黄体功能不足不孕患者子宫内膜雌孕激素受体的单克隆抗体免疫组织化学研究

采用抗人雌激素受体（ＥＲ）和孕激素受体（ＰＲ）单克隆抗体，对３０例不孕妇女分为黄体功能不足（ＬＩＳ组）１４例和对照组１６例，进行ＥＲ、ＰＲ免疫组化测定，放射免疫法（ＲＩＡ）测定黄体中期血清雌二醇（Ｅ２）、孕酮（Ｐ）水平。结果：ＬＩＳ组黄体中期Ｅ２水平明显低于对照组（Ｐ＜０．０５），孕激素水平与对照组比较，无显著差异（Ｐ＞０．０５）；两组ＥＲ、ＰＲ染色的阳性位于细胞核内，胞浆中无阳性染色；ＬＩＳ组分泌晚期子宫内膜间质细胞ＰＲ显著低于对照组（Ｐ＜０．０５），分布不均。提示：黄体功能不足不孕子宫内膜分泌不良是由子宫内膜ＰＲ含量下降所致，而卵巢雌激素分泌不足导致ＰＲ合成减少。     

17β-雌二醇对子宫内膜异位症患者在位子宫内膜问质细胞β-catenin mRNA和蛋白表达的影响

目的研究17β-立群雌二醇（17β-E2）对子宫内膜异位症（内异症）患者在位子宫内膜间质细胞β-catenin mRNA和蛋白表达的影响，探讨Wnt／β-catenin信号通路在介导雌激素促进内异症发生发展的作用。方法体外分离培养内异症患者在位子宫内膜间质细胞。用不同浓度17β-E2处理子宫内膜间质细胞48h；此后选用10^-10mol／L 17β-E2处理子宫内膜间质细胞12、24和48h，逆转录聚合酶链反应（RT-PCR）和免疫印迹法（Western blotting）检测17β-E2处理前后子宫内膜间质细胞β-catenin mRNA和蛋白的表达水平。同法分析雌激素受体拮抗剂ICI182，780（10μmol／L）对17β-E2促进β-catenin mRNA和蛋白表达的影响。免疫组织化学染色观察17β-E2作用后β-catenin在子宫内膜间质细胞中的定位。结果17β-E2能明显促进内异症患者在位子宫内膜间质细胞β-catenin mRNA和蛋白的表达，并呈剂量和时间依赖性，于10^-10mol／L作用48h最明显。雌激素受体拮抗剂ICI182，780能明显抑制17β-E2对子宫内膜间质细胞β-catenin mRNA和蛋白的表达。免疫组织化学染色发现17β-E2能促进β-catenin在子宫内膜间质细胞核内的表达。结论雌激素可能通过激活Wnt／β-catenin信号通路促进内异症在位子宫内膜的异位种植。     

雌激素对卵巢切除大鼠内源性白细胞介素-6变化的影响

目的：探讨卵巢切除后大鼠内源性白细胞介素－６（ＩＬ－６）的变化及雌激素替补治疗对此的影响。方法：通过酶联免疫吸收法检测骨髓上清的ＩＬ－６含量,通过斑点杂交和免疫组织化学方法检测骨组织ＩＬ－６的表达和分布。结果：卵巢切除导致骨髓上清ＩＬ－６含量细胞均有较强的ＩＬ－６免疫活性。结论：雌激素缺乏使骨髓内源性ＩＬ－６增加可能是由于骨细胞系ＩＬ－６ｍＲＮＡ表害升高决定的,雌激素抗骨吸收作用部分是通过降低ＩＬ     

硬膜外小剂量吗啡四种配方术后止痛的比较研究

２００例ＡＳＡⅠ－Ⅱ级，年龄１８－４４岁，４５－６８ｋｇ的子宫或卵巢全部或部分切除的患者，以单盲法分为四组，每组４０例，分别于术后自硬膜外腔注入（１）吗啡２．５ｍｇ；（２）吗啡２．５ｍｇ＋保分子右旋糖酐６ｍｌ；（３）吗啡２．５ｍｇ＋纳洛酮０４ｍｇ；（４）吗啡２ｍｇ＋５％Ｎａ Ｃｌ６ｍｌ，比较了注药前及注药后１、３、６、９、１２ｈ的ＣＯ２通气反应、止痛效应及副作用。     

