OVARIAN STEROID CELLS : I. Differentiation of the Lutein Cell from the Granulosa Follicle Cell during the Preovulatory Stage and under the Influence of Exogenous Gonadotrophins

The granulosa follicle cell of the Graafian follicle of the rabbit ovary differentiates into a lutein cell involved in steroid synthesis. Cytological events which occur within the granulosa cell of the normally stimulated follicle prior to ovulation have been duplicated by the intrafollicular injection of exogenous gonadotrophin. The luteinization of the granulosa cells involves the accumulation of 250- to 300-A, electron-opaque, spherical granules, dispersed within the cytoplasmic matrix, which have been identified as glycogen with the PAS-staining procedure. Further development of the granulosa cell following ovulation involves an increase in cell size, a decrease in the number of RNP particles, and an accumulation of an abundant system of intracellular membranes (agranular endoplasmic reticulum). Glycogen granules first appear in the granulosa cells as the separate, monoparticulate form. After follicle rupture and the formation of agranular endoplasmic reticulum, glycogen particles are present in a rosette arrangement within membrane-bounded vacuoles. The rosette arrangement of glycogen particles is also found dispersed within the cytoplasmic matrix of the lutein cell during the later stages of the cell life-span. Injection of luteinizing hormone or human chorionic gonadotrophin into a mature follicle also produces a marked accumulation of monoparticulate glycogen in the majority of granulosa cells, within 30 min. Cytoplasmic extensions which contain the glycogen masses are noticeably free of RNP particles.     

中国荷斯坦奶牛卵巢卵泡细胞的免疫组织化学研究

目的和方法应用七种特异性抗体(sGCα、sGCβ、nNOS、FOXO1、PDE4、PKB/Akt和Inhibinα)对中国荷斯坦奶牛卵巢卵泡细胞进行免疫组织化学定位。结果表明PKB主要存在于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞,黄体细胞中没有检测到;FoxO1主要位于原始卵泡、腔前卵泡和有腔卵泡颗粒细胞;PDE4仅位于部分有腔卵泡的颗粒细胞;抑制素α、nNOS、sGCα和β存在于各级卵泡的颗粒细胞层,其中sGCα和β主要存在于原始卵泡和腔前卵泡的颗粒细胞。结论这七种蛋白的阶段和细胞特异性表达表明它们与中国荷斯坦卵巢卵泡的发育、闭锁和黄体化过程有着密切的关系。     

三种鳞翅目昆虫的血球细胞和卵巢细胞的体外培养

自从Grace(1962)成功地从昆虫中建立了第一个细胞株后,到1975年已从52种昆虫中分离到115个细胞株(Maramorosch,1975)。培养的细胞株被广泛地应用于病毒学、发育生物学、细胞生物学、病理学、毒物学、生物化学和遗传学等方面的研究(Hink,1972;Weiss,1971;Vago,1971和 1972;Kurstak和Maramorosch,1976)。 为了进一步弄清昆虫病毒和寄主细胞间的相互关系及环境对它们的影响,我们在离体条件下培养了鳞翅目民虫的血球细胞和卵巢细胞。现把初步结果简述于下。     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子(PAI-1)的激素调节

哺乳动物卵巢合成两类纤溶酶原激活因子(PA),即组织型PA(tPA)和尿激酶型PA(uPA)。大鼠卵巢除主要合成tPA外,还分泌一种纤溶酶激活因子的抑制因子(PAI-1)。在促性腺激素作用下,卵巢tPA和PAI-1两种基因的协调表达是导致滤泡破裂的原因。本实验进一步证实,卵巢中的PAI-1主要由膜-间质细胞分泌,可能作为一种屏障限制颗粒细胞tPA分泌到滤泡外间质。当排卵来临时,两种细胞所分泌的tPA和PAI-1相互作用后仍发现有大量tPA活性。这可能是引起排卵的主要原因。因为成熟的卵丘细胞除分泌高量tPA外,还分泌大量PAI-1,在人工授精中两者有可能作为鉴定卵子优劣的可靠指标。     

SECRETORY CELLS OF LILY PISTILS. II. ELECTRON MICROSCOPE CYTOCHEMISTRY OF CANAL CELLS

The secretory cells which line the canal of Lilium longiflorum pistils possess, on the side facing the canal, an elaborate wall which, with associated structures, Rosen and Thomas (1970) termed the “secretion zone.” We examined the secretion zone in the electron microscope following treatment of excised pistil slices with extraction procedures which remove pectin, hemicellulose, cellulose, lipid, or protein. The outer, fibrillar wall (layer 1) of the secretion zone contains protein, pectin, and cellulose. Internal to layer 1 is a granular-fibrillar wall (layer 2) several microns thick. It consists of outer and inner regions which can be distinguished from each other cytochemically. The granular component is composed of pectin which is not esterified with methyl groups and which may be complexed with protein. The short, randomly dispersed microfibrils of layer 2 were sensitive to procedures which dissolve cellulose. The extraction procedures did not reveal the chemical nature of the “osmiophilic islands” of layer 2. Paramural body membranes appear to be composed of glycoprotein and may function in secretion by serving as sites of pinocytic interchange at the plasmalemma. The origin of stigmatic exudate and the release of canal cell secretion product are discussed.     

