一个与小鼠精子发生相关的基因—rjs 基因

应用改良的mRNA差异显示技术,对不同发育期小鼠（分别为1周龄至4周龄）睾丸曲细精管进行了差异表达的研究,并对其中一个在3周龄小鼠中具有高表达的差异表达片段进行了克隆和测序分析,其序列与Genebank中rjs基因具有高度同源性,表明rjs基因可能与小鼠精子发生相关。     

小鼠精子发生过程中差异表达基因LTBP-3的克隆

目的 :筛选与精子发生相关的基因。　方法 :应用改良的mRNA差异显示技术 ,对不同发育期小鼠 (1周龄至 4周龄 )睾丸精曲小管进行差异表达的研究 ,对其中一个在 1周龄小鼠中高表达的cDNA片段进行了克隆和测序分析 ,并与基因文库中的己知序列进行比较。　结果 :发现该片段与小鼠潜在转化生长因子 β结合蛋白 (LTBP 3)基因的部分序列 95 .4%同源。　结论 :LTBP 3基因在小鼠精子发生过程中可能起着重要的调控作用     

不同发育阶段Fmr1基因敲除小鼠睾丸组织iNOS的变化

目的:对不同周龄的Fmr1(fragile X mental retardation 1)基因敲除和野生型雄性小鼠睾丸组织诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表达进行分析比较,探讨Fmr1基因敲除小鼠睾丸组织中iNOS表达的差异,为脆性X综合征的研究提供背景资料。方法:不同周龄(4、6、8、10周)的Fmr1基因敲除型和野生型雄性小鼠各6只,先采用PCR技术对基因敲除和野生型小鼠进行基因型鉴定,之后所有小鼠麻醉取睾丸组织,采用石蜡包埋切片免疫组化染色技术对基因敲除和野生型小鼠睾丸iNOS的表达进行检测并作对比分析。结果:iNOS在4周野生型小鼠睾丸间质细胞呈弱阳性表达,在6周小鼠呈阳性表达,在8周和10周小鼠呈强阳性表达,且基因敲除小鼠睾丸的iNOS表达均弱于野生型小鼠。结论:Fmr1基因敲除小鼠在缺失FMR1蛋白(FMRP)后的睾丸组织中iNOS的表达降低。     

多配体蛋白聚糖4是糖尿病肾病的候选基因

目的 在2型糖尿病肾病(DN)白蛋白尿易感基因定位的基础上,进一步筛选白蛋白尿易感基因位点(UA-1)区域附近的候选基因.方法 提取20周龄雄性KK/Ta(n=3)和BALB/c (n=2)小鼠肾脏总RNA,应用Affymetrix Murine Genome U74Av2基因芯片检测肾脏基因表达谱.选择UA-1区域的差异表达基因多配体蛋白聚糖4(syndecan-4),竞争性RT-PCR验证基因芯片的结果.提取KK/Ta、BALB/c小鼠的基因组DNA,进行syndecan-4基因编码区和启动子区域的序列分析.结果 在2型糖尿病KK/Ta小鼠UA-1区域附近存在着约10个差异表达基因.其中syndecan-4在20周龄KK/Ta小鼠肾脏中的表达上调,为BALB/c小鼠的26.1倍.在syndecan-4基因编码区存在2个基因多态性,分别为A93C和T216C多态性,2者均为同义突变.在syndecan-4基因启动子区域存在3个基因多态性,分别为-T263C、-T396C 与-G669A多态性.TATA框位于转录起始位点上游321 bp处,-T263C处恰好为转录因子Clox 的结合位点.结论 syndecan-4基因位于2型糖尿病UA-1附近区域,在20周龄KK/Ta小鼠肾脏中的表达明显上调,是DN的候选基因.syndecan-4启动子处的基因多态性可能为其差异表达的原因.     