子宫内膜异位症患者雌、孕激素受体和Bcl-2、Bax表达的研究

目的探讨子宫内膜异位症雌孕激素受体、细胞凋亡相关基因的表达。方法用免疫组织化学方法及图像分析技术检测雌激素受体（ER）、孕激素受体（PR）、B细胞淋巴瘤／白血病基因-2（Bcl-2）、Bcl相关的X蛋白（Bax）在子宫内膜异位症患者异位及在位内膜细胞中的表达情况。结果异位内膜ER、PR、Bcl-2表达明显低于在位内膜（P〈0．05）,异位内膜ER、Bcl-2表达明显高于正常内膜（P〈0．05）,异位内膜Bax表达明显低于正常内膜（P〈0．05）,Bax在异位内膜与在位内膜表达差异无统计学意义（P〉0．05）。结论无论是增殖期还是分泌期,在位内膜ER、PR表达均高于异位内膜及正常内膜,在位内膜细胞持续低水平增殖。在位内膜及异位内膜中Bcl-2和Bax的表达均与子宫内膜周期性改变无关,不受卵巢激素调节。     

肝癌外周血T淋巴细胞丝裂原反应性和血浆白介素—2,白介素—6 …

目的 探讨肝癌发病机理。方法 应用生物测定法检测肝癌患者外周血Ｔ淋巴细胞丝裂原反应性和血浆白细胞介素－２（ＩＬ－２），白细胞介素－６（ＩＬ－６）活性及可溶性白细胞介素－２受体（ＩＬ－２Ｒ）的表达。结果 肝癌患者Ｔ淋巴细胞丝裂原反应性和血浆ＩＬ－２，ＩＬ－６活必均显著低于对照组，而ＩＬ－２Ｒ表达均显著高于对照组，结论 肝癌患者外周血Ｔ细胞功能被严重抑制，ＩＬ－２，ＩＬ－６和ＩＬ－２Ｒ可能在该病的发病     

白细胞介素－6在老年女性原发性骨质疏松症发病中的作用

为探讨白细胞介素－６（ＩＬ－６）在老年女性原发性骨质疏松症（ｏｓｔｅｏｐｏｒｏｓｉｓＯＰ）发病中的作用，本文采用ＩＬ－６依赖性细胞株ＭＨ６０．ＢＳＦ增殖反应ＭＴＴ法检测了３０例老年女性骨质疏松性骨折患者和２４例正常者以及１４例健康绝经前女性外周血单核细胞培养上清（ＰＢＭＣ）ＩＬ－６水平以及血清雌激素（Ｅ２）、骨钙素（ＢＧＰ）等水平的变化。结果：绝经后妇女ＩＬ－６水平高于绝经前，而ＯＰ组又高于ＮＯＰ组。以ＯＰ组ＩＬ－６为因变量的多元回归分析发现：ＩＬ－６与年龄无明显相关关系，与前臂骨密度（ＢＭＤ）和Ｅ２呈负相关，与ＢＧＰ和尿钙与尿肌酐比值（Ｃａ／Ｃｒ）呈正相关。结果提示老年女性骨丢失属于高转换型，雌激素水平减少使分泌ＩＬ－６细胞活化，ＩＬ－６分泌增多，从而刺激骨吸收，骨吸收超过骨形成就会导致ＯＰ的发生     

