Wnt2在植入胎盘组织中的表达及其对滋养细胞迁移和侵袭功能的影响

目的检测Wnt2在植入胎盘组织中的表达,分析Wnt2对滋养细胞凋亡、迁移和侵袭功能的影响。方法收集2020年7月至2021年1月于武汉大学中南医院妇产科行剖宫产的胎盘植入孕产妇胎盘组织15例(植入组)和正常孕产妇胎盘组织15例(对照组)。RT-PCR法和Western blot法检测胎盘组织中Wnt2表达水平,采用siRNA敲低HTR-8/Svneo细胞中Wnt2表达水平,流式细胞学法检测细胞凋亡,Transwell侵袭和迁移实验评估细胞的迁移和侵袭能力,RT-PCR法和Western blot法检测β-catenin、Bcl-2、Caspase-3、MMP-2和MMP-9表达水平。结果与对照组相比,植入组胎盘组织中Wnt2、MMP-2和MMP-9 mRNA和蛋白表达量均显著升高(均P<0.05)。转染敲低HTR-8/Svneo细胞Wnt2水平后,细胞凋亡率显著增加(P<0.05),细胞迁移和侵袭能力显著抑制(P<0.01),β-catenin、Bcl-2、MMP-2和MMP-9表达显著下降(均P<0.05),而Caspase-3表达显著升高(P<0.05)。结论植入胎盘组织中Wnt2蛋白表达显著升高,且Wnt2可能通过wnt/β-catenin信号通路参与了胎盘滋养细胞的凋亡、迁移和侵袭。     

Capsid modifications overcome low heterogeneous expression of heparan sulfate proteoglycan that limits AAV2-mediated gene transfer and therapeutic efficacy in human ovarian carcinoma

OBJECTIVES: Capsid-modified AAV vectors can mediate enhanced gene transfer to neoplasms characterized by low AAV receptor expression. Here we sought to determine the therapeutic potential of a capsid-modified AAV vector for gene therapy of ovarian carcinoma (OvCa). METHODS: We tested a panel of OvCa cell lines for AAV2-mediated gene transduction and for sensitivity to ganciclovir (GCV) following AAVHSVtk administration. Levels of AAV internalization and attachment receptor were assessed by flow cytometry and immunohistochemistry. The role of receptors in AAV-mediated gene transfer was assessed by competition assays. Finally, we examined the ability of a modified vector with an integrin-binding RGD motif inserted into the AAV capsid to improve gene delivery to OvCa and enhance AAVHSVtk/GCV-mediated killing by cytotoxicity assay. RESULTS: All OvCa cell lines were poorly transduced with AAV2 vectors and showed variably sensitive to AAVHSVtk/GCV. While OvCa cell lines expressed AAV2 internalization receptors (alphav integrins), expression of the AAV2 attachment receptor, HSPG, was variable and not detected on many lines. Analysis of archived clinical specimens showed no detectable HSPG expression on approximately 45% of primary human tumors. Gene transfer to OvCa was increased several fold using the RGD-modified vector. Gene transfer was independent of HSPG and specific to the targeted receptor. Importantly, the RGD-modified capsid markedly increased the ability of the AAVHSVtk to kill OvCa cells in the presence of GCV. CONCLUSIONS: The development of AAV vectors targeted to cell surface receptors other than HSPG will be critical to the advancement of AAV-mediated gene therapy for treating OvCa.     

Novel isolation strategy to deliver pure fetal-origin and maternal-origin mesenchymal stem cell (MSC) populations from human term placenta

The placenta is an abundant source of mesenchymal stem/stromal cells (MSC). Although presumed of translationally-advantageous fetal origin, the literature instead suggests a high incidence of either contaminating or pure maternal MSC. Despite definitional criteria that MSC are CD34−, increasing evidence suggests that fetal MSC may be CD34 positive in vivo. We flow sorted term placental digests based on CD34+ expression and exploited differential culture media to isolate separately pure fetal and maternal MSC populations. This method has considerable translational implications, in particular to clinical trials underway with “placental” MSC of uncertain or decidual origin.     

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration

IntroductionFailure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function.MethodsA transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)₂D₃. QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression.ResultsEVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p < 0.05 versus S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH)₂D₃ for 48 or 72 h reduces S1PR2 (4-fold; <0.05), but not R1 and R3, expression. Moreover, S1P did not inhibit the migration of cells exposed to 1,25(OH)₂D₃ (p < 0.05).DiscussionThis study demonstrates that although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα_12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.     

