哺乳动物基因非翻译区开放阅读框架的分析

在所分析的5’UTR序列中约19%的序列在阅读框1下含有AUG,约22%和24%的序列分别在阅读框2和阅读框3下含有AUG,而在3’UTR序列当中,无论是在哪个阅读框下,约71%含有AUG.这些AUG绝大多数都是以小ORF的形式出现的;分析小ORF序列以及与它们相应的IC序列(阅读框间序列)长度,发现虽然3’UTR序列远远长于5’UTR序列,但是它们所包含的小ORF长度几乎没有差距,只是平均来说每一条3’UTR序列所包含的小ORF数量明显多于每一条5’UTR序列所包含的小ORF数量,而且dIC(下游阅读框间序列)比uIC(上游阅读框间序列)长很多,平均长50个碱基;第二类IC序列(UTR区相邻的两个阅读框之间的序列)的平均长度比其它两类IC序列(UTR区最后一个阅读框之后的序列)的平均长度小,而且又含有相对较多的终止密码;比较uORF序列与dORF序列密码子使用偏好的CAI值发现,尽管相差不大,但是无论在哪个阅读框下,uORF序列的CAI值都显著高于dORF序列的CAI值.

过氧化氢信号途径参与哺乳动物Bax基因对长春花细胞中萜类吲哚碱生物合成的诱导作用

Bax是哺乳动物细胞凋亡基因Bcl-2家族中的一员.最近的研究结果表明小鼠Bax可以从转录水平上诱发植物细胞次生代谢产物合成积累,提示Bax及其植物体内的同源基因可能是次生代谢产物合成的一种新型调控因子.为了研究Bax诱发植物次生代谢产物合成的分子机理,文中测定了小鼠Bax对长春花细胞氧化迸发(oxidative burst)的影响,并考查了质膜NAD(P)H氧化酶抑制剂DPI和活性氧中间体淬灭剂对小鼠Bax诱发长春花碱及萜类吲哚生物碱合成的影响.实验结果表明,小鼠Bax可以诱发长春花细胞氧化迸发.DPI在抑制Bax诱发长春花细胞氧化迸发的同时,还可以阻断Bax对长春花碱及萜类吲哚生物碱合成的促进作用.实验结果说明小鼠Bax不仅可以激活长春花细胞中活性氧信号转导事件而且还依赖氧化迸发作用诱导长春花细胞次生代谢产物合成.超氧化物歧化酶(SOD)可以淬灭长春花细胞产生的超氧阴离子(O2-),但不影响小鼠Bax对长春花次生代谢产物合成的促进作用,说明由氧化迸发产生的O2-可能不是介导小鼠Bax诱发长春花碱及萜类吲哚生物碱合成必需的信号分子.而过氧化氢酶(CAT)在淬灭细胞中过氧化氢(H2O2)的同时还可以阻断小鼠Bax对长春花碱及萜类吲哚生物碱合成的诱导作用,表明H2O2可能是介导小鼠Bax诱发长春花细胞次生代谢产物合成所必需的信号分子.小鼠Bax可以诱发长春花细胞中萜类吲哚碱生物合成途径关键酶基因Tdc和Str转录上调,CAT可以抑制Bax对Tdc和Str表达的诱导作用,进一步证实小鼠Bax依赖氧化迸发所产生的H2O2激活长春花细胞中萜类吲哚碱生物合成途径并诱发长春花碱及萜类吲哚碱的合成积累.

显微注射法培育转基因哺乳动物的研究进展

加州大学医学院 Teh Ping Lin 博士早在1966年即利用小鼠受精卵原核显微注射技术,系统地研究了微注射不同量外源物质(石蜡油、牛γ-球蛋白溶液)对小鼠胚胎受体移植后的分裂、发育的影响程度,并预见该技术用于研究小鼠或其它种类哺乳动物胚胎分化及遗传效应具有潜在价值。直到1980年,随着分子生物学技术的迅速发展,Gordon 等人首先采用显微注射法育成带有人胸苷激酶基因这一外源 DNA 片断的'新型'转基因小鼠后。

哺乳动物转基因技术研究

介绍了转基因哺乳动物的研究概况,综述了哺乳动物转基因技术基本方法,外源基因的整合和表达,转基因哺乳动物的检测方法以及哺乳动物转基因技术的应用和存在的问题。

哺乳动物雷帕霉素靶蛋白及其作为骨肉瘤治疗靶点的研究进展

哺乳动物雷帕霉素靶向基因(mTOR)一直在肿瘤防治研究领域发挥着重要的作用,它具有特殊的生物学功能,对多种癌症的临床治疗具有重要意义,如骨癌、肺癌和乳腺癌等。近年来研究表明,mTOR信号通路在骨肉瘤的发生发展过程中起着重要的作用,包括细胞生存、增殖、转移以及血管再生等。关于运用mTOR抑制剂作为抗肿瘤药物已经得到广泛研究,并取得一定进展,也为骨肉瘤的防治提供了新策略。本文将综述mTOR信号通路在骨肉瘤中的作用以及目前mTOR抑制剂在临床治疗中的研究现状。