人子宫内膜植入兔眼前房模型的研究

本实验成功地将人子宫内膜植入到１５只去卵巢新西兰雌兔的眼前房，并给每只兔外源补充雌二醇（Ｅ２）１００μｇ／ｄ和孕酮（Ｐ）８ｍｇ／ｄ。在内膜植入期间每只兔皮下注射总量为９ｍｇ的霉酚酸（ｍｙ－ｃｏｐｈｅｎｏｌｉｃａｃｉｄ）以防免疫排斥反应。在眼前房的人子宫内膜存活了２８．４４±８．６５天，兔血清Ｅ２和Ｐ的平均值分别为１６１．７１±１５．１３和３８．８８±７．４５ｐｍｏｌ／Ｌ。前房液中Ｅ２和Ｐ浓度在植入人子宫内膜前后分别为２４．８０±１６．５３和１７０９．２５±４７５．１０ｐｍｏｌ／Ｌ，２．００±０．５３和１２．２２±３．８１ｎｍｏｌ／Ｌ。树脂切片验证人子宫内膜植入兔眼前房第５天就能与虹膜附贴并有血管发生。来自巩膜的血管分布在子宫内膜周围。扫描电镜观察人子宫内膜分化成分泌细胞和纤毛细胞。植入人子宫内膜后，前房液的蛋白浓度高于植入前。可以证明该模型可用来研究人子宫内膜对药物的反应和通过对前房液的测量研究子宫内膜的分泌功能。     

羟基喜树碱对膀胱癌细胞自分泌的影响

目的 检测膀胱癌细胞自分泌因子并探讨羟基喜树碱对自分泌的影响。方法 采用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术,在无血清培养情况下检测膀胱癌Ｔ２４细胞株自分泌因子ＩＧＦ－１、ＩＧＦ－２、ＩＧＦ－１受体、白细胞介素（ＩＬ）－６受体、ＩＬ－６受体表达情况,并进一步用半定量ＲＴ－ＰＣＲ方法观察膀胱灌流药物羟基喜树碱分别在０．００５、０．０１０、０．０２０ｍｇ／Ｌ浓度时对前述指标的影响。结果 经羟基喜树碱（０．０１０ｍｇ／Ｌ）处理后,ＩＧＦ－１、ＩＧＦ－２、ＩＧＦ－１受体、ＩＬ－６、ＩＬ－６受体分别下降下４４．８％,５９．７％,３９．３％,５１．５％及４６．２％,同未痉基喜树碱处理组相比差异有显著性（Ｐ〈０．０５）。结论 膀胱癌细胞可自分泌ＩＬ－６、ＩＧＦ－１、ＩＧＦ－２等自分泌因子,羟基喜树碱对膀胱肿瘤自分泌表达的     

InterleuKin-6与前列腺癌的发生发展

内分泌治疗前列腺癌（ＰＣＡ），通常会导致其复发，且复发后，ＰＣＡ不再对雌激素治疗敏感，甚至对化疗物ＣＤＤＰ、ＶＰ－１６也相对耐药。本文探讨了ＰＣＡ从激素依赖向非激素依赖过渡的分子机理，认为细胞因子ＩＬ－６和癌基因ｂｃｌ－２扮演了重要角色，ＩＬ－６和ｂｃｌ－２的表达呈正相关。抗ＩＬ－６的单抗或ＩＬ－６的嵌合毒素可为治疗ＰＣＡ开辟一辅助治疗新领域。     

c-myc对大鼠黄体细胞人绒毛膜促性腺激素诱导的孕酮和雌二醇生成的影响

应用离体细胞体外孵育法研究了反义ｃ ｍｙｃ寡脱氧核苷酸（反义ｃ ｍｙｃＯＤＮ）对大鼠黄体细胞人绒毛膜促性腺激素（ｈＣＧ）诱导的孕酮（Ｐ）和雌二醇（Ｅ２）生成的影响及其与外源性ｃＡＭＰ和Ｃａ２＋的关系。结果发现，反义ｃ ｍｙｃＯＤＮ能呈剂量相关方式抑制黄体细胞ｈＣＧ诱导的Ｐ和Ｅ２的生成。在浓度分别为１０和５μｍｏｌ／Ｌ时的抑制作用即具有显著意义（Ｐ＜０．０５）；而无义ｔａｔ寡脱氧核苷酸则无此作用。反义ｃ ｍｙｃＯＤＮ对黄体细胞ｈＣＧ诱导的Ｐ和Ｅ２产生的抑制作用能被加入１０－４ｍｏｌ／Ｌ二丁酰ｃＡＭＰ逆转，钙离子通道阻断剂维拉帕米对此种抑制作用具有协同效应。结果提示，ｃ ｍｙｃ癌基因参与黄体细胞ｈＣＧ诱导的Ｐ和Ｅ２生成的调控。     

