邻苯二甲酸二辛酯对大鼠卵巢颗粒细胞分泌功能的影响

以邻苯二甲酸二辛酯(DOP)对大鼠卵巢颗粒细胞进行体外染毒、培养来研究DOP对卵巢颗粒细胞分泌功能的影响。提取未成年大鼠卵巢颗粒细胞进行体外培养,DOP染毒后测定细胞分泌产物(雌二醇和孕酮)含量。染毒组与对照组比较,雌二醇和孕酮水平均降低,差异有统计学意义(P<0.01)。雌二醇含量与DOP剂量的相关系数为-0.68(P<0.01);孕酮含量与DOP剂量的相关系数为-0.79(P<0.01),提示在实验剂量下产生剂量-反应关系。DOP对大鼠卵巢颗粒细胞分泌功能有抑制作用。     

二噁英对大鼠卵巢颗粒细胞分泌功能的影响

分别以4．00、0．40、0．04ng/ml浓度的2,3,7,8-四氯苯二噁英（TCDD）对体外培养的大鼠卵巢颗粒细胞进行24h染毒,测定细胞活性和细胞分泌产物（雌二醇和孕酮）含量。结果显示,各染毒组与对照组比较,雌二醇和孕酮水平均有所降低,差别有统计学意义（P〈0．01）,且有剂量-反应关系。提示TCDD抑制大鼠卵巢颗粒细胞分泌功能。     

铅对大鼠原代卵巢颗粒细胞分泌雌激素能力的影响

目的检测铅对大鼠卵巢颗粒细胞分泌功能的影响。方法分别以250、50、10mg/L终浓度的醋酸铅对体外培养的大鼠卵巢颗粒细胞进行24h染毒,测定细胞活性和细胞分泌产物(雌二醇和孕酮)含量。结果各染毒组与对照组比较,细胞活性未受影响,而雌二醇和孕酮水平均有所降低,高、中剂量组与对照组比较差异有显著性(P<0.01)。雌二醇含量与醋酸铅剂量的相关系数为-0.900(P<0.01);孕酮含量与醋酸铅剂量的相关系数为-0.908(P<0.01),提示在实验剂量下产生剂量-效应关系。结论铅对大鼠卵巢颗粒细胞分泌功能有抑制作用。     

二(口恶)英对大鼠卵巢颗粒细胞分泌功能的影响

分别以4.00、0.40、0.04 ng/ml浓度的2,3,7,8-四氯苯二(口恶)英(TCDD)对体外培养的大鼠卵巢颗粒细胞进行24 h染毒,测定细胞活性和细胞分泌产物(雌二醇和孕酮)含量.结果显示,各染毒组与对照组比较,雌二醇和孕酮水平均有所降低,差别有统计学意义(P＜0.01),且有剂量-反应关系.提示TCDD抑制大鼠卵巢颗粒细胞分泌功能.     

硫酸镍对雌性大鼠卵巢的毒性作用及其机制

选健康性成熟Wistar雌性大鼠,每次以5．00mg/kg、2．50mg/kg、1．25mg/kg硫酸镍(NiSO4)腹腔注射染毒,0．2ml／100g动物体重,1次／d,连续21d。于最后一次染毒结束次日心脏采血放免法测雌二醇(E2)、孕酮(P),同时测定卵巢NOS酶活力、NO含量及卵巢和子宫镍含量。结果 染毒各剂量组动物子宫、卵巢镍含量及卵巢NOS酶活力与对照组比较差异有显著性;5．00mg/kg、2．50mg/kg染毒组血清E2、P含量,卵巢NO含量与对照组比较差异有显著性。提示腹腔NiSO4染毒引起雌性大鼠卵巢损伤,血清E2、P下降,其机制可能与卵巢镍含量升高诱导NOS、NO水平增高有关。     

