缺氧对滋养细胞VEGF分泌与基因表达调控作用的研究

目的:研究原代培养人早孕绒毛滋养细胞VEGF分泌、基因表达的特点,以及缺氧的调控作用。方法:原代培养早孕绒毛滋养细胞,研究正常和缺氧(氯化钴化学诱导)条件下细胞培养上清液中VEGF含量(ELISA法)以及滋养细胞VEGF mRNA表达(RT-PCR方法)特点。结果:(1)免疫组化技术证实培养细胞角蛋白阳性,波形蛋白阴性,纯度达95%以上;(2)正常培养的滋养细胞在培养12h后分泌产生的VEGF明显增加,至培养结束时的72h达到最高点;缺氧诱导滋养细胞大量分泌产生VEGF的时间提前至缺氧培养后6h,持续至72h无下降,VEGF水平明显高于正常,最高达到2200ng/ml,较正常上升47%;(3)滋养细胞VEGF mRNA表达于培养12h时开始明显上升,至48h时达峰值;缺氧则使VEGF mRNA表达显著上升的时间提前至6h,约12h达峰值。结论:早孕绒毛滋养细胞中存在VEGF基因转录并分泌至细胞外;缺氧诱导VEGF转录增强、分泌增加。     

VEGF及受体flk-1在子宫内膜异位症中的表达及相关研究

目的 检测血管内皮生长因子（VEGF）及其受体flk-1在子宫内膜异位症在位内膜、异位内膜中的表达，并探讨它们在子宫内膜异位症发生发展中作用。方法 利用免疫组化的方法及Western blot检测了26例正常人和31例子宫内膜异位症患者的在位内膜、异位内膜中分泌期及增殖期VEGF及其受体flk-1的表达。结果 ①VEGF在子宫内膜异位症异位内膜中呈高表达状态，正常子宫内膜、异位症在位内膜、异位内膜VEGF的表达强度依次增强，以异位内膜分泌期VEGF表达强度最高；②VEGF主要表达于子宫内膜的腺上皮细胞浆中，散在在间质中。月经周期中VEGF在子宫内膜异位症在位内膜、异位内膜腺上皮细胞中均呈周期性变化，分泌期强于增殖期，异位症内膜间质中VEGF表达无周期性变化，且与正常子宫内膜相比无显著性差别；③受体flk-1表达于异位内膜间质血管内皮细胞。异位内膜问质中血管内皮细胞表达强度增强，微血管密度（MVD）增多，腺上皮VEGF的表达与问质中的MVD呈正相关关系。结论 VEGF在子宫内膜异位症在位内膜、异位内膜腺上皮中的表达增强，间质中血管生成活动增强，血管生成可能是子宫内膜异位症发生发展的重要因素。     

肝细胞生长因子对卵巢癌血管生成因子的影响及信号传导途径

目的研究肝细胞生长因子(HGF)对人卵巢癌SKOV3细胞血管内皮生长因子的影响及其信号传导机制。方法将不同浓度、不同作用时间的HGF和核转录因子(NFkB)抑制剂PDTC刺激培养的SKOV3细胞;应用逆转录-聚合酶链反应技术(RT-PCR)检测卵巢癌细胞血管内皮生长因子(VEGF)mRNA的变化;采用蛋白印迹方法检测卵巢癌细胞VEGF蛋白和核蛋白NFkB的变化。结果HGF促进卵巢癌细胞VEGF mRNA和蛋白的表达,且随时间和浓度增加作用增强,PDTC可以抑制HGF的刺激作用;HGF促进细胞核NFkB蛋白的活化,且随时间延长作用增强,于刺激1 h达高峰,PDTC可以抑制HGF对NFkB的活化作用。结论HGF通过NFkB途径在核酸和蛋白水平调节卵巢癌细胞分泌VEGF。     