TNP2基因在小鼠睾丸组织中的表达研究

目的 筛选与精子发生相关的基因.方法 将4d、9d、18d、35d、54d和6月龄小鼠睾丸组织cDNA探针与Affymetrix全基因组芯片进行杂交,筛选出差异表达的基因.通过RT-PCR分析差异表达基因在小鼠睾丸不同发育阶段中的表达.结果 对Affymetrix全基因组芯片杂交结果分析后,筛选得到1个差异表达杂交点,通过NCBI网站与小鼠全基因组序列Blast分析可知,该差异表达基因是TNP2基因.小鼠TNP2基因全长724bp,其编码框大小为375bp.RT-PCR结果表明TNP2基因在小鼠21d龄及之前的睾丸中没有表达,在35d龄睾丸开始高表达.结论 TNP2基因为小鼠年龄依赖性表达基因,小鼠TNP2基因的表达与小鼠精子发生过程有很强的一致性,因此,可以推测该基因在精子发生中可能起重要作用.     

Daxx基因在小鼠睾丸精子发生过程中的表达特征

目的:研究死亡结构域相关蛋白(Daxx)基因在小鼠睾丸精子发生过程中的表达特征,初步探讨其在生精过程中的作用。方法:通过实时荧光定量PCR(q PCR)、Western印迹及免疫荧光等方法检测Daxx在不同周龄野生型小鼠睾丸组织以及成年睾丸支持细胞雄激素受体特异性敲除(SCARKO)和雄激素受体敲除(ARKO)小鼠睾丸中的表达特征。结果:q PCR、Western印迹和免疫荧光结果表明,Daxx基因在出生4周后小鼠睾丸中高表达,且主要定位于细胞核;与野生型小鼠相比,SCARKO小鼠睾丸中DAXX的表达差异不显著(0.853±0.058 vs1.000±0.015),但在生精细胞细胞核中呈极性分布;DAXX在ARKO小鼠睾丸表达显著降低(0.299±0.026 vs1.000±0.015,P0.01)。结论 :Daxx基因在小鼠睾丸发育中期时表达最高。ARKO小鼠中DAXX的表达与野生型相比显著降低,睾丸支持细胞中AR基因特异性敲除影响DAXX定位。DAXX可能参与调控小鼠的精子发生过程。     

自发性2型糖尿病KK/Ta小鼠肾脏基因表达谱的研究

目的:应用基因芯片技术研究自发性2型糖尿病KK/Ta小鼠肾脏基因表达谱,旨在寻找糖尿病肾病的易感基因。方法:提取20周龄雄性KK/Ta(n=3)和BALB/c(n=2)小鼠肾脏总RNA,应用Affymetrix公司生产的Affymetrix Murine Genome U74Av2基因芯片检测肾脏基因表达谱。选择差异表达基因,通过竞争性RT-PCR反应验证基因芯片的结果。结果:98个已知基因和31个表达序列标签(ESTs)在20周龄KK/Ta与BALB/c小鼠的肾脏中存在着差异表达。与BALB/c小鼠相比,KK/Ta小鼠肾脏中21个已知基因和7个EST表达上调,77个已知基因和24个EST表达下调。竞争性RT-PCR反应确认了基因芯片研究的结果。结论:KK/Ta小鼠肾脏中的差异表达基因广泛参与细胞外基质的合成与降解、信号传导、转录调节与蛋白质合成、离子转运、葡萄糖与脂类代谢等。糖尿病肾病易感基因位点UA-1区域的差异表达基因S-腺苷高同型半胱氨酸水解酶基因Ahcy为糖尿病肾病的可能易感基因。     

2058个小鼠精子发生相关基因的筛选与表达研究

目的 通过比较不同阶段的小鼠睾丸全基因组表达谱,筛选小鼠年龄依赖性表达基因.方法 采用Affymetrix全基因组表达谱芯片(版本号430.2),对日龄为4、9、18、35、54d和6月龄的小鼠睾丸组织进行全基因组表达差异分析.并采用半定量RT-PCR验证其差异表达基因.结果 经过比较,在9d和18d,18d和35d之间的小鼠睾丸基因表达谱之间,存在大量的差异表达基因,其中有2058个基因在小鼠6个日龄中表现为递增表达趋势,通过半定量RT-PCR验证了其中10个随机挑选的递增表达基因.文献检索发现目前已知功能的21个精子发生相关基因均在这2058个基因之中.因此,推测这2058个基因是小鼠精子发生相关基因.本文同时对这2058个基因的基因表达和功能分类进行了生物信息学分析.结论 本文筛选出小鼠精子发生相关基因2058个,并对其表达特征、功能分类进行了初步分析,这项研究提供了从分子水平识别小鼠睾丸组织精子发生相关基因的方法,有助于进一步阐述雄性哺乳动物生殖相关分子学基础.     