心房钠尿肽(ANP)对大鼠卵巢细胞生成雌激素与孕激素的影响

应用大鼠卵巢黄体细胞、颗粒细胞培养以及放射免疫分析法,观察了α型心房钠尿肽(α-ANP)对性甾体激素孕酮(P)和雌二醇(E_2)分泌的影响,结果发现,0.1—10ng/ml 浓度的α-ANP 促进离体培养的大鼠黄体细胞分泌孕酮,并呈量效关系。α-ANP 也促进大鼠卵泡颗粒细胞分泌孕酮,但对分泌雌二醇没有影响。说明α-ANP 也影响卵巢分泌功能。     

阿克拉霉素协同顺铂杀伤卵巢癌细胞中泛素表达

为探讨泛素在阿克拉霉素(aclarubicin,ACR)协同顺铂(cisplatin,CDDP)杀伤卵巢癌细胞中的作用,应用ACR及CDDP的药物血浆峰浓度,分别及共同作用SKOV3卵巢癌细胞株24h。采用吖啶橙、嗅乙啶双荧光染色观察细胞活率;克隆培养观察克隆形成率,以及免疫细胞化学方法检测泛素表达。结果可见:细胞活率,ACR组为78.3±3.1;CDDP组为70.1±6.6;ACR和CDDP共用组为50.8±3.2(P<0.05)。克隆培养显示,ACR和CDDP共用组能够显著抑制细胞的生存能力。同时,两药共用组细胞内泛素表达水平明显高于药物单用组。以上结果表明:ACR与CDDP合用能够提高对卵巢癌细胞杀伤效果,泛素-蛋白质降解途径在其中发挥重要作用。     

INFLUENCE OF SINUSOIDAL 50-HZ MAGNETIC FIELD ON CULTURED HUMAN OVARIAN GRANULOSA CELLS

Several studies have indicated that magnetic fields are able to influence reproduction in mammals, but very few data are available on the effect of magnetic fields on ovarian function. In this study granulosa cells isolated from follicular aspirates of 25 in vitro fertilization treated women were cultured and exposed to a sinusoidal 50-Hz AC magnetic field during the entire time of a 48-h incubation with a flux density of B(AC) = 100 μT. Progesterone (P) production by granulosa cells was determined by radioimmunoassay. Granulosa cells in culture released high amounts of P. The magnetic field induced a significant increase in P production by granulosa cells obtained from eight individuals, when comparisons were made between exposed cells and sham-exposed cells of the same patient. In 17 subjects no alteration in P production was found. The positive treatment-related hormonal response in granulosa cells obtained from 32% of patients studied indicates that extremely low-frequency magnetic fields may interfere with P biosynthesis by human granulosa cells. Considering the pivotal role of P in gonadal and uterine function, these data may help to draw attention to the role of this physical environmental factor in modulating human female reproductive function.     

阿克拉霉素对顺铂和足叶乙甙杀伤卵巢癌细胞的影响

有效化疗药物的选择对于提高卵巢癌的治疗效果至关重要。本应用MTT法,分别测定了阿拉克霉素（Aclarubicin,ACR）血浆峰浓度与不同浓度顺铂（Cisplatin,CDDP）或足叶乙甙（Etoposide,VP-16）合用对SKOV3卵巢癌细胞24h的抑制率,并分析药物合并效应。采用荧光染色观察细胞凋亡;琼脂糖凝胶电泳检测DNA断裂;并用western印迹检测TopoII表达与药物作用之关系。结果表明,ACR对VP-16具有拮抗作用;与药物单用相比,二合用后使细胞凋亡率减少、DNA断裂减少,拓扑异构酶II表达增强。CDDP单用可以促进TopoII表达。ACR与CDDP和对卵巢癌细胞的抑制具有协同效果;表现为细胞凋亡率增加、DNA断裂增加,TopoII表达降低,尤其对TopoIIβ表达的抑制作用明显。因此认为：ACP与CDDP合用能够提高对卵巢癌细胞的抑制效果,TopoII是其重要的作用靶点。该结果对于临床治疗具有一定的参考意义。     

