Effect of Baicalein on the Expression of VIP in Extravillous Cytotrophoblasts Infected with Human Cytomegalovirus In Vitro

Summary： This paper aimed to study the ability of baicalein to block human cytomegalovirus （HCMV） infection in extravillous cytotrophoblasts （EVT） and its effect on the vasoactive intestinal peptide （VIP） expression in HCMV-infected EVT in vitro. A human trophoblast cell line （HPT-8） was chosen in this study. HCMV with 100 TCIDs0was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by vi- rus titration. Real-time quantitative polymerase chain reaction （qRT-PCR） was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in in- fected HPT-8 cells was decreased （P〈0.05）, and the levels of VIP mRNA and protein, and the concen- tration were raised to the normal （P〉0.05）. Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.

Effect of Human Cytomegalovirus on Invasive Capability of Early Pregnant Extravillous Cytotrophoblasts

The effect of human cytomegalovirus （HCMV） on invasive capability of early pregnant extravillous cytotrophoblasts （EVTs） was investigated in vitro. Primary EVTs were obtained by complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women. Cytokeratin7 （CK7）, vimentin （Vim） and cerbB-2 were immunocytochemically detected to identify source of cells, and HCMVpp65 antigen was assayed to determine the infection state of primary EVTs by immunocytochemical staining. The EVTs were divided into two groups： control group and HCMV group, and the expression of c-erbB-2, matrix metalloproteinase-2 （MMP-2） and MMP-9 proteins was detected in two groups by immunocytochemistry and Western blotting. Enzymic activity changes of MMP-2 and MMP-9 were tested by gelatin zymography in primary EVTs infected with HCMV. The invasion of primary EVTs was detected by cell invasion assay in vitro after they were infected by HCMV. The cell source identification showed that the cells obtained were highly-pure primary EVTs, and primary EVTs could be infected by HCMV. Primary EVTs could express c-erbB-2, MMP-2 and MMP-9 proteins, and as compared with control group, the protein expression was decreased significantly in HCMV groups （P〈0.05）. Primary EVTs could secrete active MMP-2 and MMP-9 in vitro, and the activity of two MMPs was decreased sig- nificantly in HCMV groups （P〈0.05）. The in vitro cell invasion assay showed that the number of primary EVTs permeating Matrigel in HCMV group was decreased （P〈0.05）. We are led to conclude that HCMV can infect primary EVTs and inhibit their invasion capability, suggesting that the im- paired EVT＇s invasion capability might be related to the abnormal expression of c-erbB-2, MMP-2 and MMP-9 proteins.

Study on the Expression of Fas Ligand on the Surfaces of Human Cytotrophoblasts in Normal Pregnancy

To further study the mechanism of maternal-fetal immune tolerance, the expression of Fas ligand (FasL) on the surfaces of human cytotrophoblasts in normal pregnancy was detected by using immunohistochemical method. High precise color-image measure system for immuno-histo- chemistry was used to quantitatively analyze the expression of FasL. The results showed that FasL were expressed on the surfaces of placental cytotrophoblasts throughout normal pregnancy. The variance among the first, second and term pregnant stages was also detected. It was suggested that the expression of FasL on the surfaces of placental cytotrophoblasts might play an important role both in the maintenance of pregnancy and in the normal development of fetus. The maternal speific T cell apoptosis induced by Fas/FasL is one of the significant mechanisms of maternal-fetal immune tolerance.

Effect of Traditional Chinese Medicine on the Growth and Invasion of Human First Trimester Cytotrophoblasts in vitro

Objective To investigate the effect of Traditional Chinese Medicine（TCM） on the growth and invasion of human first trimester cytotrophoblasts in vitro.Methods Serum contained drug was collected from rats adiministered with herbs intragastricly. Serum collected from rats administered with saline was used as the control. Isolated from first trimester placental tissues, human cytotrophoblast cultured in media with 5%, 10%, 20% drug-containing serum for a 24 h, 48 h or 72 h period. Then cell morphology was measured by scanning electric microscope, cell viability by MTT assay, cell DNA content by flow cytomester, and cell invasion by transwell assay.Results After a 48 h- and 72 h-culture period in media with drug-containing rat serum, cytotrophoblast showed more and longer cell pseudopodia than control cells. In addition, cytotrophoblast exhibited more cell microvillous than control cells after a 72 h culture with drug-containing serum. Cell viability cultured with drug-containing serum was higher than control cells. Cells cultured with drug-containing serum in S phase were more, by contrast, in sub-G1 and G2/M phase were less than the control. Cells cultured with drug-containing serum for 72 h showed significantly greater invasive ability than control cells.Conclusion TCM had a beneficial role in the growth and invasion of human first trimester cytotrophoblast in vitro.