MC-LR对小鼠卵巢颗粒细胞活性及雌孕激素分泌的影响

目的研究微囊藻毒素LR(MC-LR)对小鼠卵巢颗粒细胞活性及雌孕激素分泌的影响。方法所制备21日龄雌性小鼠卵巢颗粒细胞,以0、5、10和20μg/mL MC-LR染毒,分别于24、48和72h检测其增殖活性及雌孕激素分泌情况。结果染毒24h,随着剂量增加其增殖活性上升,20μg/mL剂量组高于对照组;染毒48和72h,其增殖活性下降,20μg/mL剂量组低于对照组。染毒24h,各剂量组的雌二醇分泌量增加,均高于与对照组,随着染毒时间延长分泌量下降;至72h,各剂量组的分泌量均明显低于对照组。随着染毒剂量增加和时间延长,孕酮分泌有不同程度的上升。结论 MC-LR对小鼠卵巢颗粒细胞具有细胞毒性并影响雌二醇和孕酮的分泌。     

MIS,IGF-Ⅱ对人卵巢黄素化颗粒细胞分泌功能的影响

目的:探讨MIS,IGF-Ⅱ对人卵巢黄素化颗粒细胞分泌雌激素的影响。方法:体外受精-胚胎移植(IVF-ET)患者经阴道超声引导下取卵,剥离卵母细胞周围的颗粒细胞作体外培养,培养48h后更换无血清培养基,在有或无卵泡刺激激素(FSR)的作用下,分别加入基因重组人苗勒氏管抑制因子(rhMIS)及人胰岛素样生长因子Ⅱ(rhIGF-Ⅱ)作用于颗粒细胞,于培养后第4、6、8、10天收集培养液测定雌二醇(E2),观察rhMIS及rhIGF-Ⅱ对体外培养的颗粒细胞分泌E2的影响。结果:在无FSH作用下,rhMIS对颗粒细胞分泌E2无明显影响(P>0·05),rhIGF-Ⅱ刺激颗粒细胞分泌E2量增加(P<0·05);加入FSH后,rhMIS的明显抑制颗粒细胞E2分泌量(P<0·05),rhIGF-Ⅱ明显增加颗粒细胞E2的分泌(P<0·05)。结论:MIS和IGF-Ⅱ可单独或协同FSH刺激颗粒细胞甾体激素的分泌,对卵泡的生长发育及卵母细胞的分化成熟起着微观调控作用。     

戊酸雌二醇片联合地屈孕酮片对多囊卵巢综合征患者性激素及卵巢血流动力学的影响

目的分析戊酸雌二醇片联合地屈孕酮片治疗多囊卵巢综合征的临床疗效及对性激素、卵巢血流动力学的影响。方法选取2020年1月—2021年12月树兰(杭州)医院收治的102例多囊卵巢综合征患者为研究对象,按照数字表法分为对照组、观察组,分别予以地屈孕酮片单药治疗及戊酸雌二醇片联合治疗。治疗3月后,对比两组卵巢子宫形态、卵巢血流动力学、性激素[睾酮(T)、雌二醇(E_(2))、黄体生成素(LH)、卵泡刺激素(FSH)]及adropin蛋白(adropin)、硫酸脱氢表雄酮(DHEAS)水平,对比其临床疗效。结果治疗后,患者子宫内膜厚度增加,卵巢体积减小,相比对照组,观察组患者子宫内膜厚度较厚,卵巢体积较小,差异均有统计学意义(均P<0.05)。治疗后,患者T、LH、FSH、DHEAS水平降低,E_(2)、adropin水平升高,相比对照组,观察组T、LH、FSH、DHEAS水平较低,E_(2)、adropin水平较高,差异均有统计学意义(均P<0.05);治疗后,患者PI、PSV值升高,RI值降低,相比对照组,观察组PI、PSV值较高,RI值较低,差异均有统计学意义(均P<0.05)。相比对照组,观察排卵率、妊娠率、月经恢复率及临床治疗有效率较高,差异有统计学意义(P<0.05)。结论给予多囊卵巢综合征患者戊酸雌二醇片、地屈孕酮片联合治疗临床疗效确切,可有效改善患者机体性激素、血液状态,为卵巢排卵提供良好的环境。     

