人滋养层细胞系HTR-8细胞,均由本院西部妇幼医学研究院-分子与转化医学实验室馈赠。其中,本例CCP患者被肿瘤侵犯的胎盘组织制作组织切片后,于光学显微镜(×400)和电子显微镜(×2 000)下,观察其组织形态学、STB-EV及合体细胞滋养层(STB)旁宫颈癌细胞自噬性死亡情况等;而SiHa细胞与HTR-8细胞培养后,用于Transwell迁移实验与细胞划痕实验。将SiHa与HTR-8细胞共培养,纳入研究组;单独培养的SiHa细胞,纳入对照组。对2组细胞进行Transwell迁移实验及细胞划痕实验,2组Transwell小室穿膜细胞数、细胞迁移率等比较,采用成组t检验。本研究遵循的程序符合2013年新修订的《世界医学协会赫尔辛基宣言》要求。 结果①对本例CCP患者被肿瘤侵犯的胎盘组织制作组织切片后,于光学显微镜(×400)下观察结果显示,与STB相邻宫颈癌细胞自噬性死亡、凋亡和固缩性坏死显著。电子显微镜(×2 000)下观察结果显示,宫颈癌细胞累及胎盘绒毛组织时,STB分泌的STB-EV水平显著高于正常胎盘组织。②2组细胞Transwell迁移实验结果显示,研究组穿膜细胞数为(597.6±87.7)个/视野,显著低于对照组的(1 358.4±203.0)个/视野,并且差异有统计学意义(t＝14.490、P<0.001)。③2组细胞划痕实验结果显示,划痕后48 h时,研究组细胞迁移率为(26.6±3.8)%,显著低于对照组的(45.9±3.7)%,并且差异有统计学意义(t＝3.122、P＝0.035)。 结论STB分泌的STB-EV可能参与母体肿瘤细胞广泛自噬性死亡,从而阻止母体肿瘤细胞侵袭胎盘、影响胎儿发育。     

缺氧对滋养细胞Notch信号通路的影响

目的探讨缺氧条件下Notch信号通路在滋养细胞中的表达改变。方法利用实时荧光定量PCR(RT-qPCR)技术检测二氯化钴(CoCl2)化学缺氧条件下人早孕绒毛滋养细胞株(the human first-trimester extravillous tropho-blast cell line,TEV-1)中Notch1及Jagged1 mRNA的表达情况。结果 (1)与正常对照组相比,缺氧条件下滋养细胞Notch1 mRNA表达量无明显变化(P>0.05)。(2)随着缺氧时间的延长,滋养细胞Jagged1 mRNA表达量逐渐降低,与正常对照组相比具有统计学意义(P<0.05)。结论缺氧条件下滋养细胞Notch1及Jagged1表达平衡失调,可能参与滋养细胞功能调控。     

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.     

Autophagy protects against oxidized low density lipoprotein-mediated inflammation associated with preeclampsia

IntroductionInflammatory responses play an important role in the pathogenesis of preeclampsia. Recently, the anti-inflammatory role played by autophagy has drawn increasing attention. Our aim was to investigate variations in autophagy in preeclampsia and protection against oxidized low-density lipoprotein (oxLDL)-mediated inflammation by autophagy.MethodsWe used immunohistochemistry, immunofluorescence, quantitative real-time PCR, and western blotting to analyze the expression of autophagy proteins (beclin-1 and LC3II/LC3I) in preeclampsia placentas and in JEG-3 cells treated with oxLDL and rapamycin.ResultsWe found a decreased level of autophagy proteins in preeclampsia placentas, and oxLDL did not induce autophagy in JEG-3 cells. Furthermore, when cells were pretreated with rapamycin, autophagy was activated and expression of inflammatory factors (tumor necrosis factor-α and interleukin-6) induced by oxLDL was downregulated.ConclusionWe conclude that impaired autophagy in preeclampsia has potential to decrease trophoblast protection from oxidative and inflammatory stress, thereby contributing to the pathogenesis of preeclampsia.