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     

双炔失碳酯对离体黄体、蜕膜和滋养层细胞的影响

本文以细胞的形态和存活率为指标,用米非司酮作为阳性对照,观察了双炔失碳酯对无血清培养的大鼠黄体、人蜕膜和滋养层细胞的直接损伤作用;同时进一步观察了双炔失碳酯对大鼠黄体细胞分泌功能的影响。结果表明：（１）双炔失碳酯对大鼠黄体细胞、人蜕膜和滋养层细胞有直接损伤作用,其ＬＤ５０分别为：１４．３４±０．９μｇ／ｍｌ、１７．３３±４．１μｇ／ｍｌ和３４．８７±４．９μｇ／ｍｌ。在非致死剂量（５μｇ／ｍｌ）下双炔失碳酯未能抑制ｈＣＧ和孕烯醇酮促进大鼠黄体细胞孕酮生成作用;但能极显著地抑制Ｆｏｒｓｋｏｌｉｎ的促孕酮分泌作用。本文结果提示：溶黄体作用是双炔失碳酯抗早孕的主要机理,对于蜕膜和滋养层的直接损伤作用也是其抗早孕有效环节之一。     

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…

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）和蜕膜组织蛋白合成了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ｃ     

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。     

体外受精与胚胎移植中hCG注射前血孕酮水平与妊娠的关系

用ＧｎＲＨａ－ＦＳＨ－ｈＭＧ－ｈＣＧ方案控制性超排卵进行体外受精与胚胎移植（ＩＶＦ－ＥＴ）治疗７８例不孕患者，在ｈＣＧ注射前抽血用放射免疫法（ＲＩＡ）测孕酮（Ｐ）水平。初步了解ｈＣＧ注射时血孕酮水平与ＩＶＦ－ＥＴ结果的关系。结果，当Ｐ〈０．３５μｇ／Ｌ时９例中无１例妊娠，而０．３５≤Ｐ≤０．９μｇ／Ｌ组（５４例）与Ｐ〉０．９μｇ／Ｌ组（１５例）的妊娠率分别为２２．６％及２６．７％，但三组间无显著差     

尿促性腺激素片段在滋养细胞疾病监测中的意义

对４６例滋养细胞疾病患者尿标本２７５份进行尿促性腺激素片段（ＵＧＦ）测定，并自身对照收集同期血标本２７５份，测定绒毛膜促性腺激素（ｈＣＧ），并进行比较。另选１２例健康非妊娠妇女为对照组。结果，滋养细胞疾病患者尿ＵＧＦ阳性串（＞０．２μｇ／Ｌ）为６４．０％，血清ｈＣＧ阳性率（＞２０μｇ／Ｌ）为６６．５％，两者差异无显著性（Ｐ＞０．１）。血清ｈＣＧ阴性患者中，尿ＵＧＦ阳性者５７．６％。提示在滋养细胞疾病患者血ｈＣＧ呈高值时，尿ＵＧＦ阳性率亦高，测定意义不大。而血ｈＣＧ呈低值、ｈＣＧ阴性时，尿ＵＧＦ仍有一定的检出率。对照组１２例无假阳性。     

激活素A和卵泡休止素对人滋养层细胞增殖和激素分泌的调节作用

目的: 探讨激活素A和卵泡休止素对人早孕绒毛滋养层细胞的增殖和人绒毛膜促性腺激素及孕酮分泌的调节作用。方法: 早孕绒毛用胰蛋白酶与胶原酶联合消化后,再用小牛血清白蛋白密度梯度离心,分离纯化后所得的滋养层细胞进行体外原代培养。将不同浓度激活素A加入细胞培养液中作用48 h,观察其对滋养细胞生长和人绒毛膜促性腺激素及孕酮分泌的影响。向滋养层细胞培养液中加入激活素A和不同浓度的卵泡休止素,培养24 h,观察相互作用。结果: 激活素A和卵泡休止素都不影响滋养层细胞的增殖。激活素A以剂量依赖的方式促进滋养层细胞绒毛膜促性腺激素的分泌和孕酮的分泌,滋养细胞分别经30 ng/mL,100 ng/mL激活素A处理后,培液中绒毛膜促性腺激素和孕酮水平达到高峰(P均<0.01)。激活素A对滋养层细胞激素分泌的影响,可以被其特异结合蛋白卵泡休止素以剂量依赖方式阻断。结论: 激活素A和卵泡休止素以自分泌方式调节滋养层细胞绒毛膜促性腺激素和孕酮分泌,但并不影响细胞增殖,激活素A与卵泡休止素在人早期妊娠中起重要的生理作用。     