原位杂交检测孕酮受体mRNA在周期子宫内膜的表达

应用地高辛标记的探针进行原位杂交，检测周期内膜各组分的孕酮受体（ＰＲ）ｍＲＮＡ的表达。内膜取自子宫肌瘤患者，子宫全切术后即取内膜及部分肌层，储于液氮。据月经史及形态表现将内膜分为三期：增殖、分泌早期及中晚分泌期。结果显示，ＰＲｍＲＮＡ在子宫内膜的表达有明显的组织／细胞差异。基质细胞普遍表达ＰＲｍＲＮＡ，增殖期与分泌期比较无明显差异。中晚分泌期内膜腺体细胞ＰＲｍＲＮＡ的表达极低甚至不表达，且无明显的表浅及深层的表达差异。提示子宫内膜孕酮受体至少在腺体上可能存在转录水平的调节。     

多聚乙烯亚胺介导IL-12基因增强裸鼠抗骨肉瘤免疫

目的 :观察以多聚乙烯亚胺 (polyethylenimine ,PEI)为载体 ,体内转染小鼠白细胞介素 12 (inter leukin 12 ,mIL 12 )基因 ,治疗裸鼠骨肉瘤模型的疗效。方法 :以PEI为载体 ,体外转染mIL 12基因入人骨肉瘤细胞 ,并观察此基因表达情况。建立裸鼠骨肉瘤动物模型 ,将PEI包裹mIL 12基因直接注入肿瘤局部 ,检测此基因的蛋白表达情况和小鼠脾脏自然杀伤细胞 (NK)活性。结果 :在PEI/DNA治疗组小鼠的肿瘤局部 ,mIL 12蛋白水平明显升高 ,小鼠脾NK细胞活性增强。结论 :PEI可以成功的将mIL 12基因导入裸鼠骨肉瘤模型 ,mIL 12基因治疗可提高机体的抗肿瘤免疫应答     

促性腺激素释放激素拮抗剂对大鼠睾丸支持细胞雄激素受体和Fas配体基因表达的影响

本实验利用注射促性腺激素释放激素拮抗剂（ＧｎＲＨ Ａ）引起睾丸生精细胞程序化死亡的大鼠模型，用地高辛标记的大鼠雄激素受体和Ｆａｓ配体的ＲＮＡ探针对大鼠睾丸石蜡切片进行原位杂交，观察雄激素受体和Ｆａｓ配体基因在生精细胞凋亡中的变化。结果显示，ＧｎＲＨ Ａ处理后曲细精管有退行性变，原位杂交表明此时Ｓｅｒｔｏｌｉ细胞的雄激素受体表达明显减低，而Ｆａｓ配体表达显著升高。提示Ｆａｓ配体的表达可能与生精上皮细胞程序化死亡关系密切。     

Aging Increases the Interleukin-1β−Induced INOS Gene Expression and Nitric Oxide (NO) Production in Vascular Smooth Muscle Cells