邻苯二甲酸二丁酯对大鼠卵巢颗粒细胞中细胞凋亡因子Bcl-2、Bax表达的影响

目的研究邻苯二甲酸二丁酯(dibutyl phthalate,DBP)对大鼠卵巢颗粒细胞中细胞凋亡因子Bcl-2、Bax表达的影响。方法体外培养25 d雌性SD大鼠卵巢颗粒细胞,分别加入DBP使终浓度分别(0、5、20、80μmol/L),培养24 h。采用放射免疫方法测定雌二醇(estradiol,E2)和孕酮(progestone,P)的水平;采用实时荧光定量PCR(real-time PCR)和免疫印迹试验(Western blot)检测卵巢颗粒细胞中Bcl-2、Bax mRNA和蛋白的表达。结果 DBP染毒后颗粒细胞分泌的E2及P水平下降,各剂量DBP均可降低颗粒细胞中Bcl-2 mRNA和蛋白的表达(P0.05),且表达量与DBP剂量呈反比,同时,DBP可以增加颗粒细胞中Bax mRNA和蛋白的表达(P0.05),且表达量与DBP剂量呈正比。结论 DBP可导致大鼠卵巢颗粒细胞凋亡,且Bcl-2/Bax比例失调可能是导致其凋亡的机制。     

大豆异黄酮对去卵巢大鼠骨钙、骨强度的影响

目的　利用卵巢切除大鼠模型 ,研究大豆异黄酮对体内骨转换、雌二醇、骨钙含量及骨强度的影响 ,探讨大豆异黄酮的剂量 效应关系。方法　卵巢切除大鼠分为 3个剂量组 ,分别灌胃给与 12 . 6 5 (L SI,低剂量组 ) ,2 5 . 30(M SI ,中剂量组 ) ,37. 95mg/(kg·bw) (H SI ,高剂量组 )。同时设阴性对照组 (OVX)和假手术组 (Sham) ,以蒸馏水灌胃。喂养 12周后 ,处死大鼠 ,分离血清 ,同时剥离右侧股骨及右侧胫骨 ,进行各项指标的测定。结果　OVX组骨形成指标碱性磷酸酶 (AKP)比Sham组及H SI组显著升高 (P <0. 0 5 ) ,其骨吸收指标抗酒石酸酸性磷酸酶 (StrACP)显著性高于Sham组、M SI组及H SI组 (P <0 . 0 5 )。Sham组及各大豆异黄酮剂量组雌二醇 (E2 )有上升趋势 ,中剂量组(M SI)E2 有显著性上升 (P <0 . 0 5 )。Sham组与大豆异黄酮各剂量组内钙含量与OVX组相比显著增高 ,反映骨强度性能的指标 (最大负载、弹性系数及能量 )也较OVX组明显增强。结论　大豆异黄酮能显著改善但不能完全逆转业已形成的骨质疏松。     

Beta adrenoceptors mediate the catecholamine-induced stimulation of oxytocin secretion from cultured bovine granulosa cells.

Bovine granulosa cells were treated in culture with alpha- and beta-adrenoceptor ligands to determine the receptor subtype mediating their response to catecholamines. The secretion of oxytocin by granulosa cells in serum-free medium was measured on the fourth day of culture (during the period of acquisition of a luteal phenotype). Cultures were performed in the presence of 0.5 mM ascorbic acid, which increased hormone output and potentiated the response to catecholamines. The effects of adrenaline and noradrenaline on oxytocin secretion were concentration-dependent; maximum stimulation was over 700% with adrenalin (EC50 92 nM) and 500% with noradrenaline (EC50 87 nM). The response to noradrenaline (10(-6) M) and adrenaline (10(-6) M) could be blocked by propranolol but not by phentolamine, suggesting that beta- rather than alpha-adrenoceptors were involved. Blockade by metoprolol and practolol (beta 1-adrenoceptor antagonists) was poor and dobutamine (beta 1-agonist) was weakly stimulatory. A concentration-dependent stimulatory response (EC50 200 nM) was obtained with salbutamol (beta 2-adrenoceptor agonist) and stimulation by adrenaline or salbutamol could be blocked by a selective beta 2-adrenoceptor antagonist (ICI 118,551). It is concluded that, during luteinization, the long-term response of bovine granulosa cells to stimulation induced by catecholamines is mediated through beta- rather than alpha-adrenoceptors. Although the beta 2-subtype is probably involved, the similar potencies of adrenaline and noradrenaline are uncharacteristic of beta 2-adrenoceptors and may be peculiar to the long-term response shown by these cells.     

