IL-6、TNFα对人早孕绒毛滋养层细胞胎盘生长因子表达调控的作用

目的:探讨IL-6、TNFα对原代培养人早孕绒毛滋养层细胞胎盘生长因子(PIGF)表达的调控作用。方法:对胰蛋白酶与胶原酶Ⅰ联合消化、Percoll细胞分离液纯化而得到的滋养层细胞进行原代培养,测定100 ng/mL IL-6、50 ng/mL TNFα对滋养层细胞PIGF表达的影响。结果:酶联合消化及梯度离心能较好地分离早孕绒毛中的滋养层细胞,100 ng/mL IL-6作用于滋养层细胞后PIGF分泌增加,与对照组有显著性差异(P<0.01),TNFα作用于滋养层细胞后PIGF分泌也增加,但与对照组差异不显著(P>0.05)。结论:IL-6对PIGF分泌具有促进作用,TNFα对滋养层细胞PIGF分泌的促进作用不显著,提示VEGF与PIGF可能由母胎界面不同的细胞因子所调控。     

抑制素A、激活素A及人hCG与妊高征

研究证实,胎盘滋养细胞合成和分泌的抑制素A(INH A)、激活素A(ACT A)及人绒毛膜促性腺激素(hCG)的异常升高可能与妊娠高血压综合征(PIH)的发生发展有关,检测INH A、ACT A及hCG在血清和胎盘中浓度可能成为PIH的早期预测指标和病情监测指标.     

抑制素A、激活素A及人hCG与妊高征

研究证实，胎盘滋养细胞合成和分泌的抑制素A(INHA)、激活素A(ACTA)及人绒毛膜促性腺激素(hCG)的异常升高可能与妊娠高血压综合征(PIH)的发生发展有关，检测INHA、ACTA及hCG在血清和胎盘中浓度可能成为PIH的早期预测指标和病情监测指标。     

激活素A对早孕胎盘细胞滋养细胞凋亡的作用

激活素A是转换生长因子13家族中的一员。研究表明，激活素A在细胞增殖、凋亡以及癌变过程中都有重要的调节作用；其中激活素A对具有增殖功能的细胞可诱导其发生凋亡。人的细胞滋养细胞是具有增殖能力的正常细胞，细胞膜上有激活素A受体表达，在妊娠期与激活素A结合，继而影响细胞滋养细胞增殖与凋亡。本研究旨在探讨激活素A对细胞滋养细胞凋亡的调节作用，报道如下。     

E-钙粘素和β-连环素在妊娠滋养细胞疾病中的表达与意义

目的探讨E钙粘素和β连环素表达与妊娠滋养细胞疾病(GTD)恶性转化的相关性。方法应用逆转录聚合酶链反应技术检测12例正常早孕绒毛和35例妊娠滋养细胞疾病组织中E钙粘素mRNA和β连环素mRNA的表达。结果E钙粘素和β连环素mRNA在正常早孕绒毛与葡萄胎组织的表达差异无显著性(P>0.05);而在正常早孕绒毛、葡萄胎组织的表达量明显高于侵蚀性葡萄胎、绒癌组织(P<0.05);在侵蚀性葡萄胎组织的表达高于绒癌组织(P<0.05)。结论E钙粘素和β连环素的表达下调与妊娠滋养细胞疾病的恶性转化有关,对预测妊娠滋养细胞疾病的恶变以及评价预后有一定的参考价值。     

天花粉蛋白对体外无血清培养的人早期绒毛细胞滋养层细胞hCG,孕…

本研究以体外无血清培养的人早期绒毛细胞滋养细胞和绒毛细胞培养的模型，测定了天花粉蛋白对滋养层细胞ｈＣＧ，孕酮分泌的影响，发现体外无血清培养的细胞滋养层细胞分泌的ｈＣＧ在天花粉蛋白浓度为０．１μｇ／ｍｌ即下降５０％，而后下降缓慢８μｇ／ｍｌ以上缓慢，至８μｇ／ｍｌ以上降到零，说明体外培养的细胞滋养层细胞中有两个群体，其中一个对天花粉蛋白敏感，另一个不敏感，尽管在形态上很难区别，孕酮的反应则不同，在细     

