基因敲除技术在精子发生相关基因功能研究中的应用

从基因水平研究男性不育症发病机理将有助于发现男性不育治疗的新途径。基因敲除技术是目前研究基因功能的主流方法。筛选鉴定精子发生过程中相关基因,探索其特性和功能,对了解睾丸功能、探索男科疾病新的治疗靶点具有重要意义。本文概述基因敲除技术在精子发生相关基因功能研究中的应用。     

从基因敲除看生殖激素的作用

基因敲除技术已成为研究基因功能的一种重要手段 ,本综述主要介绍了雌激素受体、孕酮受体、前列腺素受体、促性腺激素释放激素、黄体生成素受体、卵泡刺激素受体、雄激素受体、催产素、泌乳素及其受体等主要的生殖激素或受体基因敲除后对小鼠生殖系统的发育、功能以及生殖行为的影响 ,并对这些激素可能的作用机理进行了讨论。     

采用基因表达谱芯片技术筛选新基因AngRem104的功能相关基因

目的 采用基因芯片技术来筛选基因AngRem104的功能相关基因，为进一步的基因功能研究提供线索。方法 建立新基因AngRem104的正义和反义表达的系膜细胞模型，应用含有4000个人类基因的cDNA表达谱芯片，对过量表达AngRem104基因和抑制表达AngRem104基因的人肾小球系膜细胞RNA表达进行检测。部分基因的差异表达由RT－PCR方法来验证。结果 筛选出的差异表达基因共96个，其中94个基因上调表达，2个基因下调表达。差异表达的基因涉及到细胞外基质和受体蛋白、细胞信号传导相关基因、DNA结合及转录相关蛋白、免疫相关蛋白、细胞骨架和动力蛋白和细胞合成及代谢相关蛋白等。特别是纤连蛋白（FN）及整合素β1(integrin β1）表达明显上调。RT－PCR的方法也证实了AngRem104与FN表达的相关性。结论 应用作为研究基因功能有效手段的基因表达谱芯片技术来筛选新基因AngRem104的功能相关基因，发现AngRem104与FN的表达有关，为其功能研究提供了重要线索。     

基因敲除技术在骨质疏松相关基因研究中的应用

基因敲除技术是21世纪发展最为迅速的生物高新技术之一。基因敲除是利用同源重组原理使特定的靶基因失活进而实现在其功能缺失的情况下分析靶基因功能的方法。骨质疏松是个复杂的多因素疾病,由遗传和环境因素相互作用,目前已成为世界的健康问题,骨质疏松相关基因的研究是目前研究的热点。本文就基因敲除原理及该技术在骨质疏松中的应用和进展进行综述。     

雄性不育相关基因敲除小鼠模型

男性不育的发生机制复杂,建立雄性不育的动物模型,特别是小鼠模型,为研究不育相关基因功能和分子机制提供了模型基础,目前用于生物医学研究的小鼠模型主要有3种类型,例如敲除/敲入/基因捕获方法获得小鼠,转基因小鼠和化学诱导的点突变小鼠。本文对雄性不育相关基因敲除的小鼠模型进行了总结,以期为研究男性不育的发病机制寻找合适的动物模型。     

结直肠癌肝转移相关靶基因转录调控网络的构建及基因本体论功能富集分析

目的 研究结直肠癌肝脏转移微小RNA (miRNA)表达的差异及相关特性,并对结直肠癌肝脏转移miRNA对应的靶基因和生物信息学特征进行针对性分析.方法 收集10例伴有或不伴有肝转移的结直肠癌患者肿瘤组织标本.应用miRNA芯片方法对两组样本的miRNA表达差异情况进行研究,利用软件预测靶基因.进一步构建和分析肝转移结直肠癌相关的miRNA-Target转录调控网络及基因本体论功能模块.结果 芯片结果筛选出6种结直肠癌肝转移组较非转移组表达失调的miRNA(上调的miR-224、miR-1236和miR-622;下调的miR-155、miR-342-5p、miR-363).最显著上调的miR-224不但可以与其对应靶基因行使调控作用,而且可以与其他miRNA协同作用于其下游的靶基因功能团,行使相应功能.结论 miR-224作为调控网络的核心,可同时或异时性调控相关的重要基因功能团,进而完成对结直肠癌肝脏转移的转录后水平操控,在肝转移过程中起重要作用.     