Effect of Leptin on Cytotrophoblast Proliferation and Invasion

The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that： （1） Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem- brane of cytotrophoblast; （2） Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly （P〈0.01）; （3） After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls （P〈0.05）. It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro- phoblast invasion and migration activity.

STUDY ON REPRODUCTIVE ENDOCRINOLOGY OF HUMAN PLACENTA--CULTURE OF HIGHLY PURIFIED CYTOTROPHOBLAST CELL IN SERUM-FREE HORMONE SUPPLEMENTED MEDIUM

A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta.

Expression of matrix metallo-proteinase-28 in human normal cytotrophoblast cells and a choriocarcinoma cell line,JEG-3

There are striking similiarities present between the behavior of invasive placentaL cells and that of invasive cancer cells. Matrix metalloproteinases (MMPs) are one of the most important mediators. MMP-28, the new member of MMPs, was sequenced and identified recently. Expression of MMP-28 mRNA and protein in the cytotrophoblast cells and a choriocarcinoma cell line, JEG-3 cell, was conducted by zymography, RT-PCR and Northern blot. There is MMP-28 mRNA expression in both the cytotrophoblast cells and JEG-3 cells by RT-PCR. The activity of MMP-28 in cytotrophoblast cells was significantly weaker than that in JEG-3 (P 【 0.01) by zymography. Furthermore, mRNA expression of MMP-28 was significantly stronger (P 【 0.001) in JEG-3 than in human cytotrophoblast cells in a time-dependent way by Northern Blot. Our results suggest that MMP-28 may play a role in some of the tissue-remodeling events associated with normal pregnancy and tumor progression.

Lipopolysaccharide induces apoptosis of cytotrophoblasts by activating an innate immune reaction in vitro

Background Enhanced apoptosis of cytotrophoblasts in early pregnancy is associated with high risk of intrauterine growth retardation and preeclampsia, which are two common pregnant complications. Its etiological factors remain unclear. Cytotrophoblasts share some traits with innate immune cells and may show response to lipopolysaccharide. This study was conducted to demonstrate whether lipopolysaccharide has apoptosis-inducing effects on cytotrophoblast and the role of innate immune reaction in this process. Methods Cytotrophoblasts were isolated from eady pregnant villous tissues and cultured with serum-free medium. Subsequently, cytotrophoblasts were treated with lipopolysaccharide at the concentrations of 0 （control）, 25, 50, 100 and 200 ng/ml for 24 hours. Apoptosis of cytotrophoblasts was determined by light microscopy, Hoechst 33258 DNA staining with a fluorescent microscope, transmission electron microscope and annexin V-fluorescein isothiocyanate-cenjugated / propidium iodide （PI） staining with flow cytometry. Then expression of caspase-3 was detected by Western blot. Confocal immunofluorescence technique was used to detect tumor necrosis factor a expression in cytotrophoblasts. The levels of tumor necrosis factor a in the culture medium were detected by enzyme-linked immunosorbent assay. Results Under light, fluorescence microscope and transmission electron microscope, characteristic altemations of apoptosis in cytotrophoblasts were observed after lipopolysaccharide treatment. Flow cytometry results showed that lipopolysaccharide significantly increased apoptosis indexes of cytotrophoblasts. Significant statistical differences were found in the above groups （P≤0.01）. The mean relative densities of bands corresponding to caspase-3 were significantly increased in groups treated with lipopolysaccharide, as compared with the normal control （P〈0.001）. Tumor necrosis factor a expression was found to increase in cytotrophoblasts by confocal immunofluorescence technique and in culture medium by enzyme-linked immunosorbent assay after lipopolysaccharide treatment. A positive correlation was found between tumor necrosis factor a expression and apoptosis indexes of cytotrophoblasts （r=0.747, P〈0.001）. Conclusion Apoptosis of cytotrophoblasts could be induced by lipopolysaccharide, in which innate immune reaction is the important mechanism.