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。     

白细胞介素6在滋养层细胞胎盘生长因子表达调控中的作用

目的：探讨原代培养人早孕绒毛滋养层细胞中,胎盘生长因子（PLGF）的表达及白细胞介素（IL）6对体外培养的滋养层细胞PIGF表达的调控作用。方法：将胰蛋白酶与胶原酶联合消化,再与Percoll细胞分离液纯化而得到的滋养层细胞进行原代培养,采用逆转录聚合酶链反应（RT-PCR）方法,检测体外培养人滋养层细胞PIGF表达;观察100、10、1及0.1μg/L浓度的IL-6作用12h及100μg/L浓度的IL-6作用6、12、24及48h时,对滋养层细胞PIGF表达的影响。结果：显示PIGF为185bp、248bp的阳性条带;随IL-6浓度的增加,滋养层细胞PIGF分泌也相应增加;当100μg/L浓度的IL-6作用于滋养层细胞6h后,PIGF分泌开始增加,12h达到高峰。结论：PIGF的分泌具有对IL-6的时间与浓度的依赖性,并在早孕中发挥生理作用。     

妊娠中晚期妇女血清hCG水平的临床研究

ｈＣＧ(绒毛膜促性腺激素 )由胎盘绒毛的合体滋养层细胞合成分泌 ,在妊娠 8～ 10周时达高峰 ,1～ 2周后迅速下降 ,中晚期妊娠时稳定于一定浓度。妊娠早期血清ｈＣＧ异常升高与葡萄胎、双胎有关 ,而中、晚期ｈＣＧ升高的临床意义尚不十分清楚 ;许多妊娠并发症可能涉及到胎盘的功能状态 ,而ｈＣＧ又是反映胎盘分泌状态的一个重要生化指标。本研究通过放免测定 (ＲＩＡ)妊娠中、晚期血清ｈＣＧ水平 ,观察妊娠中期ｈＣＧ升高者晚期是否仍高 ,以及整个妊娠过程中持续的ｈＣＧ升高与妊高征、早产、胎儿宫内生长迟缓 (ＩＵＧＲ)等妊娠并发症的关系…     

Differentiation and secretory activities of cultured human placental cytotrophoblast

Ultrastructural, autoradiographic, immunofluorescent and biochemical techniques were used to characterize primary cultures of term placental cytotrophoblast in order to gain insight into the differentiation and secretory capacities of the cellular component of human trophoblast. Trypsin treatment of placental villi allowed isolation of a predominantly cytotrophoblast cell population that maintained viability up to 13 weeks in monolayer culture. Autoradiographic studies of tritiated thymidine incorporation identified a smaller diameter mononucleated cell population that was mitotically active and developed into larger diameter mononucleated cells and into multinucleated cells during culture. Ultrastructurally, cultured cells formed desmosomes, had an extensive network of cytoplasmic microfilaments and contained the organelles for hormone synthesis and secretion. These cells secreted steroid hormones, secreted Schwangerschafts protein I, actively incorporated tritiated glycoprotein precursors and expressed surface immunoreactivity for the beta-subunit of human chorionic gonadotrophin (hCG). However, medium concentrations of hCG and human placental lactogen dropped rapidly to undetectable levels after 14 days in primary culture. Cells grown beyond confluence differentiated into 1 to 2 mm structures with a villus-like histology. Our studies indicate that cytotrophoblast can secrete steroids, cytotrophoblast differentiation occurs in vitro in the absence of maternal tissues, hCG synthesis occurs in cultured cytotrophoblast and medium concentrations of placental protein hormones are not the best indicators of cell viability for cultures of cytotrophoblast.     

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.     

Effect of gonadal steroids on gonadotropin-releasing hormones stimulated human chorionic gonadotropin release by trophoblast cells

The effect of gonadal steroids on basal and GnRH-stimulated hCG release was studied using collagenase-dispersed trophoblast cells from early pregnancy. Both GnRH and a GnRH superagonist, Buserelin, stimulated hCG release with a similar dose dependency. Progesterone (0.1 to 10 micrograms/ml) inhibited GnRH-stimulated hCG release in a dose dependent manner as well as basal hCG release. Relatively high concentrations of estradiol (10 micrograms/ml) stimulated both basal and GnRH-mediated hCG release and antagonized the inhibitory effect of progesterone on hCG release at 1 micrograms/ml as well as RU486 (1 microgram/ml). These results indicate that progesterone has an important role in both basal and GnRH-mediated hCG regulatory system in the placenta.     