补肾益气方激活ERK通路影响人早孕细胞滋养细胞生长

目的:研究补肾益气方对人早孕细胞滋养细胞增殖和早期凋亡的影响及其可能的信号转导通路。方法:体外分离培养人早孕细胞滋养细胞,用四甲基偶氮唑盐(MTT)比色法测定细胞的增殖活力,流式细胞术增殖细胞核抗原(PCNA)表达检测细胞增殖、AnnexinⅤFITCPI双染色法检测细胞早期凋亡,Western blot法检测细胞外信号调节蛋白激酶1/2(ERK1/2)磷酸化。结果:与20%对照血清组比较,10%及20%补肾益气方含药血清作用48h,上调细胞滋养细胞增殖活力,促进PCNA表达、降低早期凋亡细胞百分率、诱导细胞ERK磷酸化,上述指标均呈量-效关系;ERK通路的特异性抑制剂U0126几乎完全抑制20%含药血清诱导的细胞ERK1/2活化,下调20%含药血清诱导的细胞增殖活力及PCNA蛋白表达、增加早期凋亡细胞百分率。结论:补肾益气方对人早孕细胞滋养细胞的生长具有促进作用,ERK1/2信号通路可能是补肾益气方促进细胞滋养细胞增殖、减少细胞凋亡的主要信号转导通路之一。     

The proprotein convertase furin in human trophoblast: Possible role in promoting trophoblast cell migration and invasion

Furin, a proprotein convertase (PC), is ubiquitously expressed and implicated in many physiological and pathological processes. This study is aimed to identify the role of furin in human trophoblast invasion and migration. Furin was found to be highly expressed in placental villi of both rhesus monkeys and human beings during early pregnancy. Specifically, furin was found in trophoblast column and trophoblast shell, regions where highly invasive cytotrophoblast cells invade the maternal decidua during human placentation. To determine whether furin plays any role in trophoblast invasion and migration, we employed human extravillous HTR8/SVneo cells in Matrigel invasion and transwell migration assays. Knocking-down furin expression by siRNA significantly inhibited invasion and migration of HTR8/SVneo cells (P < 0.01), with corresponding decrease of matrix metalloproteinase-9 (MMP-9) activities. In contrast, over-expression of furin markedly increased cell invasion and migration (P < 0.01), accompanied by significant increase of MMP-9 activities. Furthermore, furin siRNA significantly increased the levels of both tissue inhibitors of MMPs (TIMP)-1 and -2. Our results suggest that furin may play an important role in the invasion and migration of human trophoblast cells during early pregnancy.     

Hypoxia inhibits invasion of extravillous trophoblast cells through reduction of matrix metalloproteinase (MMP)-2 activation in the early first trimester of human pregnancy

During early pregnancy, extravillous trophoblast (EVT) cells are exposed to very low pO₂ values. In this study, we investigated the proteolytic functions and invasiveness of human primary EVT cells under hypoxic conditions to show the early placental pathophysiology.Placental samples (from 5 to 10 weeks gestation) were obtained at termination of pregnancy. Cytotrophoblast cells were separated by Percoll^® gradient method and cultured on Matrigel^® to obtain an invasive phenotype (similar to EVT). The invasion capacity (Matrigel-coated invasion assay), migration of the cells (wound healing assay), activity and expression of matrix metalloproteinase (MMP)-2 and tissue inhibitor for MMP (TIMP)-2 (gelatin gel zymography, ELISA, and quantitative RT-PCR), and expression of membrane-type (MT)1-MMP (western blot) were investigated. All cultures (except for quantitative RT-PCR) were performed under 20% oxygen, 5% oxygen, and 5% oxygen with 3 repetitions of 0.1% oxygen hypoxic stimulation for 1 h.Invasion and MMP2 activity of the cells were significantly increased in 20% and decreased in 0.1% oxygen. There was no significant difference in cell migration among the oxygen environments. Concentrations of MMP2 in the supernatant and expression of MT1-MMP were increased in both the 0.1% and 20% oxygen environments. The MMP2 mRNA level was increased after 1-h stimulation with 0.1% oxygen. The TIMP2 concentration was increased only in 20% oxygen, but the mRNA level was decreased in 0.1% oxygen.These results suggested that hypoxia might inhibit the invasive capacity and MMP2 activation of EVT cells in the early first trimester of pregnancy. Decrease in TIMP2 production may reduce the MMP2/TIMP2/MT1-MMP complex and lead to this unique behavior of EVT cells under hypoxic conditions.     