Introduction : Studies in animals have indicated that increased production of nitric oxide (NO) from an inducible isoform of nitric oxide synthase (iNOS) contributes to the vascular abnormalities of endotoxin‐ and cytokine‐induced shock. Objective: The aim of this study was to determine if the cytokine interleukin‐1β (IL‐1β) depresses vasocontractions induced by an α‐adrenergic agonist in aortic rings of control C57BL/6J (wild‐type) mice and if mice lacking iNOS gene expression (using iNOS‐knockout mice) completely lacks the vasodepressant effect of IL‐1β. Methods: Thoracic aortas were removed from wild‐type and knockout mice, and setup in isolated organ baths with Krebs solution and 95% oxygen and 5% carbon dioxide. After equilibration, contractions by the α‐adrenergic agonist phenylephrine, over a complete concentration‐response range, were first measured before adding IL‐1β. Rings were then incubated with 100 ng/ml IL‐1β for 2 hr, followed by replacing with fresh Krebs solution every 20 min over another 3 hr. Contractile responses to phenylephrine, over a complete concentration‐response range, were again measured. Results: There was no significant difference between the contractile response to phenylephrine at the beginning and the end of the 5 hr incubation in all of the time controls (i.e. both wild‐type and iNOS‐knockout mouse aortic rings, both with and without endothelium) that were not exposed to IL‐1β. In aortic rings incubated with IL‐1β, contractions induced by phenylephrine were significantly attenuated both in endothelium‐preserved and endothelium‐denuded rings of wild‐type mice. In endothelium‐denuded aortic rings of iNOS‐knockout mice, IL‐1β had no effect on phenylephrine‐induced contractions, indicating a complete lose of the vasodepressant actions of IL‐1β. In contrast, in endothelium‐preserved aortic rings of iNOS‐knockout mice, IL‐1β significantly enhanced the contractions caused by phenylephrine. Conclusions: The present data demonstrate that the IL‐1β?induced depression of vasocontractions in mouse aortic rings is completely dependent on the expression of iNOS and increased production of NO in vascular smooth muscle cells. Aortic rings with intact endothelium but lacking iNOS expression showed enhanced vasocontractions following incubation with IL‐1β. The data suggest that, when iNOS is absent, IL‐1β induced another endothelium‐dependent pathway that potentiates contractile responses of α‐adrenergic agonists. (Supported by a Direct Grant for Research)     

Effect of Bisphenol A on steroid production in human granulosa cells in vitro

Objective:To study the effects of environmental estrogen-like chemical bisphenol A(BPA)on ovarian function with the model of the cultured human granulosa cells in vitro.Methods:The granulosa cells from healthy women were collected and cultured in DMEM with 10% heat-inacti-vated fetal bovine serum.BPA,final concentration 10-7 to 10-4 mol/L,was added to the cell-culture medium and treat cells for 48 hours.The levels of estrogen and progesterone in the supernatant of the cultured cells were meas-ured by DELFIA.Total RNA was extracted from the cultured cells.The expression levels of P450 scc and P450 arom mRNA were measured by RT-PCR.Results:The cultured human granulosa cells could express high levels of P450scc and P450arom.The levels of estrogen and progesterone increased after BPA treatment,expression of P450 arom mRNA was reduced significantly at 10-5 to 10-4mol/L of BPA,but the expression of P450 scc mRNA was increased at 10-6 to 10-5 mol/L of BPA.The levels of estrogen and progesterone in the supernatant of the cultured cells were not affected significantly by BPA at the same concentrations incubated for 48 hours.Conclusion:BPA could affect steroid hormone synthesis and transformation in granulosa cells,such as the ex-pression of P450 scc and P450 atom,so it can affect ovarian function.Although we did not find a significant effect of BPA on the final estrogen and progesterone levels in this studying model,noxious effects of BPA on ovarian function may be exist in the human granulose cells.     