γ-干扰素对卵巢癌细胞株血管内皮生长因子表达的影响

目的　研究γ 干扰素 (IFN γ)对人卵巢癌细胞株SKOV3细胞血管内皮生长因子 (VEGF)表达的影响。方法　将IFN γ以不同浓度 (1、10、10 0、10 0 0、10 0 0 0U/ml)、不同作用时间 (12、2 4、3 6、48、60h)作用于SKOV3细胞 ,在相差显微镜下观察细胞形态的变化 ;用四甲基偶氮唑蓝 (MTT)比色法测定细胞增殖 ;用逆转录聚合酶链反应 (RT PCR)半定量法测定细胞VEGFmRNA含量 ;用双抗体夹心酶联免疫吸附试验 (ELISA)和免疫细胞化学方法测定细胞培养上清液和细胞浆中VEGF蛋白含量。结果　 (1) 10 0 0U/mlIFN γ作用后SKOV3细胞形态无明显变化。 (2 )不同浓度 (1、10、10 0、10 0 0、10 0 0 0U/ml)IFN γ作用不同时间 (12、2 4、3 6、48、60h)后 ,SKOV3细胞增殖活性比较 ,差异无显著性 (P>0 0 5 )。 (3 )IFN γ作用后 ,SKOV3细胞VEGFmRNA和蛋白含量明显减少 (P <0 0 1)。IFN γ浓度达到 10U/ml后起作用 [抑制率为 (5 2± 0 6) % ],其后随着浓度的增高 ,IFN γ对VEGFmRNA的抑制作用增强 ,在浓度达到 10 0 0U/ml时 ,抑制作用达到高峰 [抑制率为 (2 1 6± 2 6) % ]。但IFN γ的抑制作用并不存在时间依赖关系 ,用药 2 4h后出现抑制作用 ,并立即达到高峰 [抑制率为 (2 2 4± 1 7) % ],其后抑制作用不随时间的延长而     

Vascular endothelial growth factor is a chemoattractant for trophoblast cells

A role for angiogenic growth factors in trophoblast invasion has been postulated. Directional motility (chemotaxis) is an important function of trophoblast cells. We have previously shown that vascular endothelial growth factor (VEGF) increases the random movement of trophoblast cells although placental growth factor (PlGF) has no effect. Heparin inhibited this effect of VEGF. Motility of trophoblast cells has been proposed to be mediated by a nitric oxide (NO) pathway. We hypothesized that VEGF but not PlGF would be chemotactic for trophoblast cells. Chemotaxis of a first trimester extravillous trophoblast cell line, SGHPL-4, and primary isolates of first trimester and term trophoblast cells was measured using a Boyden chamber. Initial experiments to optimize the time of the experiment and identify a positive control were performed. Subsequent experiments ran for 20 h, used 0.5 per cent FBS or 10 ng/ml PDGF as negative and positive controls and were performed in triplicate. VEGF (1, 10 and 100 ng/ml+/-1 microg/ml heparin or +/-100 microM L-NAME) and PlGF (1, 10, 100 ng/ml) were tested. The chamber was placed in a 5 per cent CO(2) in air, 37 degrees C incubator. The number of cells in the lower chamber were counted. There was a dose dependent increase in chemotactic motility of the SGHPL-4 cell line and term trophoblast cells in response to VEGF. PlGF had no effect on the movement of the first trimester trophoblast cell line but did increase the motility of the term trophoblast cells in a dose dependent manner. Heparin increased the cellular motility of both cell types alone. It also further enhanced the chemoactivity of VEGF on the term trophoblast cells but not the cell line. L-NAME did not affect the VEGF-stimulated motility of the first trimester cell line. However, in the term trophoblast cells L-NAME increased the directional cellular motility in the absence of, or in the presence of low concentrations of VEGF. In conclusion, the first trimester and term trophoblast cells appeared to respond differently to the various factors tested in the present study that may reflect differential cellular function as gestation progresses.     

Use of Matrigel in culture affects cell phenotype and gene expression in the first trimester trophoblast cell line HTR8/SVneo

There is inconsistent use of Matrigel for experiments with the HTR8/SVneo first trimester trophoblast and other cell lines. We quantified the effects of Matrigel on the expression of genes considered to be markers of extravillous cytotrophoblast (EVT) differentiation and invasive potential. Culture on Matrigel promoted formation of "endothelial-like" tubes and reduced mRNA expression of matrix metalloproteinase 2 (MMP2), cytokeratin 7 (KRT7) and integrin alpha 1 (ITGA1), while increasing VE-cadherin (CDH5) expression consistent with a vascular phenotype. This process may constitute part of the endothelial cell mimicry exhibited by endovascular EVTs invading the maternal spiral arteries. HTR8/SVneo appears to be phenotypically polymorphic and adopt endovascular morphology on Matrigel.     

miR-219a suppresses human trophoblast cell invasion and proliferation by targeting vascular endothelial growth factor receptor 2 (VEGFR2)

ObjectiveVascular endothelial growth factor (VEGF) plays a critical role in regulating trophoblast cell invasion and proliferation, involved in a variety of pregnancy complications, such as spontaneous abortion and pre-eclampsia. Numerous studies have revealed that microRNAs (miRNAs) are participated in a series of molecular processes that regulate cell function, such as cell invasion, proliferation, and apoptosis. Vascular endothelial growth factor receptor 2 (VEGFR2), a receptor of VEGF, has been shown to be involved in trophoblast function. However, the relation between miRNA and VEGFR2 and their role in trophoblast function remain to be elucidated.MethodsThe effect of miR-219a on the trophoblast function has been explored using luciferase reporter, transwell, qRT-PCR, western blot, bromodeoxyuridine (BrdU), ELISA, immunofluorescent staining, and tube formation assays.ResultsIn the current study, we observed that through targeted inhibition of VEGFR2 expression by miR-219a, the function of VEGFR2 as well as the downstream PI3K/AKT/NF-κB signaling pathway were suppressed, leading to suppression of trophoblastic proliferation and invasion. Moreover, upregulation of VEGFR2 restored the miR-219a–inhibited cell proliferation, invasion, and tube formation.ConclusionsThese results revealed that miR-219a played crucial roles in negatively regulating trophoblastic proliferation and invasion by suppression of the PI3K/AKT/NF-κB signaling pathway by targeting VEGFR2, therefore serving as a potential treatment method for the complications of pregnancy caused by trophoblastic dysregulation.     