MICROBODIES IN EXPERIMENTALLY ALTERED CELLS : II. The Relationship of Microbody Proliferation to Endocrine Glands

The liver cells of intact male rats given ethyl-α-p-chlorophenoxyisobutyrate (CPIB) characteristically show a marked increase in microbodies and in catalase activity, while those of intact female rats do not. In castrated males given estradiol benzoate and CPIB the increase in catalase activity and microbody proliferation is abolished, while in castrated females given testosterone propionate and CPIB the livers show a marked increase in microbodies and in catalase activity. No sex difference in microbody and catalase response is apparent in fetal and neonatal rats. Both sexes show a sharp rise in catalase activity on the day of birth, with a rapid decline at 5 days after birth. Thyroidectomy abolishes the hypolipidemic effect of CPIB in rats, but microbody proliferation and increase in catalase activity persists in thyroidectomized male rats, indicating that microbody proliferation can be independent of hypolipidemia. Adrenalectomy does not alter appreciably the microbody-catalase response to CPIB. These experiments demonstrate that (1) in adult rats, hepatic microbody proliferation is dependent to a significant degree upon male sex hormone but is largely independent of thyroid or adrenal gland hormones; (2) hepatic microbody proliferation is independent of the hypolipidemic effect of CPIB; (3) displacement of thyroxine from serum protein may not be sufficient cause for stimulation of microbody formation.     

山溪鲵卵巢滤泡细胞的显微与超微结构

对两栖类卵巢滤泡细胞的研究已有一些报道。Ｔｈｏｒｎｔｏｎ等 ( 1973)用电镜观察比较了蟾蜍 (Ｂｕｆｏｂｕｆｏ)的成熟滤泡细胞和排卵后滤泡细胞 ,认为两栖类没有膜细胞和颗粒细胞的分化。Ｄｕｍｏｎｔ等 ( 1978)较为系统地观察并描述了光滑爪蟾 (Ｘｅｎｏｐｕｓｌａｅｖｉｓ)卵泡壁的超微结构。Ｋｗｏｎ等( 1994)对黑斑蛙 (Ｒａｎａｎｉｇｒｏｍａｃｕｌａｔａ)卵巢组织细胞的离体培养研究表明 ,其类固醇激素的生成是在卵泡壁上完成的 ,并提出了两栖类卵泡类固醇生成的两类细胞模型。但目前有关有尾两栖类滤泡细胞结构与功能的研究仍少见…     

卵巢癌细胞与腹膜间皮细胞相互作用对VEGF和bFGF表达的影响

目的探讨卵巢癌细胞与腹膜间皮细胞(HPMC)相互作用对血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)表达的影响。方法用Millicell将卵巢癌细胞SKOV3与HPMC进行非接触性共培养,用RT-PCR检测细胞VEGF及bFGF mRNA表达,ELISA检测细胞条件培养液中VEGF及bFGF蛋白水平。结果共培养后,SK-OV3 VEGF及bFGF mRNA表达分别为3.62±0.23及3.41±0.25,条件培养液中VEGF及bFGF蛋白水平分别为(523.5±24.9)pg/ml及(156.4±17.3)pg/ml,与SKOV3单独培养时相比,差别均有显著性(P<0.01)。HPMC共培养后VEGF及bFGF mRNA表达分别为3.96±0.09及3.54±0.21,条件培养液中VEGF及bFGF蛋白水平分别为(1567.62±45.42)pg/ml及(682.9±33.7)pg/ml,均明显高于HPMC单独培养时的水平(P<0.01)。结论卵巢癌细胞与HPMC均可合成VEGF及bFGF;二者共培养时,相互刺激表达更高的VEGF及bFGF。     

Toxoplasma gondii-Vertebrate Cell Interactions. II. The Intracellular Reproductive Phase

SYNOPSIS.
The reproduction of Toxoplasma gondii RH-strain in vertebrate cells was studied in a controlled-environment culture system. The lag period before reproduction and the doubling time of individual parasites were determined using a least-squares linear regression method of analysis which does not artificially constrain the data. In the majority of cases, the time intercept of the linear regression line was either zero, implying the lack of a lag phase before reproduction, or negative, implying the parasite had completed part of its reproductive cycle before entering the host cell. The mean doubling time of T. gondii is 10.9 h in bovine embryo skeletal muscle cells and 8.3 h in HeLa cells. This difference is not significant at the 5% level. The population doubling times of mouse-derived parasites is best described by a gamma distribution.     