哺乳动物STE20相关激酶1基因在肝癌组织的表达及其与乙型肝炎病毒的关系

目的 探讨哺乳动物STE20相关激酶1（MST1）基因在肝癌中的表达水平及与乙型肝炎病毒（HBV）感染及乙型肝炎病毒X蛋白（HBx）的关系.方法 收集临床肝癌组织标本及培养肝癌细胞株,采用Western blot和实时定量聚合酶链反应（Real-time PCR）方法检测MST1的蛋白与mRNA水平及与HBV及HBx的关系.提取感染HBV的细胞检测MST1启动子区甲基化,加入甲基化抑制剂后MST1的变化.结果 32对组织中4对未检测出MST1的表达,其余28对癌组织的MST1 mRNA水平和蛋白水平均明显低于癌旁的表达（P＜ 0.05）.且HBV感染时MST1表达（HepG2 mRNA相对值为13％,HepG2.215 mRNA相对值为7％）明显低于对照组（P＜0.05）.HBV感染细胞后MST1启动子区发生甲基化率为39％,非HBV感染细胞甲基化率为17％,且加入甲基化抑制剂后MST1随抑制剂浓度的升高而增加（P＜0.05）.结论 HBV可能通过甲基化MST1启动子区抑制MST1基因的表达从而进一步导致肝癌发生发展.

17例原发性肺黏液表皮样癌的病理分析与文献复习

目的 探讨原发性肺黏液表皮样癌的组织病理、临床特征及CRTC1/3-MAML2融合基因的情况。方法 回顾分析2019-2021年中国科学技术大学附属第一医院17例原发性肺黏液表皮样癌,并对其临床信息、病理组织学形态、免疫组化及CRTC1/3-MAML2融合基因进行检测。结果 患者平均年龄为51.3±17.3岁。男女人数比例相仿。瘤体大小为3~97mm(平均值为27.4±22.6mm)。左右肺发病比例相仿。低级别黏液表皮样癌14例(82.4%),高级别黏液表皮样癌3例(17.6%)。免疫组化中,CK7和CK5/6表达率均为100%。MAML2基因重排与患者的年龄、性别、瘤体直径、部位及病理分化程度均无显著相关性(P>0.05)。结论 CRTC1/3-MAML2融合基因重排在黏液表皮样癌中的发生率高,因此MAML2基因重排可以成为原发性肺黏液表皮样癌的分子辅助诊断指标之一。

Mammalian Models Based on RCAS-TVA Technique

The retroviral vector （RCAS） has been widely used in avian system to study development and diseases, but is not suitable for mammals which do not produce the retrovirus receptor TVA. In this review, we trace the current uses of RCAS-TVA approach in mammalian system with improved strategies, including generation of tv-a transgenic mice, use of soluble TVA receptor and retroviral receptor-ligand fusion proteins, improvement of RCAS vectors, and compare a series of mammalian models in variant studies of gene function, development, oncogenesis and gene therapy. All those studies demonstrate that the RCAS-TVA based mammalian models are powerful tools for understanding the mechanisms and target treating of human diseases.

金柚提取物对雌激素受体调节作用的研究

目的研究金柚提取物对雌激素受体(ER)的调节作用,并鉴定其功效成分。方法利用哺乳动物报告基因系统检测金柚总提取物及高效液相色谱(HPLC)分离的单一化学成分对ERα和ERβ的调节作用。通过与标准品的紫外可见吸收光谱及HPLC图谱鉴定其活性成分,并与雌二醇、染料木苷等ER激活剂比较活性。结果金柚提取物能特异性激活ERβ,对ERα无影响,活性成分主要为柚皮素和柚皮苷(部分激活剂),与雌二醇、染料木苷活性比较:雌二醇>染料木苷>柚皮素>柚皮苷。结论金柚含有ERβ特异性激活成分,能用作ERβ相关的药物治疗及保健品。

Metallothionein:An overview

Metallothioneins （MTs） were discovered in 1957 by Margoshes and Vallee and identified as low-molecular weight and sulphydryl rich proteins. It is not surprising that most mammalian tissues contain age related basal levels of MTs since they are involved in metalloregulatory processes that include cell growth and multiplication. In an effort to understand the biology of this intriguing tumor, various biomarkers such as oncogenes, p53 tumor suppressor gene, war 1 protein, proliferating cell nuclear antigen, telomerase, microsatellite markers and cytogenetic changes have been examined. One biomarker which has recently shown to be expressed in various human tumors but still less reported in carcinoma is MT. Immunohistochemical detection of MT proteins in cold acetone-fixed paraffin embedded liver sections was performed by the streptavidin-avidin-biotin immunoperoxidase complex method.