胰岛素样生长因子-Ⅰ对体外人滋养层细胞孕酮分泌的影响

目的:探讨胰岛素样生长因子-I(IGF-I)对人早孕绒毛滋养层细胞孕酮(P)的合成和调节 作用。方法:将胰蛋白酶和胶原酶联合消化人早孕绒毛滋养组织,Percoll密度梯度分离纯化后得 到的人早孕胎盘滋养层细胞进行原代培养。以终浓度为0.1μg/L、1μg/L、10μg/L、100μg/L　IGF-I分 别对其作用12h,以及100μg/L浓度IGF-I作用12h、24h、48h、72h时,放免法检测滋养细胞 分泌P 的含量,RT-PCR法检测低密度脂蛋白受体(LDLR)mRNA的表达。结果:滋养层细胞P 的 分泌量随着IGF-I的浓度升高而增加;同时100μg/L浓度的IGF-I作用于滋养层细胞12h 后,P 分泌开始增加,48h达到高峰,以后逐渐下降。半定量RT-PCR均显示LDLRmRNA阳性条带,且表 达规律与P一致。结论:滋养层细胞P的分泌具有对IGF-I的时间和浓度依赖性,并且IGF-I能 上调LDLRmRNA的表达,对促进滋养细胞P分泌的调节起重要作用。     

抑制素A和激活素A与妊娠

妊娠期间母体血清、羊水、脐血中的抑制素A和激活素A主要来源于胎盘.抑制素A、激活素A的合成分泌可能与胎盘功能有关.在一些妊娠相关疾病,如妊娠滋养细胞疾病、流产、异位妊娠、妊娠期高血压疾病、胎儿缺氧和21三体综合征中,抑制素A和激活素A有不同的变化.测定其在血清、羊水中的表达,可能对这些妊娠相关疾病的预防、诊断、预后评估...     

激活素A和卵泡休止素与先兆子痫的相关性研究

目的　探讨先兆子痫患者血清中激活素A和卵泡休止素水平及其mRNA在胎盘组织中的表达 ,及其与先兆子痫发病的关系。方法　 2 0例足月妊娠先兆子痫孕妇作为先兆子痫组 ,2 0例足月妊娠血压正常孕妇作为对照组。应用酶联免疫吸附试验 (ELISA)检测两组孕妇血清中激活素A和卵泡休止素水平。应用半定量逆转录 聚合酶链反应 (RT PCR)技术检测两组孕妇分娩后胎盘组织中激活素AmRNA和卵泡休止素mRNA的相对表达强度。将两组孕妇的胎盘组织激活素AmRNA的表达强度与血清激活素A水平进行直线相关分析。结果　 (1)先兆子痫组孕妇血清中激活素A水平为 (33 7± 6 6 ) μg/L ,明显高于对照组的 (9 9± 2 1) μg/L(P <0 0 1)。先兆子痫组孕妇血清中卵泡休止素水平为 (5 1± 0 6 ) μg/L ,与对照组的 (4 7± 0 3) μg/L比较 ,差异无显著性 (P >0 0 5 )。 (2 )先兆子痫组胎盘组织中激活素AmRNA为 1 11± 0 2 1,明显高于对照组的 0 6 1± 0 17(P <0 0 1)。先兆子痫组胎盘组织中卵泡休止素mRNA为 0 5 7± 0 31,与对照组的 0 5 4± 0 2 7比较 ,差异无显著性 (P >0 0 5 )。(3)在先兆子痫组和对照组孕妇中 ,血清中激活素A水平与胎盘组织激活素AmRNA相对表达强度呈正相关 [相关系数 (r) =0 89,P <0 0 1]。结论     