RU_（486）对大鼠早、中期妊娠影响的研究

应用光、电镜方法观察ＲＵ４８６对早期和中期妊娠大鼠脑垂体、卵巢、子宫、胎盘及肝脏的影响。妊娠大鼠分别在受孕第７天（ｄ７）和第１０天（ｄ１０）给药１．２５ｍｇ／只。中期妊娠大鼠给药后２４ｈ全部流产，蜕膜坏死；４８ｈ子宫内膜修复正常。黄体细胞内脂滴增多、退变。血清孕酮水平低于对照组。早期妊娠大鼠给药后４８ｈ，个别胚胎发育与对照组相似，其余胚胎和胎盘均解体，其中２例子宫已修复。黄体和脑垂体促性腺激素细胞的结构及血清孕酮水平与对照组比较无明显差异。提示：ＲＵ４８６对大鼠抗中期妊娠效果比抗早孕好。     

The effect of magnesium sulfate on the placental corticotropin-releasing factor (CRF) and CRF binding protein mRNA expression in perfused human placental cotyledon

Objectives: Stress stimuli and inflammation influence the secretion of the placental corticotropin-releasing factor CRF (CRF) that has a significant role in controlling the timing of birth. The CRF-binding protein (CRF-BP) binds CRF with high affinity and inhibits its activity. Magnesium sulfate (MgSO₄) has been known to ameliorate maternal, fetal and gestational tissue-associated inflammatory response. We aimed to study the effect of MgSO₄ on the CRF and CRF-BP mRNA expression levels in perfused human cotyledon.

Methods: Placentas from elective caesarean section were obtained and selected cotyledons were cannulated and dually perfused ex-vivo within 30?min. MgSO₄ (7?mg/dl) was added to the maternal reservoir. Each perfusion experiment was conducted for 180?min. At the end of the experiment, RNA was extracted from the perfused cotyledon, and RT-PCR was performed to quantify the expression of CRF and CRF-BP. Human HPRT gene served as a reference gene.

Results: Perfusion with MgSO₄ (n?=?3) induced a significantly lower CRF and higher CRF-BP mRNA expression compared to placentas perfused only with medium (n?=?3).

Conclusion: In the human placenta, MgSO₄ possibly exerts its action through different modulation on the CRF and CRF-BP expression.     

Glucose transporter 3 (GLUT3) protein expression in human placenta across gestation

Conflicting information regarding expression of GLUT3 protein in the human placenta has been reported and the localization and pattern of expression of GLUT3 protein across gestation has not been clearly defined. The objective of this study was characterization of syncytial GLUT3 protein expression across gestation. We hypothesized that GLUT3 protein is present in the syncytial microvillous membrane and that its expression decreases over gestation. GLUT3 protein was measured in samples from a range of gestational ages (first to third trimester), with human brain and human bowel used as a positive and negative control respectively. As an additional measure of specificity, we transfected BeWo choriocarcinoma cells, a trophoblast cell line expressing GLUT3, with siRNA directed against GLUT3 and analyzed expression by Western blotting. GLUT3 was detected in the syncytiotrophoblast at all gestational ages by immunohistochemistry. Using Western blotting GLUT3 was detected as an integral membrane protein at a molecular weight of ∼50 kDa in microvillous membranes from all trimesters but not in syncytial basal membranes. The identity of the primary antibody target was confirmed by demonstrating that expression of the immunoblotting signal in GLUT3 siRNA-treated BeWo was decreased to 18 ± 6% (mean ± SEM) of that seen in cells transfected with a non-targeting siRNA. GLUT3 expression in microvillous membranes detected by Western blot decreased through the trimesters such that expression in the second trimester (wks 14-26) was 48 ± 7% of that in the first trimester and by the third trimester (wks 31-40) only 34 ± 10% of first trimester expression. In addition, glucose uptake into BeWo cells treated with GLUT3 siRNA was reduced to 60% of that measured in cells treated with the non-targeting siRNA. This suggests that GLUT3-mediated uptake comprises approximately 50% of glucose uptake into BeWo cells. These results confirm the hypothesis that GLUT3 is present in the syncytial microvillous membrane early in gestation and decreases thereafter, supporting the idea that GLUT3 is of greater importance for glucose uptake early in gestation.     