CatSper基因家族在人和小鼠组织中的表达特征

目的研究CatSper基因家族在人和小鼠组织中的分布特点。方法将不同发育阶段的小鼠睾丸组织cDNA与Affymetrix全基因组芯片探针进行杂交,筛选出差异表达基因CatSper基因家族。RT—PCR验证差异表达基因在不同发育阶段的小鼠睾丸组织的表达特征,及其在人和小鼠不同组织中的分布。结果基因芯片分析发现CatSper1、CatSper2和CatSper3在小鼠睾丸的表达呈阶段特异性。RT-PCR结果表明该基因家族在睾丸的表达丰度明显高于其它组织;CatSper1和CatSper4在人体睾丸组织中特异性表达。结论CatSper基因家族在人和小鼠中呈现睾丸特异性表达或高表达,可能在精子发生中发挥重要功能。     

睾丸特异性基因TSF22在小鼠中的表达研究

目的 筛选与精子发生相关的基因。方法 将4、9、18、35、54日龄和6月龄小鼠睾丸组织cDNA探针与Affymetrix全基因组芯片进行杂交，筛选出差异表达的基因。利用网络信息资源对该基因进行生物学信息分析。RT-PCR分析该基因在小鼠睾丸不同发育阶段及小鼠不同组织中的表达。结果 芯片结果分析筛选出一个差异表达杂交点（GenBank登录号：NM_199034），生物信息学分析发现该基因全长1597bp，含有570bp的完整ORF，编码190个氨基酸、分子量为22．106kDa的蛋白质，我们将其命名为TSF22。RT-PCR分析表明TSF22基因特异性表达于睾丸组织及18日龄小鼠睾丸中。结论 TSF22基因的表达与小鼠精子发生的过程一致，可能在精子发生中起重要作用。     

Nonviral gene transfer for cancer gene therapy

Although the pre‐clinical and clinical results of gene therapy have shown promise for some cancers, cancer gene therapy is still at an early stage of clinical development. Due to the complexity of targeted vector delivery to the tumor, our strategy for gene therapy is focussed on the development of local non‐viral gene transfer to treat tumors. The local application of non‐viral gene therapy is of particular value in the context of pre‐ or intraoperative application of therapeutic genes. This ensures accessibility of targeted tumor areas and will contribute to better local control of the disease. In this regard, applicable transfer technologies are needed in gene therapy. Different physical procedures, such as in vivo electroporation, sonoporation, ballistic transfer etc. are employed to deliver naked DNA into the target cells or tissues in vitro and in vivo. Among the various non‐viral gene delivery technologies jet‐injection is gaining increasing acceptance, since this technique allows gene transfer into different tissues with deep penetration of naked DNA. The jet‐injection technology is based on low‐volume jets of high‐velocity to penetrate skin and deeper tissues associated with efficient transfection of the affected area. For non‐viral in vivo gene transfer a jet‐injector prototype was created and tested. The beta‐galactosidase (LacZ), green fluorescence protein reporter gene constructs were successfully jet‐injected into different syngeneic mouse and patient‐derived xenotransplanted human tumor models of colon‐ or mammary carcinoma and malignant melanoma. Qualitative and quantitative expression analysis of jet‐injected tumor tissues revealed the efficient expression of these genes. Therapeutic in vivo experiments using the jet‐injection transfer of the cytosine deaminase suicide gene in tumors demonstrated antitumor effects with significant growth inhibition of the jet‐injected xenotransplanted colon carcinomas. Furthermore, jet‐injection was also successfully used for the application of a heat‐inducible TNF‐α expressing vector system leading to efficient in vivo tumor growth inhibition in the combined non‐viral TNF‐α gene transfer and hyperthermia approach. Based on our pre‐clinical experiments for non‐viral gene transfer, a phase I clinical trial has been conducted at the Clinic for Surgery and Surgical Oncology, Charité, Berlin to evaluate the feasibility, efficiency, and safety of jet‐injection aided LacZ‐reporter gene transfer in patients with cutaneous metastases from breast cancer and malignant melanoma. In this study naked GMP‐plasmid DNA was applied intratumorally by jet‐injection. The jet‐injection was well tolerated by all patients and no side effects have been experienced. The study clearly demonstrated that the single application of plasmid‐DNA is safe and leads to the expression of the LacZ‐reporter gene in the tumor tissue, as shown at mRNA‐ and at protein level.     