激活素A对人早孕细胞滋养层细胞凋亡的调节

目的探讨激活素A(Activin A,ActA)对细胞滋养层细胞凋亡的调节作用。方法用ActA刺激无血清培养的7~8周人绒毛细胞滋养层细胞后,激光共聚焦和蛋白印迹分析检测凋亡信号通路相关蛋白的表达变化,TUNEL检测细胞凋亡情况。结果高浓度ActA(50 ng/ml)作用24 h后,人绒毛细胞滋养层细胞中TUNEL阳性信号显著增强;同时Caspase-3,-9表达量明显提高(对照组:0.13±0.048和0.26±0.004;50 ng/ml组:0.34±0.068和1.54±0.062;100 ng/ml组:0.45±0.091和0.58±0.008),Caspase-8表达亦略有增加;凋亡相关蛋白P53(对照组:5.2±1.02;50ng/ml组:12.3±1.91;100 ng/ml组:11.5±1.73)和Bax(对照组:0.09±0.021;50 ng/ml组:0.46±0.065;100 ng/ml组:0.68±0.142)表达水平显著增加。结论高浓度ActA可诱导人细胞滋养层细胞发生凋亡,其作用可能主要是通过细胞凋亡的线粒体途径来实现的。     

Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A

AIM: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. RESULTS: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 microg/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. CONCLUSIONS: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.     

Trophic effects of first-trimester human trophoblasts and human chorionic gonadotropin on lymphocyte proliferation

We examined the effects of pure first-trimester human trophoblast cells grown in long-term cultures or their secreted products on the proliferation of human peripheral blood lymphocytes cultured alone, under allogeneic stimulation, or in the presence of concanavalin A. Both trophoblasts and their culture supernatants stimulated lymphocyte proliferation. Culture supernatant had a moderate enhancing effect on lymphocyte mitogenesis in mixed lymphocyte cultures and in the presence of concanavalin A. Anti-human chorionic gonadotropin antibody suppressed the proliferative effect of trophoblast cells and their supernatants in the above experiments in a dose-dependent manner. At physiologic concentrations, both pure and impure forms of human chorionic gonadotropin enhanced (in a dose-dependent manner) proliferation of lymphocytes cultured alone (peak stimulation index, 6 to 7.8 at approximately 7 IU/ml). At higher concentrations (20 to 400 IU/ml) the proliferative effect was abolished. Trophoblast culture supernatant induced the expression of interleukin-2 receptors on lymphocytes after 48 hours of incubation. The supernatant also stimulated proliferation of human colon carcinoma cells. Thus trophoblast or trophoblast-derived human chorionic gonadotropin has a lymphocytotrophic function that may have implications for fetal survival.     

Characterization of pure human first-trimester cytotrophoblast cells in long-term culture: growth pattern, markers, and hormone production

Pure long-term cytotrophoblast cultures were established from human first-trimester placentas by growing chorionic villus explants without enzymatic digestion. Cytoplasmic human chorionic gonadotropin was detectable in all (100%) cells in culture when labeled with a polyclonal anti-human chorionic gonadotropin antibody and in 71% to 83% of cells labeled with a monoclonal anti-alpha-human chorionic gonadotropin antibody. Most of the cells expressed cytokeratin and surface Trop-1 and Trop-2 antigens (89% to 95%), but none expressed cytoplasmic vimentin or surface 63D3 antigens. Study of the ultrastructure of the cells demonstrated epithelial morphologic features. The average doubling time of the trophoblast was 48 to 96 hours. Some of the lines have been continuously propagated for 8 months. They produced variable amounts of human chorionic gonadotropin (50 to 710 mIU/ml per 10(5) cells per 24 hours). The basal level of progesterone secreted by trophoblast (444.4 +/- 32.4 pg/ml per 10(5) cells per 24 hours) doubled in the presence of pure human chorionic gonadotropin (100 ng/ml). They produced small amounts of 17 beta-estradiol (less than 20 pg/ml per 10(5) cells per 24 hours); human chorionic gonadotropin had no effect on the estradiol production. Trophoblast-derived human chorionic gonadotropin acted as a growth factor because trophoblast proliferation (measured by uptake of thymidine labeled with tritium) was reduced by 60% in the presence of an anti-human chorionic gonadotropin antibody. Availability of pure, functionally competent human cytotrophoblast in long-term cultures is relevant for further studies in reproduction biology.     

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary.

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10, 100 and 1000 ng/ml). Following 48 hours of incubation, the levels of progesterone, 20 alpha-hydroxy-4-pregnene-3-one (20 alpha-OH-P) and adenosine 3',5'-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20 alpha-OH-P in a dose-dependent manner (p < 0.01 and 0.05, respectively, ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each, ANOVA). Conversely, LH-stimulated 20 alpha-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20 alpha-OH-P production in LH-stimulated cells, PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.     