Decreased expression of phosphorylated placental heat shock protein 27 in human and ovine intrauterine growth restriction (IUGR)

Introduction

Intrauterine growth restriction (IUGR) has been documented to increase placental apoptosis at term. HSP27 has been shown to be involved in the control of apoptosis. Our objective is to determine the expression of phosphorylated HSP27 (p-HSP27) in human IUGR, and to determine the role of HSP27 during gestation in an ovine hyperthermia induced model of IUGR.

Methods

Human placenta tissue samples were collected at term to quantify p-HSP27. Pregnant sheep were placed in hyperthermic (HT) conditions to induce IUGR. Placental tissues were collected at 55 (early), 95 (mid-gestation) and 130 (near-term) days gestational age (dGA) to determined phosphorylated and total HSP27 across the development of IUGR.

Results

Phosphorylated HSP27 was significantly reduced in human placenta IUGR compared to controls at term. HSP27 was increased throughout gestation during the development of IUGR in the sheep. P-HSP27 was increased in early gestation (55 dGA), and decreased near term (130 dGA). The near term decrease was localized to the trophoblast cells of the placenta.

Discussion and conclusion

We conclude that decreased p-HSP27 at term is present when placental apoptosis is increased during IUGR. This could be a factor leading to the decreased placental weight observed during IUGR.     

Decreased expression of complement 3a receptor (C3aR) in human placentas from severe preeclamptic pregnancies

Objectives

The aim of this study was to determine the expression of the anaphylatoxin receptors complement C3a receptor (C3aR) and C5a receptor (C5aR) in the placentas of pregnancies complicated by severe early onset preeclampsia.

Study design

We recruited women with pregnancies complicated by severe early-onset preeclampsia (n = 19, 11 of which were further complicated with IUGR) and women with preterm pregnancies not affected by preeclampsia (n = 8). Gene and protein expression of C3aR and C5aR was analysed by quantitative RT-PCR and Western blotting, respectively.

Results

C3aR was detected in the Hofbauer cells in the villous stroma of the placenta. C5aR staining was detected in the syncytiotrophoblast and endothelial cells. We found significantly decreased expression of C3aR mRNA and protein expression in placentas with preeclampsia compared to controls. However, C5aR expression was not significantly different between preeclamptic and control placentas at either the mRNA or protein level.

Conclusions

Decreased C3aR expression indicates a dysregulation of the complement system in the placentas of preeclamptic women. Further studies would elucidate the exact mechanisms that complement has in preeclampsia.     

High-mobility group protein B1 (HMGB1) is a novel biomarker for human ovarian cancer

Background

High mobility group box l (HMGB1), a nuclear and extracellular protein, is implicated in some physiologic and pathologic conditions. In this study, we investigated the expression and function of HMGB1 in ovarian cancer.

Methods

cDNA microarray analysis was performed to compare gene expression profiles of the highly invasive and the low invasive subclones derived from the SKOV3 human ovarian cancer cell line. Immunohistochemistry (IHC) staining was performed to investigate HMGB1 expression in a total of 100 ovarian tissue specimens. In functional assays, effects of HMGB1 knockdown on the biological behavior of ovarian cancer cells were investigated.

Results

HMGB1 was overexpressed in the highly invasive subclone compared with the low invasive subclone. High HMGB1 expression was associated with poor clinicopathologic features. Knockdown of HMGB1 expression significantly suppressed ovarian cancer cell proliferation accompanied by decreased cyclin D1 and PCNA expression, and inhibited cell migration and invasion accompanied by decreased MMP2 and MMP9 activities.

Conclusion

HMGB1 is a newly identified gene overexpressed in ovarian cancer and associated with poor clinicopathologic features. HMGB1 may serve as a new biomarker and a therapeutic target for ovarian cancer in the future.     