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。     

基因芯片在膀胱癌基因研究中的应用

膀胱癌是泌尿系统最常见的恶性肿瘤,以空间上的多中心与时间上的反复发作为其生物学特点,其发生由多个基因控制、多步骤进行.基因芯片技术是一种高通量的基因分析平台,现已广泛用于疾病机制的研究、疾病的分类和诊断、疾病的预测和治疗,并用于膀胱癌发生、发展相关基因的筛选、分子治疗靶点的筛选、寻找膀胱癌亚型的分子标记以及肿瘤预后分类的研究.     

Human insulin-receptor gene

The human insulin-receptor (hINSR) gene spans a region of greater than 120,000 base pairs (bp) on the short arm of chromosome 19. It is comprised of 22 exons or coding regions that vary in size from 36 to greater than 2500 bp. To a large degree, the introns appear to divide the hINSR gene into segments that encode structural and/or functional elements of the hINSR protein. The exon-intron organization of the hINSR gene provides a clue to the evolutionary history of this gene and suggests that it is a mosaic constructed from protein-coding regions recruited from other genes. Eight mutations in the hINSR gene that result in expression of structurally abnormal proteins have been described. These mutations are associated with insulin resistance and provide insight into the role of the hINSR gene in the development of diabetes mellitus.     

Cardiac gene therapy

Heart failure is a chronic progressive disorder in which frequent and recurrent hospitalizations are associated with high mortality and morbidity. The incidence and the prevalence of this disease will increase with the increase in the number of the aging population of the United States. Understanding the molecular pathology and pathophysiology of this disease will uncover novel targets and therapies that can restore the function or attenuate the damage of malfunctioning cardiomyocytes by gene therapy that becomes an interesting and a promising field for the treatment of heart failure as well as other diseases in the future. Of equal importance are developing vectors and delivery methods that can efficiently transduce most of the cardiomyocytes that can offer a long-term expression and that can escape the host immune response. Recombinant adeno-associated virus vectors have the potential to become a promising novel therapeutic vehicles for molecular medicine in the future.     

Hepatocyte-based gene therapy

Hepatocyte-based gene therapy may be used to replace a missing gene product, confer proliferating ability to cultured hepatocytes, prevent allograft rejection, massively repopulate the host liver, or grow xenogeneic hepatocytes in mammalian liver. Gene transfer into isolated hepatocytes can be accomplished via nonviral or viral vectors, the viral vectors being more useful at this time. Common recombinant viruses that integrate into the host genome include murine leukemia retroviruses and lentiviruses, adenoassociated virus, and the T-antigen-deleted SV40 virus. Episomal viruses, such as adenoviruses, permit efficient gene transfer, but the transgene is lost upon proliferation of the transplanted hepatocyte in the host. Hybrid viruses that combine the high transduction efficiency of adenoviral vectors and the integrative capacity of other vectors, such as adenoassociated viruses, have been designed. Massive repopulation of the liver by transplanted hepatocytes can be achieved if a mitotic stimulus to the transplanted cells is combined with prevention of proliferation of the host hepatocytes. Treatment with a plant alkaloid or retrorsine, or preparative irradiation of the liver can be used to inhibit host hepatocellular proliferation, while partial hepatectomy, expression of Fas ligand, or thyroid hormone administration can be used as a mitotic stimulus to the transplanted cells. Received: July 4, 2000 / Accepted: October 13, 2000