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O₂) yet PIGF expression decreased 73 ± 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of functional flt-1 on normal trophoblast suggests that VEGF/P1GF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.     

Expression of serum amyloid A4 in human trophoblast-like choriocarcinoma cell lines and human first trimester/term trophoblast cells

Trophoblast invasion into uterine tissues represents a hallmark of first trimester placental development. As expression of serum amyloid A4 (SAA4) occurs in tumorigenic and invasive tissues we here investigated whether SAA4 is present in trophoblast-like human AC1-M59/Jeg-3 cells and trophoblast preparations of human first trimester and term placenta. SAA4 mRNA was expressed in non-stimulated and cytokine-treated AC1-M59/Jeg-3 cells. In purified trophoblast cells SAA4 mRNA expression was upregulated at weeks 10 and 12 of pregnancy. Western-blot and immunohistochemical staining of first trimester placental tissue revealed pronounced SAA4 expression in invasive trophoblast cells indicating a potential role of SAA4 during invasion.     

子宫腺肌病患者在位内膜中KDR表达的研究

目的 探讨子宫腺肌病（腺肌病）患者在位子宫内膜中血管内皮生长因子受体（VEGFR）KDR的表达及意义。方法 采用免疫组化（SABC法）方法，检测11例腺肌病患者在位子宫内膜组织（腺肌病组）及30例非内异症患者的正常子宫内膜组织（正常内膜组）中VEGF受体KDR的表达差异。结果 （1）腺肌病在位内膜及正常内膜KDR受体阳性表达率分别为81．81％、76．67％。（2）腺肌病在位内膜腺上皮细胞在增生期、分泌期阳性表达强度分别为（＋＋）和（＋＋＋），较正常内膜组相应时期的（＋～＋＋）和（＋＋～＋＋＋）有一定增高。结论 腺肌病组中KDR受体的明显表达可能与KDR受体参与腺肌病血管生成，促使子宫内膜侵入基层生长有关；也提示基底层子宫内膜侵入基层与卵巢激素相关。     

Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro.

OBJECTIVE: The object of this study was to determine the effect of epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) on the differentiation of first-trimester and term cytotrophoblasts. METHODS: The first-trimester trophoblasts were isolated from villous tissue obtained at suction termination (n = 5), and the term trophoblasts were isolated from placentas (n = 6) at elective cesarean. Cultured cells were stimulated with EGF, VEGF, or PlGF at 0.5, 5, and 50 ng/mL, in the presence or absence of N(G)-nitro-L-arginine methyl ester hydrochloride (10(-4) M). Syncytialized trophoblasts were identified by immunostaining with antidesmosomal protein and anti-cytokeratin-7, whereas nuclei were counted in each syncytia using hematoxylin. RESULTS: Without treatment, background levels of syncytialization were significantly higher in term preparations than first-trimester cells. With VEGF and EGF, the number and size of syncytia increased significantly for the first-trimester cytotrophoblasts (P <.05). Neither VEGF nor EGF had any effect on the syncytialization of cultured cells at term. Nitric oxide showed no involvement in syncytial induction, and PlGF had no effect on syncytialization of cytotrophoblasts, from either the first or third trimester. CONCLUSION: Both EGF and VEGF appeared to enhance the in vitro syncytialization of first trimester cytotrophoblasts.     

血管内皮生长因子在异位和在位子宫内膜中的表达

目的研究血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)在子宫内膜异位症(endometriosis,EM)患者的异位和在位内膜中的表达。方法用免疫组化SP法分别检测45例EM患者(研究组)的异位和在位内膜及32例子宫肌瘤(对照组)的增殖期和分泌期内膜中VEGF的表达强度。结果在整个月经周期中,研究组异位和在位内膜中VEGF表达均持续高于对照组(P<0.01)。结论VEGF在EM患者异位和在位内膜中的持续过度表达可能与EM的发生发展有关。在位内膜VEGF高表达可望成为诊断EM的新方法。     

Glycodelin A and differentiation of first trimester trophoblast cells in vitro

Aim The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro.Methods Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry.Results Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid.Conclusions The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.