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 3 5 %、4 5 %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以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。

人早孕期绒毛和绒毛外细胞滋养细胞的分离、培养及鉴定

目的:建立人早孕期绒毛细胞滋养细胞(villous cytotrophoblasts,VCT)及绒毛外细胞滋养细胞(extravillous cytotrophoblasts,EVCT)的分离、培养方法。 方法:利用不同的胰酶消化条件,收集人早孕期绒毛组织的VCT及EVCT并分别培养。倒置显微镜、扫描电镜观察VCT及EVCT的形态学特征;免疫细胞化学鉴定细胞来源及纯度。 结果:胰酶短期、温和消化获得的EVCT,可生长于Matrigel包被的培养器皿上,并表达特异性标志物c-crbB-2;延长消化时间、增加胰酶浓度获得的VCT,种植于塑料或玻璃培养器皿上可聚集并融合形成合体滋养细胞。VCT不表达c-crbB-2。结论:采用不同的胰酶消化及体外培养条件,可简单、快捷地分别获得高纯度人早孕期VCT及EVCT。

细胞过度凋亡是脂多糖抑制细胞滋养细胞侵入能力的重要机制

目的:探讨脂多糖(LPS)对细胞滋养细胞侵入能力的影响及细胞凋亡在此过程中的作用。方法:对体外培养的早孕细胞滋养细胞,给予不同浓度的LPS(0ng/ml、25ng/ml、50ng/ml、100ng/ml、200ng/ml),采用Transwell检测侵入能力的改变,荧光显微镜、电镜观察凋亡细胞的形态学改变,流式细胞仪定量检测细胞凋亡指数,western blot检测Caspase-3、MMP-2和MMP-9的表达。结果:LPS诱导细胞滋养细胞出现过度凋亡,抑制其侵入能力,促进Caspase-3的表达,抑制MMP-2、MMP-9的表达。以上各指标组间差异显著,P值均＜0．01。细胞凋亡指数与其侵入能力以及MMP-2和MMP一9的表达呈负相关,P值均＜0．01。结论:LPS对细胞滋养细胞的侵入能力有显著的抑制作用,细胞过度凋亡是这一作用发生的重要机制。

菟丝子总黄酮对早孕期人细胞滋养细胞增殖能力的影响及其信号机制

目的:探讨菟丝子总黄酮对早孕期人细胞滋养细胞增殖的影响及其信号转导机制。方法:应用硅化聚乙酞胺吡咯烷酮(Percoll)密度梯度离心法,分离纯化早孕期人细胞滋养细胞,经免疫细胞化学法鉴定其分子表达特征和纯度;采用MTT法评价早孕期人细胞滋养细胞活力;流式细胞术分析细胞滋养细胞PCNA的表达;用Western blotting法观察pERK1/2的水平。结果:菟丝子总黄酮呈时间和剂量依赖性增强早孕期人细胞滋养细胞活力,促进细胞滋养细胞ERK1/2的磷酸化。结论:菟丝子总黄酮可能通过活化MAPK/ERK1/2信号通路促进人细胞滋养细胞增殖。

MMP-26在人正常胎盘滋养层细胞中的表达及激活素A对其表达的调节

胚胎植入和胎盘形成涉及细胞外基质的降解和重建,以及细胞的增殖、凋亡、迁移和分化,基质金属蛋白酶(MMPs) 是参与这些事件的主要蛋白水解酶系统. MMP-26是近年来发现的MMPs家族的新成员,但其功能所知甚少. 通过半定量RT-PCR、免疫组织化学、荧光免疫细胞化学等手段,发现人胎盘中MMP-26主要定位于绒毛滋养层细胞,在绒毛间质细胞中也有少量表达. 妊娠早期,胎盘中MMP-26表达水平较高,至妊娠中期降至最低,但在足月胎盘中其表达又有显著提高,提示MMP-26可能参与妊娠早期滋养层细胞的侵润和分娩时的胎盘剥离. 体外培养的妊娠早期人细胞滋养层细胞能产生一定水平的MMP-26,而其表达受到激活素A的剂量依赖性刺激,表明滋养层细胞中存在MMP-26表达的自分泌/旁分泌调节.

