基因敲除技术在妇科领域的应用

基因敲除技术是运用胚胎干细胞及分子生物学方法去除生物体内某一特定基因,制成基因敲除的动物模型,借此研究该基因的生理及病理功能.这类动物模型是探讨基因在疾病中作用机制的一个非常有效的研究工具.目前在妇科领域主要是利用此项技术敲除性腺激素或相关受体的基因,然后研究该类基因在生殖生育、妇科疾病、肿瘤发生及药物治疗的影响机制.     

基因敲除技术在妇科领域的应用

基因敲除技术是运用胚胎干细胞及分子生物学方法去除生物体内某一特定基因，制成基因敲除的动物模型，借此研究该基因的生理及病理功能。这类动物模型是探讨基因在疾病中作用机制的一个非常有效的研究工具。目前在妇科领域主要是利用此项技术敲除性腺激素或相关受体的基因，然后研究该类基因在生殖生育、妇科疾病、肿瘤发生及药物治疗的影响机制。     

卵泡发育中相关基因的调控

卵泡发育是一个受内分泌、旁分泌及基因调节的复杂的生理过程。基因通过表达产物反馈调节卵泡发育,但它们各自对卵泡发育的调节作用有所不同。FOXL2基因能促进卵泡的生长、发育及抗凋亡,AMH基因、GDF-9基因、INH基因与卵泡早期的生长和发育有关。原癌基因c-myc促进卵泡发育和黄体形成,c-mos基因在卵母细胞减数分裂中呈现特异作用,而bcl-2和bax分别参与了卵巢颗粒细胞的增殖和凋亡的调节。由于各基因调控的相互协调,才使卵泡得以正常生长、发育和成熟。     

卵泡生长发育调控机制的研究进展

近年卵泡生长发育调控机的研究取得了显著的进展。卵泡的生长发育可能是一严格的程控过程，其主要调控因素是ＦＳＨ，但也受到卵巢局部非类固醇网络的调控，卵泡闭锁的本质可能是细胞凋亡，而分子设计及基因重组等技术的引入不仅有可能为临床诊治表达具有特定生物学特性的促排卵制剂，同时也将深化卵泡生长发育调制机的研究。     

βB2晶状体蛋白对小鼠卵巢发育及动情周期的影响

目的:初步探讨βB2晶状体蛋白(Crybb2)参与调节生殖过程的作用机制。方法:选取11-13周龄βB2基因敲除(KO组,n=19)与野生型C57BL/C(WT组,n=23)雌性小鼠,阴道涂片观察动情周期变化;HE染色观察卵巢病理变化;光学显微镜下计数卵巢最大切面原始卵泡、初级卵泡、闭锁卵泡;Western blotting和免疫组织化学确定βB2晶状体蛋白在卵巢组织中的表达与定位。结果:βB2晶状体蛋白主要表达在WT组小鼠卵巢颗粒细胞内。与WT组小鼠相比,KO组小鼠卵巢相对重量减轻,动情周期紊乱,原始卵泡、初级卵泡减少,闭锁卵泡增多。结论:βB2基因敲除小鼠的动情周期及卵巢发育异常,βB2晶状体蛋白对小鼠卵巢的发育有重要影响。     

哺乳动物卵泡发育的调控机制

哺乳动物卵泡发育是一个极为复杂的过程。卵泡主要是由一个生殖细胞及外围众多的颗粒细胞和卵泡膜间质细胞等组成。在促性腺激素等的作用调控下,卵泡中颗粒细胞增殖、分化,产生分化程度不一的颗粒细胞群。颗粒细胞又通过旁分泌和间隙连接的通讯方式控制卵母细胞的生长和成熟,因而卵泡发育是处在一个庞大的调控系统严格控制之下。本文综述了生长期和成熟期调控机制及人工诱导卵泡发育、成熟和卵成熟的一些思路。     

人卵丘细胞与卵母细胞发育及成熟的关系

卵丘细胞与卵母细胞共处于同一个卵泡液微环境中,卵丘细胞与卵母细胞之间复杂的"对话机制"调控着卵母细胞的成熟和卵丘细胞的增殖延伸。在窦卵泡阶段,卵丘细胞由颗粒细胞分化而来,通过缝隙连接与卵母细胞共同形成一个结构和功能上的合胞体。卵泡发育不同时期,卵丘细胞对卵母细胞的代谢调控主要表现为:在窦卵泡期,卵丘细胞为卵母细胞发育提供必需的营养,而卵母细胞分泌的信号因子亦调控着卵丘细胞的增殖和延伸;在排卵前卵泡中,卵丘细胞主要通过调控卵母细胞中cAMP水平,促使卵母细胞恢复减数分裂;在排卵后卵泡中,卵丘细胞亦影响着精-卵结合及胚胎发育的过程。另外,伴随卵泡内微环境的变化,卵丘细胞与卵母细胞间发生着复杂的信号传递,从而对卵母细胞的发育实现分子水平的调控,其中部分基因可能作为卵母细胞发育成熟、胚胎发育及妊娠结局的分子标志物。     