The relationship between trophoblast differentiation and the production of bioactive hCG

We previously showed that a significant number of failing pregnancies are associated with production of human chorionic gonadotropin (hCG) having relatively low bioactivity. The present study was designed to compare the secretion of intact, immunoreactive hCG to the secretion of bioactive hCG during trophoblast differentiation, and to test the hypothesis that the lower bioactive: immunoreactive hCG ratios in failing pregnancies are related to reduced or impaired trophoblast differentiation. Cytotrophoblast cells were isolated from term placentas and cultured under conditions that induced or did not induce syncytiotrophoblast formation. Culture media were collected at regular intervals up to 72 h and levels of immunoreactive and bioactive hCG were measured. The differentiation of cytotrophoblast cells to multinucleated syncytiotrophoblast was monitored by immunocytochemistry and electron microscopy. During the 72 h culture period, concentrations of immunoreactive and bioactive hCG increased in both differentiating and non-differentiating cells. However, the concentrations of immunoreactive and bioactive hCG were higher under culture conditions that promoted trophoblast differentiation. Furthermore, the ratio of bioactive hCG to immunoreactive hCG was higher in differentiating cultures. When differentiation was inhibited by dimethyl sulfoxide, the secretion of bioactive hCG was reduced and the bioactive: immunoreactive hCG ratio did not change. These findings are consistent with the idea that production of bioactive hCG accompanies syncytiotrophoblast formation.     

催乳素及多巴胺对大鼠卵巢颗粒细胞产生甾体激素的影响

使用含有FSH和雄烯二酮的DME-F 12培养液培养未成熟大鼠的卵巢颗粒细胞,观察了不同剂量的催乳素和多巴胺对颗粒细胞合成孕酮和雌二醇的影响。结果表明,低浓度的催乳素(0.5 μg/ml)促进孕酮产生,高浓度时(5 μg/ml)抑制孕酮产生。催乳素能抑制雌二醇产生。低浓度的多巴胺(0.5 μg/ml)不影响这两种甾体激素的产生。当浓度为5μg/ml时能明显抑制这两种激素的产生。但是,多巴胺对颗粒细胞发挥作用的机制需进一步研究。     

Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy

Problem: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8–13 weeks of gestation (WG)] and term placentae (TP; 38–42 WG).Methods: Serum levels of these hormones were determined in 15 paired maternal (7–41 WG) and fetal (37–41 WG) samples. Cytotrophoblast cells were isolated from term (TP; 38–42 weeks) and early (EP; 8–13 weeks) placentae by enzymatic digestion and subsequent purification on a Percoll gradient. These cells were cultured for 6 days. The production of the hormones hPL, hCG, and leptin was determined as release during culture + cell content after culture – cell content before culture.Results: Serum levels (mean ± SD; n ± 15) at 7–12 and 37–41 WG were 89,652 ± 21,431 and 13,620 ± 5854 mIU/ml for hCG, 400 ± 182 and 7088 ± 2030 ng/ml for hPL, and 12,675 ± 4266 and 32,236 ± 10,961 pg/ml for leptin, respectively. For cultured cells from EP and TP, hCG and hPL showed different patterns of release during the first 2–3 days. While the release of these two hormones by EP cytotrophoblast cells continued during 6 days in culture, their concentrations reached a plateau for TP cytotrophoblasts between 4 and 6 days. Leptin was undetectable (<15 pg/ml) in TP cell cultured media, while for EP all three hormones showed the same release profiles. Production calculated for 30,000 TP trophoblast cells cultured for 6 days (n = 8) was 2–31 mIU for hCG and 0.5–2 ng for hPL. For EP (n = 11), it was 50–1070 mIU for hCG, 15–323 ng for hPL, and 137–580 pg for leptin. Net synthesis of hCG and hPL for TP was >10-fold and <1-fold, respectively. In contrast, the production of all three hormones for EP was at least 100 times the initial cell content.Conclusions: These results demonstrate that trophoblasts from early pregnancy show much higher production rates of hCG, hPL, and leptin than at term. However, the in vitro findings are difficult to be reconciled with the different serum concentrations of the two hormones hPL and leptin observed during the course of pregnancy.     

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.