人重组γ－干扰素（hrIFN－γ）对人胎盘滋养层hCG分泌和蜕膜蛋白质合成的影响

研究证明人重组γ－干扰素（ｈｒＩＦＮ－γ）对人胎盘滋养层组织绒毛膜促性腺激素（ｈＣＧ）和蜕膜组织蛋白质合成在体外实验中均有抑制作用。结果指出ｈｒＩＦＮ－γ在剂量为２５０Ｕ／ｍｌ培液和２５００Ｕ／ｍｌ培液时明显地抑制滋养层组织ｈＣＧ分泌，与对照组比较Ｐ值分别为０．０５和０．０１，并呈剂量相关反应，同时ｈｒＩＦＮ－γ在剂量范围为１０Ｕ～１０００Ｕ／ｍｌ培液时，明显抑制蜕膜蛋白质合成３Ｈ亮氨酸掺入的ｃｐｍ值，在对照组和实验组之间无论是细胞内或是分泌蛋白都有明显差别（Ｐ＜０．０５），而且抑制作用呈剂量相关反应，ＩＦＮ－γ抗血清能阻断其抑制作用，但ＩＦＮ－α对蜕膜组织蛋白质合成无作用。     

The interplay of human chorionic gonadotropin (hCG) with basic fibroblast growth factor and adipokines on angiogenesis in vitro

Introduction

Human chorionic gonadotropin (hCG) is suggested to regulate placental angiogenesis, however, its role is incompletely understood. hCG may directly stimulate angiogenesis or influence the effect of other angiogenic factors. We examined the effect of hCG and the interplay of hCG with basic fibroblast growth factor (bFGF) and with various adipokines on proliferation of vascular endothelial cells in vitro.

Methods

Human umbilical vein endothelial cells (HUVEC) were incubated for 2 days with combinations of hCG, bFGF, leptin, resistin, adiponectin, IL6 and TNFα. Incorporation of radiolabelled thymidine was used to assess cell proliferation. Immunofluorescence and flow cytometry were used to examine activation of p44/42 mitogen-activated kinase (MAPK).

Results

hCG induced proliferation of HUVEC alone and in combination with bFGF. Cells exposed to both hCG and bFGF displayed increased activation of p44/42 MAPK as compared to hCG or bFGF alone. Increased HUVEC proliferation was observed in the presence of increasing concentrations of leptin, resistin, adiponectin, and IL6, whereas HUVEC proliferation decreased in the presence of TNFα. hCG in combination with leptin, resistin, adiponectin or IL6 stimulated HUVEC proliferation beyond the effect of hCG alone.

Discussion

An interplay of hCG with adipose tissue-derived factors with angiogenic properties is plausible. Thus, maternal obesity may affect placental angiogenesis in pregnancy.

Conclusions

hCG may directly stimulate angiogenesis. Also, hCG may indirectly stimulate angiogenesis through interplay with bFGF and adipokines.     

妊高征胎盘组织PKC活性变化的初步研究

目的　研究胎盘组织中蛋白激酶Ｃ（ＰＫＣ）与妊高征的关系。　方法　采用改良Ｔａｋａｉ法检测了１８例妊高征患者和２０例正常孕妇的胎盘组织ＰＫＣ比活。　结果　妊高征组胎盘组织质膜ＰＫＣ 活性（１１．６４±４．６６ ｐｍ ｏｌ·ｍ ｉｎ－ １ ／ｍ ｇ ｐｒｏｔｉｅｎ）明显低于对照组（２０．６２±１５．５６ ｐｍ ｏｌ·ｍ ｉｎ－ １／ｍ ｇｐｒｏｔｉｅｎ）（Ｐ＜ ０．０１）,而胞液ＰＫＣ活性（２４．６０±６．５７ ｐｍ ｏｌ·ｍ ｉｎ－ １／ｍ ｇ ｐｒｏｔｉｅｎ）显著高于对照组（１９．０２±６．０１ ｐｍ ｏｌ·ｍ ｉｎ－ １ ／ｍ ｇ ｐｒｏｔｉｅｎ）（Ｐ＜ ０．０１）;且重度妊高征组胎盘组织质膜ＰＫＣ活性（８．８１±２．６８ｐｍ ｏｌ·ｍ ｉｎ－ １ ／ｍ ｇ ｐｒｏｔｉｅｎ）较轻、中度妊高征组（１４．１５±４．７０ ｐｍ ｏｌ·ｍ ｉｎ－ １／ｍ ｇ ｐｒｏｔｉｅｎ）更低（Ｐ＜ ０．０１）;但胞液ＰＫＣ活性无明显差异（Ｐ＞ ０．０５）。妊高征组胎盘组织质膜ＰＫＣ活性与平均动脉压呈负相关（ｒ＝ － ０．４８８,Ｐ＜ ０．０５）。　结论　胎盘组织ＰＫＣ活性与妊高征发生及发展有关,胎盘组织ＰＫＣ活性与全身血管阻力和灌流状态可能相关