性激素对人卵巢颗粒细胞凋亡的作用

目的通过探讨雌、雄激素对人卵巢颗粒细胞凋亡的作用,阐明其调节人类卵泡闭锁的分子机制。方法应用细胞培养、选择性ＤＮＡ抽提和琼脂糖凝胶电泳技术分析闭锁卵泡和发育中卵泡颗粒细胞的凋亡状况,及雌激素（１μｇ／ｍｌ）、雄激素（１μｇ／ｍｌ）对体外培养发育中的卵泡颗粒细胞凋亡的作用;用Ｎｏｒｔｈｅｒｎ印迹杂交技术检测经雌、雄激素刺激后发育中的卵泡颗粒细胞ｂｃｌ－２基因ｍＲＮＡ表达的变化。结果闭锁卵泡颗粒细胞均出现凋亡,ｂｃｌ－２基因ｍＲＮＡ表达下降,雌激素则可致相反的效应。结论卵泡闭锁的本质可能是其细胞发生凋亡;雄激素促进卵泡闭锁、雌激素对抗雄激素作用及促进卵泡发育的机理,可能与调节内源性核酸内切酶的活化和ｂｃｌ－２基因表达有关     

卵泡液促血管生成因子在卵泡发育中的作用的研究进展

卵泡液由血浆渗出物和卵巢局部分泌物构成,卵泡液不仅为卵母细胞和卵巢颗粒细胞的生存提供适宜的生物环境,并且对卵泡发育、卵母细胞成熟起着直接调控作用。卵泡液中存在大量的细胞因子,它们都通过各自特有的机制作用于女性生殖过程,且随着辅助生殖技术的不断发展,卵泡液中各种细胞因子在女性生殖系统的作用逐渐被重视。本文以近年来生殖医学领域对卵泡液微环境的研究为基础,结合血管生成的机制和卵泡液中参与血管生成过程的各种细胞因子对女性生殖过程的调节,综述了卵泡液中血管生成相关因子在卵泡发育、卵子成熟及胚胎种植等女性生殖过程中的作用。     

卵巢机能调控研究的现代进展

<正> 近年来,由于近代科学技术的发展,生殖内分泌学的研究已经从激素的定性分析发展到定量测定;由卵泡发育的形态学描述发展到动力学分析;由激素的提纯和分离发展到在受体水平上对激素作用原理进行探讨,尤其对卵巢机能调控的研究取得了较大的进展。本文对这方面问题作一概述。卵泡发育的调控及生化改变卵泡发育经原始卵泡、初级卵泡、次级卵泡、成熟卵泡四个阶段。卵泡在发育的各个阶段呈现一系列超微结构和生化方面的变化。     

Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in rats

Over the past 25 years, the reverse genetic approach including precise and conditional replacement or loss of gene function at a specific locus was considered possible only in mice due to the absence of embryonic stem (ES) or induced pluripotent stem (iPS) cell lines in other species. Recently, however, stem cell technology in rats has become available for biomedical research. In this paper we overview the recent progress of rat ES and iPS cell technology. Starting from the establishment of rat ES cells, the use of ES cells for foreign gene transfer and endogenous gene knock-out is discussed, followed by the successful establishment of rat iPS cells and the generation of an iPS cell-derived organ via interspecific blastocyst complementation. Finally, the possible contribution of rat stem cell technology to reproductive medicine is described.     

Models of aromatase insufficiency

The recent studies on humans with a defective gene that encodes aromatase p450 (p450arom) and aromatase knockout (ArKO) mice with a disrupted p450arom gene uncovered many interesting aspects of physiology. Aromatase-deficient humans and ArKO mice continue to serve as invaluable models for us to study the roles of estrogen in normal and abnormal functions of several tissues. These roles will be discussed in detail in the following review article.     

Gene knockout mice in the study of parturition

OBJECTIVE: To review recent studies of parturition control in mice with relevance to understanding the control of human parturition. METHODS: Assimilation of published studies of gene knockout mice with mutations in neuropeptides, prostaglandin synthetic enzymes and receptors, and other molecules implicated in parturition. RESULTS: The central role of prostaglandins in murine labor is demonstrated by mice with gene mutations at multiple levels of the prostaglandin synthetic pathway. In addition, novel molecules such as steroid 5 alpha-reductase are found to play an essential role in the progression of labor. Surprisingly, deficiency of neuropeptides such as oxytocin and corticotropin-releasing hormone have little effect on parturition. CONCLUSION: Molecular genetic analyses in mice provide an efficient way to define molecules critical for murine parturition. Extrapolation of the importance of these molecules to human parturition provides the next challenge.     

Nitric oxide synthase gene knockout mice do not become hypertensive during pregnancy.