过氧化物酶体增殖物激活受体γ及其配体对早期细胞滋养细胞MMP-2、MMP-9表达的调控

目的:探讨过氧化物酶体增殖物激活型受体γ(PPARγ)及其配体(15-脱氧-前列腺素J2,15-d-PGJ2)对细胞滋养细胞表达基质金属蛋白酶-2(MMP-2)和MMP-9的调控作用。方法:采用免疫荧光细胞化学方法检测细胞滋养细胞中PPARγ的表达;利用免疫荧光共聚焦技术观察15-d-PGJ2作用前后细胞滋养细胞MMP-2和MMP-9表达强度的变化;通过荧光定量PCR(Real-timePCR)和Westernblot方法定量检测MMP-2和MMP-9mRNA和蛋白的表达变化。结果:在细胞滋养细胞中有PPARγ蛋白表达,且主要定位在细胞滋养细胞核中;15-d-PGJ2作用后细胞滋养细胞中MMP-2和MMP-9的表达明显下降,与对照组相比差异显著(P<0.01);15-d-PGJ2对MMP-2的作用强于MMP-9。结论:PPARγ及其配体15-d-PGJ2调节滋养细胞浸润作用可能是通过调节MMP-2和MMP-9的表达实现的。

早孕期人细胞滋养层细胞的分化及分离、培养、鉴定

目的:分离正常早孕期人细胞滋养层细胞并分析其分化功能。方法:用不同的消化方法分离正常早孕期人细胞滋养层细胞,分别在体外培养24h,用相差显微镜、Giemsa染色、免疫细胞化学法、扫描电镜、Transwell分析其形态及分化功能。结果:①采用低浓度胰酶一次性消化法获得绒毛外滋养层细胞,特异表达细胞角蛋白7,不表达波形蛋白,细胞互相聚集但不聚合,具有高度增殖活性和侵袭能力;②采用高浓度胰酶、长时间连续消化法分离得到绒毛滋养层细胞,具有高度融合、分泌hCG-β的合体滋养层细胞特性;③采用低浓度胰酶连续消化法获得的细胞包含绒毛外滋养层细胞和绒毛滋养层细胞,其共性是表达细胞总角蛋白,不表达波形蛋白。结论:利用不同的分离方法可有效、经济地获得具有不同分化功能的早孕期人细胞滋养层细胞。

早孕绒毛细胞滋养层细胞分离培养的实验研究

目的 改善体外分离和培养人早孕绒毛细胞滋养层细胞 (cytotrophoblast,CTB)的方法 ,探讨CTB的一些生物学特性。方法 取人工流产 6～ 8周妊娠绒毛 ,机械法分离 ,酶消化 ,经Ficoll分离介质离心得到单个核细胞进行培养并鉴定 ,测定其生长曲线并观察其体外转化、细胞周期和激素合成。结果 分离培养的CTB经免疫细胞化学染色鉴定 ,接种后2h开始贴壁 ,第 5天融合 90 %以上 ,细胞周期显示约有 79%细胞处于G0 G1 期 ,激素分泌表现出随CTB的转化而变化。结论 成功分离培养了人早孕绒毛CTB ,方法简便易行 ,获得的细胞形态单一、生长稳定、定向转化 。

人类白细胞抗原G、E在人胎盘组织中的表达及其意义

目的:探讨人类白细胞抗原G、E(HLA-G、E)在人胎盘组织中的表达及意义。方法:用原位杂交及免疫组化法分别检测HLA-G、E mRNA及蛋白质在人正常早孕绒毛组织、晚孕胎盘组织中的表达。结果:在人正常早孕绒毛组织中的绒毛细胞滋养细胞、合体滋养细胞及绒毛外滋养细胞(EVCT)内均有HLA-G、E mRNA及HLA-E蛋白质表达,而HLA-G蛋白质仅在EVCT内表达;晚孕胎盘组织中HLA-G、E mRNA及蛋白质表达于蜕膜板内的EVCT及羊膜上皮细胞。结论:HLA-E表达于人正常早孕绒毛组织中所有类型的滋养细胞及晚孕胎盘组织中的EVCT和羊膜上皮细胞。抗人HLA-G单克隆抗体4H84仅能检测出人胎盘组织中EVCT及羊膜上皮细胞内的HLA-G蛋白。