内皮素—1对大鼠排卵前卵泡颗粒细胞产生孕酮的影响

本文用离体细胞体外孵育法研究了内皮素－１（ＥＴ）对大鼠排卵前卵泡颗粒细胞孕酮生成的影响及其作用机理。结果发现,ＥＴ能显著抑制ｈＣＧ刺激下的孕酮产生,抑制作用在浓度为１０－８ｍｏｌ／Ｌ时,即有显著意义（Ｐ＜０．０５,ｎ＝６）,至１０－７ｍｏｌ／Ｌ时则有非常显著的意义（Ｐ＜０．０１,ｎ＝６）;不同浓度ＥＴ（１０－７—１０－７ｍｏｌ／Ｌ）,对颗粒细胞基础孕酮的产生无明显影响。进一步研究表明,ＥＴ对ｈＣＧ刺激下孕酮生成的抑制作用,在用免抗人内皮素抗血清（ＥＴ－Ａ）１：１０００及ｃＡＭＰ后能明显被逆转。实验中还观察到,ＥＴ使颗粒细胞ＬＨ／ｈＣＧ受体数下降,亲和力降低。本文结果提示,ＥＴ可能为卵巢内的一种局部调节肽,通过作用于ＥＴ受体,干扰ＬＨ／ｈＣＧ受体功能和ｃＡＭＰ生成而抑制颗粒细胞孕酮的产生。     

多囊卵巢综合征模型鼠颗粒细胞凋亡及TRAIL蛋白的表达

目的通过观察卵巢颗粒细胞凋亡及TRAIL(肿瘤坏死因子相关凋亡诱导配体)蛋白的表达情况,探讨颗粒细胞凋亡与PCOS发病的相关性及凋亡调控蛋白TRAIL在PCOS颗粒细胞凋亡中的作用。方法采用硫酸普拉睾酮钠诱导大鼠PCOS模型,3’-末端原位标记法(TUNEL)检测大鼠卵巢颗粒细胞凋亡情况,免疫组化染色及RT-PCR分析检测TRAIL蛋白及TRAIL mRNA在颗粒细胞的表达。结果PCOS组大鼠卵巢窦状卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达较对照组明显增强(P<0.01,P<0.05),窦前卵泡颗粒细胞凋亡发生率及TRAIL蛋白的表达两组无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未发现凋亡征象及TRAIL蛋白表达。PCOS组大鼠卵巢颗粒细胞TRAIL mRNA的表达较对照组明显增强(P<0.01)。结论PCOS大鼠卵巢窦状卵泡颗粒细胞凋亡明显增强,TRAIL在PCOS大鼠卵巢颗粒细胞凋亡调控中发挥了作用。     

LIPOSOMAL DAUNORUBICIN OVERCOMES DRUG RESISTANCE IN HUMAN BREAST,OVARIAN AND LUNG CARCINOMA CELLS

ABSTRACT

Multi-drug resistance due in part to membrane pumps such as P-glycoprotein (Pgp) is a major clinical problem in human cancers. We tested the ability of liposomally-encapsulated daunorubicin (DR) to overcome resistance to this drug. A widely used breast carcinoma cell line originally selected for resistance in doxorubicin (MCF7ADR) was 4-fold resistant to DR compared to the parent MCF7 cells (IC₅₀ 79 nM vs. 20 nM). Ovarian carcinoma cells (SKOV3) were made resistant by retroviral transduction of MDR1 cDNA and selection in vinblastine. The resulting SKOV3MGP1 cells were 130-fold resistant to DR compared to parent cells (IC₅₀ 5700 nM vs. 44 nM). Small-cell lung carcinoma cells (H69VP) originally selected for resistance to etoposide were 6-fold resistant to DR compared to H69 parent cells (IC₅₀ 180 nM vs. 30 nM). In all three cases, encapsulation of DR in liposomes as Daunoxome (Gilead) did not change the IC₅₀ of parent cells relative to free DR. However, liposomal DR overcame resistance in MCF7ADR breast carcinoma cells (IC₅₀ 20 nM), SKOV3MGP1 ovarian carcinoma cells (IC₅₀ 237 nM) and H69VP small-cell lung carcinoma cells (IC₅₀ 27 nM). Empty liposomes did not affect the IC₅₀ for free DR in the three resistant cell lines, nor did empty liposomes affect the IC₅₀ for other drugs that are part of the multi-drug resistance phenotype (etoposide, vincristine) in lung carcinoma cells. These data indicate the possible value of liposomal DR in overcoming Pgp-mediated drug resistance in human cancer.     

Cell Synchrony Techniques. II. Analysis of Cell Progression Data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times T_G1, T_s and T_G2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G₁ state have a 14-hr cycle time with T_G1, T_s and T_G2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G₂M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G₁ population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.