雌、孕激素作用后小鼠子宫内膜细胞中核因子-κB的表达

目的　探讨不同浓度雌、孕激素对小鼠子宫内膜细胞核因子 κB(NF κB)表达的影响。　方法　采用去势雌性昆明小鼠模型 ,分为假手术、雌激素、孕激素及雌、孕激素联合组 ,采用免疫组织化学方法检测给药后小鼠子宫内膜NF κB的表达。　结果　雌激素浓度为 30及 10 0 μg·kg-1·d-1时均可促进小鼠子宫内膜细胞NF κB的表达 (P <0 .0 1) ,但无明显的量效相关性 (P >0 .0 5 )。单用孕激素降低子宫内膜细胞胞浆中NF κB的表达 (P <0 .0 1)。雌、孕激素联合促进细胞浆及细胞核的NF κB表达 (P <0 .0 1)。　结论　雌激素可上调小鼠子宫内膜细胞NF κB的表达 ,而孕激素对其表达呈降调节作用 ;NF κB途径可能参与了雌、孕激素对子宫内膜容受性建立的调节     

Prostatic neoplasia in transgenic mice with prostate-directed overexpression of the c-myc oncoprotein

BACKGROUND: Promoter elements within the 5' DNA region of the rat C(3)1 gene have been shown to direct prostate-specific expression of gene products when they are fused through recombinant DNA procedures and used to produce transgenic mice. In order to test the in vivo effects of chronic overexpression of the mouse c-myc protooncogene on the prostate glands of transgenic mice, we created several lines of C(3)1-c-myc transgenic mice and then examined the phenotype of males with this genetic alteration. METHODS: The modified promoter and 5' region of the rat C(3)1 gene was fused to the coding region of the mouse c-myc gene using recombinant DNA techniques. This DNA was used to create three different founder lines of transgenic mice. Tissues from males and females heterozygous for the transgene were examined for expression of the recombinant mouse c-myc mRNA by an RNase protection assay. Prostates from males were examined for expression of recombinant c-myc mRNA by in situ hybridization. Thin sections of fixed ventral prostates from males were analyzed by microscopy for histological abnormalities. RESULTS: Three different lines of transgenic mice were obtained from these procedures. These mice demonstrated expression of recombinant mouse c-myc mRNA in the testis and ventral prostates of males and in the uterus of females. In situ hybridization demonstrated that the epithelial cells were the source of recombinant c-myc expression in the ventral prostates of the transgenic lines. Microscopic analysis of the ventral prostates from these mice demonstrated abnormalities in epithelial cell morphology seemingly typical of an intraepithelial neoplasia-like phenotype. However, none of the males of any of the lines developed overt prostatic adenocarcinoma over their lifetimes. CONCLUSIONS: Chronic overexpression of c-myc in the ventral prostate epithelial cells of C3(1)-c-myc transgenic mice leads to the development of epithelial cell abnormalities similar to those seen in low-grade prostatic intraepithelial neoplasia in humans. These abnormalities were not found to progress to adenocarcinoma over the lifetimes of the transgenic mice, suggesting the need for additional oncogenic changes in the pathway to prostatic adenocarcinomas. Furthermore, our cumulative experience with the use of the C3(1) gene promoter in the generation of transgenic mice suggests that the probasin promoter element provides a much more specific and effective means to target transgenes to the prostate glands of mice.     

表皮生长因子与人绒毛膜促性腺激素共同作用对大鼠卵巢颗粒细胞功能的影响

为阐明表皮生长因子（ＥＧＦ）对卵巢功能的调节作用，本实验用培养的大鼠卵巢颗粒细胞观察了ＥＧＦ与人绒毛膜促性腺激素（ｈＣＧ）共同作用对细胞增殖、甾体激素与ｃＡＭＰ生成的影响。结果：ＥＧＦ可明显抑制ｈＣＧ刺激的孕酮生成，并呈剂量依赖性，而对ｈＣＧ刺激的雌二醇生成无影响；ＥＧＦ对ｈＣＧ刺激的颗粒细胞ｃＡＭＰ生成具有增强作用，使ｃＡＭＰ生成增加；ＥＧＦ能刺激颗粒细胞增殖，使３Ｈ ＴｄＲ掺入增加，ｈＣＧ不影响ＥＧＦ的这种作用。提示ＥＧＦ与ｈＣＧ对卵巢功能的影响既有相互作用，又有各自的作用。