补肾益气方对正常早孕期人细胞滋养层细胞生物学行为的影响

目的探讨补肾益气方对正常早孕期人细胞滋养层细胞增殖、侵袭、分化等生物学行为的影响。方法体外培养正常早孕期人细胞滋养层细胞 ,分别设空白对照组、5 %含药血清组、10 %含药血清组、2 0 %含药血清组 ,用扫描电镜、噻唑蓝 (MTT)法、流式细胞仪、Transwell侵袭实验检测含药血清作用 2 4h、4 8h、72h后细胞的形态、增殖活性及侵袭力的变化。结果含药血清培养 2 4h、4 8h、72h后 ,细胞滋养层细胞微绒毛变丰富 ,伪足增多、延长 ;细胞在酶联免疫分析仪 4 90nm波长的吸光度值增加 (P <0 0 1) ;sub G1期细胞减少 (P <0 0 1) ,S期细胞增加 (P <0 0 1) ,G2 /M期细胞明显减少 (P <0 0 1) ;每视野穿过PET膜的细胞数明显增多 (P <0 0 1)。结论补肾中药促进人细胞滋养层细胞增殖及侵袭力 。

菟丝子提取物含药血清对人早孕滋养层细胞增殖及凋亡的影响

目的探讨菟丝子提取物的含药血清对正常人早孕细胞滋养层细胞(cytotrophoblast,CTB)增殖及凋亡的调节作用。方法雌性SD大鼠随机分为8组,每组5只,包括空白血清对照组,减味寿胎丸组,菟丝子总提物高、低剂量组,菟丝子总黄酮高、低剂量组,菟丝子总多糖高、低剂量组,各组灌胃予对应药物,2次/天,连续3 d后腹主动脉采血分离血清。上述空白血清或各提取物血清以不同体积(5%、10%、20%)分别培养CTB细胞,噻唑蓝(MTT)法检测细胞增殖活力,流式细胞仪分析细胞周期。结果 MTT结果显示,各提取物高、低浓度含药血清培养细胞48 h后,吸光度值均一定程度增加,除5%菟丝子总提物高剂量组及5%菟丝子总多糖低剂量组外,余各组5%、10%、20%含药血清与空白对照组比较差异均有统计学意义(P<0.05,P<0.01)。细胞增殖活性随含药血清浓度升高而增加,呈明显剂量依赖性。流式细胞仪分析结果显示,与空白血清组比较,各组S期细胞百分比均显著增加(P<0.05,P<0.01);而G2/M期细胞百分比显著减少(P<0.05,P<0.01)。干预48 h后,细胞周期分布变化趋势与24 h一致,而且变化更明显。结论菟丝子各提取物高、低剂量含药血清均可增加CTB细胞的增殖活性,降低细胞凋亡的发生。药效最佳剂量分别为:菟丝子总提物低剂量、总黄酮高剂量及总多糖高剂量。

母-胎免疫调节机制在妊娠高血压综合征病理过程中的作用

目的:分析妊娠高血压综合征(PIH)患者外周血中免疫细胞因子水平变化及滋养层细胞中免疫相关转录因子的基因、蛋白表达情况,探讨母-胎免疫调节机制在妊娠高血压综合征病理过程中的作用。方法:纳入PIH患者50例作为观察组,选择同期分娩的正常妊娠妇女40例作为对照组。ELISA法检测两组孕妇外周血中IFN-γ、IL-4、IL-17及TGF-β水平;实时荧光定量PCR(RT-q PCR)法和蛋白质印迹法(Western blot)分别测定两组孕妇胎盘滋养层细胞中转录因子T-bet、GATA-3、RORC、Foxp3的mRNA及蛋白表达情况。结果:(1)观察组外周血中IFN-γ和IL-17水平明显高于对照组,TGF-β水平明显低于对照组(P<0.05);两组间IL-4水平无明显差异(P>0.05);观察组IFN-γ/IL-4、IL-17/TGF-β比值均明显大于对照组(P<0.05)。(2)与对照组比,观察组T-bet、RORC的mRNA及蛋白表达水平均显著升高(P<0.05),而GATA-3、Foxp3的mRNA及蛋白表达水平均显著降低(P<0.05),T-bet/GATA-3及RORC/Foxp3比值均显著增大(P<0.05)。结论:PIH具有明显的Th1/Th2及Th17/Treg比例失衡现象,其病理机制可能与Th细胞介导的母-胎免疫耐受机制功能紊乱有关。