基因一环境相互作用与男性生殖功能

由于遗传因素无法解释短时期内发生的改变,环境和生活方式相关因素被认为是引起与时间相关的男性生殖功能衰退的原因。但是,鉴于不同人群之间以及相同人群内部男性生育力存在很大差别,遗传因素可能极大地影响到人们对环境或生活方式的不利影响的个体易感性。尽管我们对这种和生殖系统相关的相互作用的机制还知之甚少,最近已经有研究表明一些特异的基因型会使得男性在接触某些环境因素后生殖疾病风险大大提高。本文对一些基因如何调节环境对男性生殖功能影响的人类和动物学研究进行了回顾和评论。虽然已经发现一些证据支持这一说法,但是研究数量依然有限。这类基因一环境相互作用的研究可以帮助我们更好地了解正常生理学,并且确定男性生殖疾病的风险因素。我们还简短讨论了与生殖相关的其它基因一环境相互作用方面的话题,即环境和生活方式因素引起精子DNA损伤。这些基因到底改变到什么程度,是通过自然方式,还是辅助生殖技术的应用传给下一代,从而引起子代发病率上升？这些问题还有待于研究探讨。     

RNA干扰技术与肝细胞癌基因治疗

肝细胞癌已被证实为一种基因病,相关基因的异常表达引起细胞生物学特性改变,导致细胞永生化和癌变,因此,深入探讨肝细胞癌发病的分子机制以寻求新的诊治方法是提高疗效的关键.RNA干扰作为一种高效特异性抑制基因表达的新技术,广泛应用于研究基因功能和基因治疗,本文就近年来RNA干扰技术应用于肝细胞癌基因治疗领域的研究进展予以综述...     

雷公藤多苷诱导小鼠睾丸生殖相关基因异常表达及补肾中药的干预作用

目的:探讨雷公藤多苷诱导小鼠睾丸生殖相关基因的表达及补肾中药的干预作用。方法:成年Balb/C雄性小鼠,以雷公藤多甙30 mg/(kg.d)灌胃3周,造成生殖减退模型。随后,分别予生理盐水0.25 ml/d、雷公藤多甙30 mg/(kg.d)、肉苁蓉煎液[相当于生药10 g/(kg.d)]、熟地煎液[相当于生药10 g/(kg.d)]、肉苁蓉、熟地等比例的煎液[相当于生药20 g/(kg.d)]每日1次灌胃进行干预;另设肉苁蓉预处理组持续给予雷公藤多甙30 mg/(kg.d)和肉苁蓉煎液[相当于生药10 g/(kg.d)],每日1次灌胃,灌胃3周。检测各组睾丸生殖相关基因Dzip1、Fas、c-jun、Wnt4的表达量的变化。结果:雷公藤模型小鼠Y染色体微缺失相关基因Dzip1表达下调,生殖细胞凋亡相关基因Fas表达上调,原癌基因c-jun表达上调,信号转导相关基因Wnt4表达上调,补肾中药干预后均有不同程度改善,其中以补益肾阳药物肉苁蓉与补益肾阴药物熟地联合应用效果最显著。结论:雷公藤多苷对生殖相关基因的表达有影响;补益肾阳药物肉苁蓉能够部分拮抗雷公藤生殖毒性,补益肾阴药物熟地亦表现出一定的拮抗雷公藤生殖毒性作用,联合应用肉苁蓉和熟地则加强了拮抗雷公藤生殖毒性的作用,揭示了阴中求阳、阴阳互根互用的现代药理基础。肉苁蓉有一定的预防雷公藤生殖毒性的作用。     

丝状真菌基因敲除技术研究进展

丝状真菌在自然界分布广泛，与人类的生产、生活密切相关。近年来，对于其基因功能的研究取得了较大的进展，一系列转化和基因操作技术已在不同的丝状真菌中得到运用。许多对于工业、农业和医药卫生具有重要意义的丝状真菌已经完成或正在进行全基因组序列测定。作者简述了丝状真菌基因敲除的历史，重点介绍了近年来基因敲除技术在丝状真菌研究中的进展，并对冀在丝状真菌基因功能研究、工业菌株改良等方面进行了展望。     

3ioinformatics for spermatogenesis： annotation of male ＇eproduction based on proteomics

Proteomics strategies have been widely used in the field of male reproduction, both in basic and clinical research. Bioinformatics methods are indispensable in proteomics-based studies and are used for data presentation, database construction and functional annotation. In the present review, we focus on the functional annotation of gene lists obtained through qualitative or quantitative methods, summarizing the common and male reproduction specialized proteomics databases. We introduce several integrated tools used to find the hidden biological significance from the data obtained. We further describe in detail the information on male reproduction derived from Gene Ontology analyses, pathway analyses and biomedical analyses. We provide an overview of bioinformatics annotations in spermatogenesis, from gene function to biological function and from biological function to clinical application. On the basis of recently published proteomics studies and associated data, we show that bioinformatics methods help us to discover drug targets for sperm motility and to scan for cancer-testis genes. In addition, we summarize the online resources relevant to male reproduction research for the exploration of the regulation of spermatogenesis.     

Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models

Gene disruption technology has long been beneficial for the study of male reproductive biology. However, because of the time and cost involved, this technology was not a viable method except in specialist laboratories. The advent of the CRISPR/Cas9 system of gene disruption has ushered in a new era of genetic investigation. Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost. This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.Mice are one of the most ideal organisms to study mammalian reproduction. This is because of their relatively fast reproductive cycle coupled with their similarity to the human genome.1 However, reproduction remains one of the most complex yet poorly understood biological processes, despite decades of dedicated research. Numerous genes have been thought to play essential roles in fertilization, because of their localization or specific expression in the male and/or female gonads, yet analysis of their specific roles has been problematic due to the difficulty in maintaining gametes and embryos in vitro.2Thus, gene manipulation experiments in animal models have played an essential role in the investigation of reproductive processes. Reproduction is arguably one of the best-suited biological systems to which gene knockout (KO) can be applied, for several reasons. First, genes essential for fertility are most often highly specific to the gonads, eliminating the need for conditional gene knockout models to be utilized. Often spermatogenic or haploid male germ cell genes are comprised of a single exon, eliminating the issues of alternate splicing. Another benefit of utilizing gene-disrupted mice is the incidental discovery of genes involved in reproduction by groups investigating other body systems.3,4 The breeding schemes involved in producing homozygous genetic manipulations for any target gene automatically highlight genes involved in gametogenesis, fertilization and pregnancy. Whatever the reason, there is a higher level of specific homologues in reproductively related genes than in those coding for somatic cell phenotypes, making the use of gene-disruption essential to uncover the truly essential factors of fertility. This review will look at the history of gene manipulation techniques for the study of mammalian reproduction, with a focus on discoveries in the male system and analyze the application of the latest in these technologies, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated (Cas) protein number 9) system.     

生精障碍相关基因单核苷酸多态性研究进展

生精相关基因遗传多态性是生精障碍的一个重要的遗传病因.通过基因敲除技术现已鉴定出大量与精子发生密切相关基因.此类生精障碍基因包括表达酶类、受体类、细胞凋亡类、转录调控类等基因.上述基因的遗传易感性、感染和环境等因素共同作用导致男性非梗阻性无精子症和少精子症.生精障碍相关基因单核苷酸多态性(SNP)分析可从分子水平上阐述...     

SRY and AZF gene variation in male infertility: a cytogenetic and molecular approach

Aim The aim of this study was to identify the genetic effects of Y chromosome and azoospermia factor (AZF) gene variation in men with infertility and to elucidate the molecular mechanism responsible for the identified point mutation. Methods Chromosome analysis was performed according to standard methods on lymphocyte cultured cells and genomic DNA was extracted from the peripheral blood. Three sets of primers were used encompassing the AZFb, AZFc and SRY14 gene regions. Products were genotyped with single-strand comformational polymorphisim (SSCP) analysis. Results The profiles of the mutated genes were detected in five of three azoospermic and two oligoasthenozoospermic infertile males. The SSCP variability of the AZFc gene was detected in all of the cases, while sex-determining region Y (SRY) gene variation was detected in two of the current cases. Three cases with oligoasthenozoospermia showed mutated SSCP profiles in both their SRY and AZFc gene regions. No AZFb variation was detected in the presented cases. Conclusion The AZF locus is assumed to contain the genes responsible for spermatogenesis in human. Deletions in these genes are thought to be involved in male infertility associated with azoospermia, oligozoospermia and/or both. AZF microdeletions and variations that are seen in infertile males suggest the need for molecular screening of such cases. Advance studies are also needed to detect of these variations and their relevance to male infertility before using assisted reproduction techniques in such cases.     

电磁辐射致雄性生殖损伤机制的研究进展

电磁辐射对雄性生殖影响的研究已有50多年的历史,越来越多的结果显示,一定剂量的电磁辐射具有明显雄性生殖损伤效应,可引起生精细胞结构与功能的损伤,其发生机制与能量代谢障碍、脂质过氧化、凋亡相关基因及蛋白异常表达、DNA损伤等相关,本文就此方面的研究进展进行了综述。     

Gene expression patterns in bone following mechanical loading

The advent of high‐throughput measurements of gene expression and bioinformatics analysis methods offers new ways to study gene expression patterns. The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone‐formation process, including expression of matrix‐related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading‐induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time‐dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading‐induced bone formation were identified within the clusters, including AP‐1‐related genes in the early‐response cluster, matrix‐related genes in the upregulated gene clusters, and Wnt/β‐catenin signaling pathway inhibitors in the downregulated gene clusters. Several novel gene groups were identified as well, including chemokine‐related genes, which were upregulated early but downregulated later in the time course; solute carrier genes, which were both upregulated and downregulated; and muscle‐related genes, which were primarily downregulated. © 2011 American Society for Bone and Mineral Research.     

人类Y染色体的遗传特性

Y染色体为男性特有 ,因此其与男性特有的遗传性状密切相关。根据Y染色体特性和功能特点 ,可将Y染色体分区 ,为基因的定位打下了基础。随着人类基因组计划研究的深入 ,Y染色体基因组结构也逐渐得到认识 ,但由于Y染色体较小 ,所携带的基因也较少 ,因此至今尚无遗传图谱 ,物理图谱也不完善。已发现了 376个STS片段 ,部分STS已用于睾丸功能基因的研究。Y染色体上的主要基因有睾丸决定基因和精子发生相关基因。