The biphasic modulation of inhibin mRNA levels and secretion by PMSG in rat granulosa cells in vitro

Granulosa cell cultures derived from diethylstilboestrol-treated immature rats were used to study the in vitro effect of pregnant mare serum gonadotrophin (PMSG) on steady state mRNA levels for the inhibin alpha and beta A subunits and the secretion of immunoreactive inhibin and progesterone. After 48 h treatment the dose-response curve of PMSG revealed a maximum stimulation (2.5-3.5 fold) of cytosolic alpha and beta A mRNAs over the range of 1 to 10 mU PMSG mL-1, with corresponding stimulation of inhibin secretion. A high dose of PMSG (160-500 mU mL-1) clearly suppressed inhibin alpha mRNA levels as well as inhibin secretion, whereas progesterone (P) was maximally stimulated (up to 600 fold). Although the level of cytosolic inhibin beta A subunit mRNA was also down-regulated by a high concentration of PMSG in the culture medium, the doses required to suppress its mRNA level to less than those of the control varied. These data demonstrate that low doses of follicle stimulating hormone/luteinizing hormone (FSH/LH)-like (PMSG) activity enhances and high doses decrease the steady-state mRNA levels of inhibin in rat granulosa cells in vitro; this biphasic regulation in vitro reflects the differential regulation of inhibin secretion observed during the rat oestrous cycle.     

Effect of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture

ABSTRACT

Information on the role of resistin on steroidogenesis is limited to animal studies. The aim of this study was to investigate the effect of various doses of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture. Granulosa cells were obtained from follicular fluid aspirated from 50 women undergoing in vitro fertilization (IVF) treatment. The cells were cultured for 48 h after a 24 h pre-incubation period. The effect of resistin at dosages 1, 10 and 100 ng/ml alone or in combinations with FSH (10 and 100 ng/ml) on steroidogenesis was investigated. Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. FSH treatment increased both estradiol and progesterone secretion. Resistin suppressed basal estradiol (at 1 ng/ml) and progesterone secretion (at all concentrations tested). When resistin (all concentrations) was combined with FSH (100 ng/ml), it eliminated the stimulatory effect of FSH on the secretion of estradiol and progesterone. This study indicates an inhibitory effect of resistin on the secretion of estradiol and progesterone by human luteinized granulosa cells in vitro. It is likely that this adipokine locally affects ovarian function in women.

Abbreviations: 3β-HSD: 3β-hydroxysteroid dehydrogenase; CAP1: cyclase-associated protein 1; DCN: decorin; FIZZ: Found in Inflammatory Zones; hCG: human chorionic gonadotropin; IGF1: insulin-like growth factor type 1; IVF: in vitro fertilization; PCOS: polycystic ovary syndrome; RIA: radioimmunoassay; ROR1: receptor tyrosine kinase-like orphan receptor-1; TLR4: Toll–like receptor 4     

Effect of hyperglycemic condition on proteoglycan secretion in cultured human endothelial cells

Background Proteoglycans (PGs) are important constituents of the plasma membrane and of the basement membrane supporting the endothelial cell layer. Changes in the amounts or the structures of PGs in the endothelium may affect important functions such as turnover of lipoproteins, filtration properties, and regulation of chemokines during inflammation, which are all relevant in diabetes. Aim of the study The purpose of this study was to investigate if hyperglycemic conditions would affect the biosynthesis and secretion of PGs in cultured primary human endothelial cells. Methods Primary human umbilical cord vein endothelial cells were established and cultured in vitro. The cells were cultured either in medium with low glucose (LG) (1 g/l) or high glucose (HG) (4.5 g/l). From day 3–4 cells were labeled with ³⁵S-sulfate for 24 h. ³⁵S-Labeled macromolecules (medium) were purified by gel chromatography, and isolated macromolecules were analyzed by gel chromatography after different types of treatment, electrophoresis, and immunoprecipitation. Results Lower levels of secreted PGs were found in human endothelial cells exposed to HG. The major part of the PGs released was of the heparan sulfate (HS) type, and immunoprecipitation experiments showed that one such PG was syndecan-1. However, there was no difference in the ratio between HS and chondroitin sulfate (CS) under the different experimental conditions. Further, the PGs expressed neither differ with regard to molecular size of the glycosaminoglycan (GAG) chains, nor were their polyanionic properties affected by the different experimental conditions. Conclusion: The results obtained suggest that treatment of primary human endothelial cells with hyperglycemia leads to a decrease in PG secretion in primary cultures of human endothelial cells.     