OBJECTIVE: The purpose of this study was to test whether omitting the vasodilator nitric oxide that is derived from any 1 of the 3 isoforms of nitric oxide synthase results in hypertension during pregnancy. STUDY DESIGN: We measured systolic blood pressure before, during, and after pregnancy using an automated tail cuff method in 3 mutant (gene knockout) mouse strains in which the gene for neuronal nitric oxide, inducible nitric oxide, or endothelial nitric oxide was disrupted by gene targeting. RESULTS: In neuronal nitric oxide gene knockout mice (n = 10), blood pressure was 100 +/- 3 mm Hg, not significantly different from 101 +/- 3 mm Hg in matched wild-type control mice (n = 10). Pregnancy did not change blood pressure or heart rate in either group. In inducible nitric oxide gene knockout mice (n = 9), blood pressure was 110 +/- 3 mm Hg, the same as in the wild-type control mice (110 +/- 2 mm Hg; n = 14). Blood pressure was unaffected by pregnancy in either group of mice. However, heart rate was significantly less in knockout mice (647 +/- 11 beats/min vs 666 +/- 9 beats/min; P <.005); this difference persisted through pregnancy. In endothelial nitric oxide gene knockout mice (n = 8), blood pressure was higher before pregnancy (114 +/- 4 mm Hg vs 103 +/- 4 mm Hg; P <.05) than in wild-type control mice (n = 9), but this difference disappeared during pregnancy, returning only after delivery. Heart rates were not different before pregnancy and were unaffected by pregnancy. CONCLUSION: There was no apparent increase in systolic blood pressure in any of the 3 nitric oxide synthase gene knockout strains during pregnancy compared to the wild-type control mice. This suggests that, at least in the mouse, genetic deficiency of any 1 isoform of nitric oxide synthase does not result in pregnancy-induced hypertension.     

Current concepts of human azoospermia and its causes

Infertility is a serious social problem in advanced nations today. One of the most important causes is the male factor. Striking progress has been achieved in recent years in elucidating the mechanisms of spermatogenesis in mice by experimental methods represented by the knockout mouse. Although many factors associated with male infertility are known in mice, the translation of this information to people has been slow. This is because the knockout mouse phenotype cannot necessarily be reproduced faithfully in humans. However, it is known that environmental factors, chromosomal defects and several specific gene mutations result in human male infertility. In this review, we first discuss the environmental factors considered likely to be involved in male infertility, and secondly we describe the Y chromosome and several important genes on the Y chromosome that play critical roles in spermatogenesis in humans. Then, we demonstrate the three critical genes identified in our laboratory in autosomes involved in human spermatogenesis, the SYCP3, MEI1 and PARP-2. Finally, we explain the future directionality and possibilities of research in this field.     

Hsp90β1 knockout targeted to male germline: a mouse model for globozoospermia

To study the role of Hsp90β1, an endoplasmic chaperone, we have built a conditional knockout by crossing Hsp90β1(flox/flox) with the Vasa-Cre transgenic line. Spermatozoa deficient in Hsp90β1 could not naturally fertilize oocytes and exhibited large and globular heads with abnormal intermediate pieces, a phenotype reminiscent of human globozoospermia.     

宫颈癌相关显著突变基因研究进展

宫颈癌是最常见的妇科恶性肿瘤,在发展中国家,宫颈癌的发病率与死亡率常年居高不下,虽然关于宫颈癌的研究已广泛开展,但其具体发生机制及协同因素至今尚未阐明。近年来,基因突变与宫颈癌的关系逐渐受到重视。众多研究表明,癌基因、抑癌基因及相关调节基因的改变与宫颈癌发生及病情的进展密切相关。随着基因组测序技术在恶性肿瘤中的应用,更多基因的突变情况在宫颈癌中被发现,其中显著突变基因的确定可帮助进一步锁定恶性肿瘤驱动基因,故显著突变基因可能是发现恶性肿瘤内在机制的关键所在。对宫颈癌中新发现的显著突变基因ErbB-2受体酪氨酸激酶3(ErbB-3)、人类白细胞抗原A(HLA-A)、半胱氨酸天冬氨酸特异性蛋白酶8(CASP8)、转化生长因子βⅡ型受体(TGFBR2)的研究现状进行综述,可能为宫颈癌的机制研究、诊断及靶向治疗提供新的思路。     

Reproductive tract gene transfer

OBJECTIVE: Gene therapy is a rapidly evolving novel treatment for human disease. This review discusses the latest development in gene transfer technology and its potential use in the female reproductive tract. METHODS: A comprehensive search using the MEDLINE database was performed to review current, innovative trends in gene transfer technology. In addition, articles on reproductive tract gene transfer were reviewed. CONCLUSION(S): Recent developments, such as the Human Genome Project, have generated great interest in the genetic basis of human health and disease. Gene therapy is a rapidly evolving field that uses gene transfer to treat disease. Ongoing research in the field focuses on improving vector technology to enable efficient in vivo gene transfer. Although multiple techniques for gene transfer have been described, no single technique can be used in all instances. The human female reproductive tract is easily accessible and can be readily transfected. In vivo gene transfer has resulted in successful alteration of implantation rates and has demonstrated potential for use in treatment of ovarian cancer.