Benefit of everolimus as a monotherapy for a refractory breast cancer patient bearing multiple genetic mutations in the PI3K/AKT/mTOR signaling pathway

A postmenopausal patient with a diagnosis of estrogen receptor(ER)(+), progesterone receptor(PR)(+), and human epidermal growth factor receptor-2(HER2)(-) breast cancer was reported. The patient refused surgery and was resistant to conventional chemotherapy regimens. Computed tomography and the circulating tumor cell test indicated that the patient's tumor burden increased rapidly even after several chemotherapy sessions. Multiple genetic aberrances in the phosphatidylinositol3-kinases(PI3 K) signaling pathway were detected via next-generation sequencing(NGS)-based liquid biopsy, including a p. G1007 R missense mutation in exon 21 of PIK3 CA(33.61%), a p.L70 fs frameshift mutation in exon 3 of phosphatase and tension homolog deleted on chromosome ten(PTEN)(49.14%), and a p. D1542 Y missense mutation in exon 32 of mammalian target of rapamycin(m TOR)(1.66%). Therefore, only the m TOR inhibitor everolimus was administered to the patient. Partial remission(PR) was observed after 2 months, and sustained stable disease(SD) was observed after a year and a half. Subsequent sequencing showed that the mutation ratio of PIK3 CA decreased to 4.17%, and that the PTEN and m TOR mutations disappeared, which revealed the significant curative effect of everolimus. We report the first case of successful monotherapy treatment using everolimus in a patient with advanced breast cancer bearing mutations in genes involved in the PI3 K/ARK/m TOR signaling pathway. The success of this case highlights the invaluable clinical contribution of NGS-based liquid biopsy, as it successfully provided an optimal therapeutic target for the patient with advanced breast cancer.

The Function of SeMNPV IAP3 in Mammalian Cells

The baculoviral inhibitors of apoptosis play a significant role in infectivity and viral host-range, which make them potential candidates for the engineering and improvement of baculovirus insecticidal. The iap3 gene of Spodoptera exigua nucleopolyhedrovirus （SeMNPV）, amplified by PCR, was 939 bp encoding IAP3. The PCR product was cloned into EcoR I/Barn H I of the plasmid pEGFP-C1. GFP was fused to the N-terminaus of IAP3 to study distribution in HEK293. It was observed that the plasmid expressing IAP3 significantly inhibited apoptosis induced by cisplatin in HEK293 cells. We conclude that the IAP3 of SeMNPV is functional in mammalian cells.

mTOR、eIF4E在原发性肝细胞癌组织中的表达及意义

目的研究m TOR和e IF4E因子在原发性肝细胞癌中的表达及意义。方法采用免疫组织化学SP法分析45例原发性肝癌中m TOR和e IF4E的表达情况,实验结果图像采用Biosens Digital Imaging System v1.6图像分析系统进行定量分析,免疫组化结果的染色标记强度用平均灰度值表示,应用医学统计学方法对m TOR与e IF4E蛋白的相关性进行分析。结果 m TOR和e IF4E在组织中的表达均为远癌肝组织组<癌旁组<肝癌组,两两比较,差异具有统计学意义(P<0.05);m TOR和e IF4E在组织中表达均呈正相关性(P<0.05)。结论 m TOR和e IF4E的异常表达可能与原发性肝癌的发生、发展有关,并在肝癌细胞的增殖与侵袭中起着协同作用,共同参与了原发性肝癌的侵袭性生长。

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules

AIM： To construct and produce a recombinant bispecific humanized single-chain Fv （sFv） /Interleukin-2 （IL-2） fusion protein by using mammalian cells. METHODS： The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. RESULTS： The fusion protein was constructed and high efficiently expressed with yields up to 102 ±4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy （VH） and light （VL） domains of monoclonal antibody （mAb） 520C9 directed against the human HER-2/neu （c-erbB2） proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL- 2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. CONCLUSION： The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means.of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

Overview of macroautophagy regulation in mammalian cells

Macroautophagy is a multistep, vacuolar, degradation pathway terminating in the lysosomal compartment, and it is of fundamental importance in tissue homeostasis. In this review, we consider macroautophagy in the light of recent advances in our understanding of the formation of autophagosomes, which are double-membrane-bound vacuoles that sequester cytoplasmic cargos and deliver them to lysosomes. In most cases, this final step is preceded by a maturation step during which autophagosomes interact with the endocytic pathway. The discovery of AuTophaGyrelated genes has greatly increased our knowledge about the mechanism responsible for antophagosome formation, and there has also been progress in the understanding of molecular aspects of autophagosome maturation. Finally, the regulation of autophagy is now better understood because of the discovery that the activity of Atg complexes is targeted by protein kinases, and owing to the importance of nuclear regulation via transcription factors in regulating the expression of autophagy genes.

Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line

RNA interference(RNAi) is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the capsid protein gene of potato virus Y(CP-PVY) was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.

Novel Functions of Interleukin-1 Beta in Spotted Halibut(Verasper variegatus) at Its Embryonic and Metamorphic Stages

Interleukin-lbeta （IL-1β） is a cytokine involved in the control of inflammatory responses. The functions of IL-1β have been extensively studied in many vertebrates. However, few studies focused on the functions of IL-1β in the spotted halibut （Verasper variegatus）, an endangered aquatic fish that easily suffers from bacterial and viral infections. We first cloned and sequenced the IL-1β gene from V. variegatus （VvIL-1β）. Its conservative interleukin-1 propeptide and interleukin-1/18 domains corresponding regions, characteristics of the IL-1 protein family and TA-rich motifs relating to inflammatory responses were similar to those of the IL-1β genes from other teleost and mammals. The phylogenetic analysis showed that VvIL-1β was homologous to the IL-1β gene of other vertebrates. Treatment with pathogen-associated molecular patterns （PAMPs） induced a significant and rapid up-regulation of VvIL-1β expression in the peripheral blood leukocytes （PBLs）, revealing that VvIL-1β is actively involved in the host ilmnune responses against bacterial and viral pathogens. The expression level of VvIL-1β was relatively high in the PBLs, gill, and intestine, indicating the immunological function of VvIL-1β . VvIL-1β also had interesting expression patterns at specific developmental stages, implying that the IL-1β of flatfishes may have some novel functions during embryonic development and metamorphosis. The 3D structure prediction also provided information about how VvIL-113 interacts with its receptor protein. These results suggested that VvIL-1β may have multiple functions in V. variegatus and provided us valuable information for understanding the novel functions of IL- 1β.

microRNAs regulation and its role as biomarkers in diseases

MicroRNAs （miRNAs）, approximately 21 to 23 nucleotides （nt） in length, belong to a set of smal non-coding RNA molecules that were not thought to be functional until the recent decades. miRNAs play important roles in many diseases such as various kinds of cancers and immune disorders. Many studies have focused on the relationship between miRNAs and diseases. miRNAs are significant mediators in human growth and development and in the genesis and development of diseases. Almost 30% of the activity of protein-coding genes is forecasted to be regulated by miRNAs in mammals, and some miRNAs are regarded as potential therapeutic targets for various diseases. In this review, we outline some functions of miRNAs, especialy those related to diseases.

Construction and identification of a vector expressing TRAM siRNA in mammalian cells

Objective:To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods:It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264.7 cell by using Lipofectamine TM2000, and the expression of TRAM was detected by Western blotting. Results:Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion:The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.

mTOR 基因多态性与肝细胞癌发病关联性＊

目的探讨广西地区人群mTOR基因的潜在生物学功能单核苷酸多态性(SNP)位点rs2536和rs1883965与肝细胞癌(HCC)发病的关系,为HCC的预防控制提供科学依据。方法采用以医院为基础的病例对照研究方法,抽取2007年1月—2011年4月在广西医科大学第一附属医院和广西医科大学附属肿瘤医院就诊的1 048例HCC患者和1 052例非肿瘤患者分别作为病例组和对照组人群进行问卷调查和基因分型,应用logistic回归模型分析各SNP位点及其与环境因素交互作用与HCC发病的关系。结果病例组人群mTOR基因rs2 536位点TT、TC、CC和TC+CC基因型者分别占81.01%、17.75%、1.24%和18.99%,与对照组人群的80.80%、17.87%、1.33%和19.20%比较,差异均无统计学意义(P>0.05);病例组人群mTOR基因rs1883965位点GG、GA、AA和GA+AA基因型者分别占84.06%、15.74%、0.19%和15.94%,与对照组人群的83.84%、15.21%、0.95%和16.16%比较,差异均无统计学意义(P>0.05);分别按年龄、性别、民族、吸烟史、饮酒史和乙型肝炎病毒感染进行分层分析,结果表明,mTOR基因rs2536和rs1883965位点多态性与HCC发病风险均无统计学关联(P>0.05)。结论 mTOR基因rs2536和rs1883965位点基因多态性与HCC发病无关。