阿特拉津对大鼠支持细胞凋亡的影响

目的探讨阿特拉津对雄性SD大鼠睾丸支持细胞凋亡的影响及其作用机制。方法选用18~21日龄雄性SD大鼠,建立支持细胞原代培养模型。设溶剂对照组和阿特拉津10、50、100和200μmol/L 4个染毒剂量组,对支持细胞染毒24 h后,采用MTT法检测细胞活性,免疫荧光法观察波形蛋白的表达,流式细胞术测定细胞凋亡率,Real time-PCR与ELISA分别检测Fas L、caspase-3与caspase-8的mRNA表达及蛋白含量。结果阿特拉津50、100和200μmol/L染毒组细胞活力分别为82.47%、75.23%和53.76%,与溶剂对照组比较分别降低17.53%(P<0.05)、24.77%(P<0.05)、46.24%(P<0.01)。波形蛋白表达受抑制。阿特拉津50、100和200μmol/L染毒组细胞凋亡率分别为8.53%、13.07%和14.45%,与溶剂对照组比较分别升高86.86%(P<0.01)、186.13%(P<0.01)、216.06%(P<0.01)。Fas L、caspase-3、caspase-8的mRNA表达及蛋白含量上调(P<0.05或P<0.01)。结论阿特拉津可影响大鼠支持细胞活性,可通过Fas L途径诱导支持细胞凋亡。     

Effects of metformin and vanadium on leptin secretion from cultured rat adipocytes

OBJECTIVE: We have reported that glucose utilization regulates leptin expression and secretion from isolated rat adipocytes. In this study, we employed two antidiabetic agents that act to increase glucose uptake by peripheral tissues, metformin and vanadium, as pharmacological tools to examine the effects of altering glucose utilization on leptin secretion in primary cultures of rat adipocytes. RESEARCH METHODS AND PROCEDURES: Isolated adipocytes (100 microL of packed cells per well) were anchored in a defined matrix of basement membrane components (Matrigel) with media containing 5.5 mM glucose and incubated for 96 hours with metformin or vanadium. Leptin secretion, glucose utilization, and lactate production were assessed. RESULTS: Metformin (0.5 and 1.0 mM) increased glucose uptake in the presence of 0.16 nM insulin by 37 +/- 10% (p < 0.005) and 62 +/- 8% (p < 0.0001) over insulin alone, respectively. Metformin from 0.5 to 5.0 mM increased lactate production by 105 +/- 43% (p < 0.025) to 202 +/- 52% (p < 0.0025) and at 1.0 and 5.0 mM increased the proportional rate of glucose conversion to lactate by 78 +/- 18% (p < 0.005) and 166 +/- 41% (p < 0.0025), respectively. At concentrations less than 0.5 mM, metformin did not affect leptin secretion, but at 0.5 mM, the only concentration that significantly increased glucose utilization without increasing glucose conversion to lactate, leptin secretion was modestly stimulated (by 20 +/- 9%; p < 0.05). Concentrations from 1.0 to 25 mM inhibited leptin secretion by 25 +/- 8% (p < 0.005) to 89 +/- 4% (p < 0.0001). Across metformin doses, leptin secretion was inversely related to the percentage of glucose taken up and released as lactate (r = -0.74; p < 0.0001). Vanadium (5 to 20 microM) increased glucose uptake from 20 +/- 7% (p < 0.01) to 34 +/- 13% (p < 0.02) and increased lactate production at 5 microM by 17 +/- 8% (p < 0.025) and 10 microM by 61 +/- 20% (p < 0.02) but did not alter the conversion of glucose to lactate. Vanadium (5 to 50 microM) inhibited leptin secretion by 33 +/- 6% (p < 0.0025) to 61 +/- 8% (p < 0.0001). DISCUSSION: Both metformin and vanadium increase glucose uptake and inhibit leptin secretion from cultured adipocytes. The inhibition of leptin secretion by metformin is related to an increase in the metabolism of glucose to lactate. The inhibition by vanadium most likely involves direct effects on cellular phosphatases. We hypothesize that the effect of glucose utilization to stimulate leptin production involves the metabolism of glucose to a fate other than anaerobic lactate production, possibly oxidation or lipogenesis.     