In vitro recovery of human chorionic gonadotropin-stimulated cyclic adenosine 3',5'-monophosphate production in desensitized human granulosa-luteal cells

Human granulosa-luteal cell production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone (P) were studied in response to purified human chorionic gonadotropin (hCG) in cultured cells from hyperstimulated follicles of in vitro fertilized patients. The hCG injection given to the patients 36 hours before laparoscopy caused partial desensitization of adenylate cyclase of these cells to gonadotropins. Preincubation of the cells in hormone free medium for 2 to 3 days significantly increased their cAMP responsiveness to hCG. P production was stimulated initially by hCG and showed no desensitization. In the cells preincubated for 72 hours without hCG, a subsequent stimulus of 50 ng/ml of hCG elicited maximal cAMP response, whereas 1 ng/ml of hCG was sufficient to bring about maximal P secretion. Time-course studies indicated that maximal cAMP response to hCG was obtained in 1 to 3 hours. Both basal and hCG-stimulated P accumulation continued to rise for up to 24 hours. Preincubation of granulosa-luteal cells from hyperstimulated follicles improves the cells' cAMP responsiveness to hCG, whereas P response remains unaltered.     

Human amniotic fluid glycoproteins expressing sialyl Lewis carbohydrate antigens stimulate progesterone production in human trophoblasts in vitro

BACKGROUND: Progesterone is thought to mediate immune modulator effects by regulating uterine responsiveness. The aim of the study was to clarify the effect of transferrin and glycodelin A (former name PP14) as sialyl Lewis X-expressing glycoproteins on the release of progesterone by trophoblast cells in vitro. METHODS: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a Percoll gradient centrifugation step. Trophoblasts were incubated with varying concentrations (50-300 microg/ml) of human amniotic fluid- and serum-transferrin as well as with glycodelin A. Culture supernatants were assayed for progesterone, human chorionic gonadotropin (hCG) and cortisol by enzyme immunometric methods. RESULTS: The release of progesterone is increased in amniotic fluid transferrin- and glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. There is no relation between transferrin and the hCG or cortisol production of trophoblast cells. CONCLUSION: The results suggest that sialyl Lewis carbohydrate antigen-expressing amniotic fluid glycoproteins modulate the endocrine function of trophoblasts in culture by upregulating progesterone production.     

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-μn pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of a5 and a6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly (P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG (P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl CAMP (db cAMP) and the CAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell (JEG-3) invasion. The collagenolytic activity of trophoblastic cells (IEG-3) was increased by hCG stimulation. No changes were shown in the expression of α5 and α6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.     

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10 ,100 and 1000 ng/ml). Following 48 hours of incubation ,the levels of progesterone ,20α-hydroxy-4-pregnene-3-one (20α-OH-P) and adenosine 3′ ,5′-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20α-OH-P in a dose-dependent manner (p < 0.01 and 0.05 ,respectively ,ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each ,ANOVA). Conversely ,LH-stimulated 20α-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20α-OH-P production in LH-stimulated cells ,PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.     

Progesterone production in cultured human granulosa cells: correlation with follicular fluid hormone levels

STUDY OBJECTIVE: To examine the progesterone (P) production by cultured granulosa cells and the hormonal content in the follicular fluid (FF) of ovarian-hyperstimulated women. DESIGN: Retrospective. SETTING: Private Fertility Clinic and National Research Institute. PATIENTS: Eighteen patients undergoing in vitro fertilization or gamete intrafallopian transfer programs. RESULTS: Progesterone levels Measured in the culture medium of granulosa cells decreased sixfold with culture time. Human luteinizing hormone (LH) increased P production only when basal P production was less than 1 microgram/mL. Granulosa cell P production in culture was negatively correlated with FF LH-human chorionic gonadotropin (hCG) levels. Follicular fluid follicle-stimulating hormone (FSH) levels were positively correlated with FF P and 17 beta-estradiol (E2) concentrations. Similar results were found between FF LH (hCG) and E2 levels, but there was no relationship between FF LH (hCG) and FF P values. CONCLUSION: The high dose of hCG administered during gonadotropin treatment could induce a decrease in the in vitro granulosa cell P production.