软骨藻酸对原代培养大鼠神经胶质细胞膜的影响

目的 研究软骨藻酸(domoic acid,DA)对原代培养大鼠神经胶质细胞膜的损伤作用.方法 6.4×10-2、6.4×10-3,6.4×10-4 μmol/L的DA作用于原代培养的大鼠神经胶质细胞24 h后,分别测定细胞Na+-K+-ATPase和Ca2+-Mg2+ATPase的活力、细胞膜流动性和通透性的变化.结果 神经胶质细胞经DA处理后,Na+-K+-ATPase和Ca2+-Mg2+-ATPase的活力均明显受到抑制,细胞膜的流动性降低、通透性升高,低、中、高剂量DA染毒组荧光偏振度分别为0.0626±0.0051、0.0685±0.0097、0.0648±0.0086,微黏度分别为0.3154±0.0298、0.3510±0.0571、0.3286±0.0504,与对照组(荧光偏振度为0.0481±0.0069,微黏度为0.2338±0.0372)相比,差异均有统计学意义(P＜0.01).结论 DA能明显损害神经胶质细胞膜的功能,进而可能引发细胞的其他损伤.     

室内主要挥发性有机物对离体培养胚胎发育的影响

目的探讨室内主要挥发性有机物对大鼠胚胎发育的影响。方法采用植入后全胚胎培养模型,将9.5dSD大鼠胚胎与吸入染毒的大鼠即刻离心血清共培养48h,观察空气染毒大鼠血清对离体培养胚胎生长发育和组织器官形态分化的影响。结果大鼠在模拟小室中染毒10d,染毒浓度约为现场检测最高值的2、4、8、16倍。在16倍剂量组,胚胎的平均头长为2.07mm、干重为0.53mg、发育评分为26.1,均显著低于空白对照组(P<0.05),并出现畸形胚胎。结论高浓度挥发性有机物对大鼠胚胎具有胚胎毒性和致畸性。     

大鼠骨髓来源树突状细胞的体外诱导扩增及其功能鉴定

目的探讨体外诱导和扩增大鼠骨髓来源树突状细胞（DC）的方法，并观察其表型及功能特性。方法取得大鼠骨髓细胞后，去除红细胞，加入重组大鼠粒细胞／巨噬细胞集落刺激因子（rrGM-CSF）、重组大鼠白细胞介素4（rrIL-4）培养2周；在光镜和扫描电镜下观察培养DC的形态学特征，流式细胞仪检测DC表面MHC-Ⅱ、CD80、CD86的表达水平，混合淋巴细胞反应检测其刺激同种T细胞增殖能力。结果细胞培养8d后，经倒置显微镜和电镜观察DC出现典型形态，培养6d的DC具有不成熟表型，流式细胞仪检测MHC-Ⅱ为（29．03±4．39）％、CD80为（21．98±7．08）％，CD86为（25．94±6．80）％，刺激同种T细胞增殖能力极低，而培养12dDC的MHC-Ⅱ为（74．05±5．97）％、CD80为（79．85±6．53）％，CD86为（81．00±7．47）％，刺激同种T细胞增殖能力明显增强。结论大鼠骨髓来源干细胞可经联合应用细胞因子诱导培养为具有典型形态特征及功能的DC，为进一步研究其在移植免疫学中的应用奠定基础。