同时检测小鼠微小病毒(MVM)和小鼠细小病毒(MPV)的荧光定量PCR的建立及应用

目的 建立同时检测小鼠微小病毒(MVM)和小鼠细小病毒(MPV)的荧光定量PCR方法,并进行初步应用。方法 比对NCBI上发表的MVM和MPV基因组序列,设计1对引物和探针,可同时检测MVM和MPV。考察MVM-MPV引物探针的特异性和灵敏度,并对178份清洁级小鼠粪便DNA样本进行检测。结果 MVM-MPV荧光定量PCR方法最佳线性范围为109～104拷贝/μL,标准曲线的线性关系良好,R2值可达0.99,灵敏度为101拷贝/μL,特异性强。应用MVM-MPV探针对178份小鼠粪便DNA检测,结果为2份阳性样本,经MVM、MPV特异性探针鉴定,2份阳性样本均为MPV感染。阳性样本经全基因组测序后与NCBI网站上MPV(NC_001630.1)序列比对,一致率为96%。结论 建立的MVM-MPV荧光定量PCR方法,能够有效快速地同时检出小鼠细小病毒和小鼠微小病毒。     

致癌基因密码子使用频数统计

本文在最新的致癌基因核酸序列数据库的基础上,对各258个致癌基因序列的密码子使用频数进行统计,并且分析统计结果,认为翻译的选择、变异和碱基成分可能是造成致癌基因序列的密码子使用偏好的主要原因。     

小鼠细小病毒抗体的检测

<正> 小鼠细小病毒(MVM)普遍存在于开放饲养的实验小鼠群中,呈隐性感染状态,在某些条件下,可诱发此病,以致影响实验结果。由于小鼠呈隐性感染时,无临床症状,也无病理形态学的改变,很难分离到病毒,所以血清抗体的检测在诊断上有着重要的意义。我们采用血凝抑制试验(HAI)、免疫荧光(IFA)和酶免疫吸附试验(ELISA)方法来检测开放鼠群中的MVM抗体。材料和方法一、MVM标准株:由中国医学科学院医学实验动物研究所提供。二、MVM抗原的制备: 1.用于HAI抗原:MVM感染二代小     

小鼠细小病毒VP2蛋白在昆虫细胞-杆状病毒系统中的表达和免疫原性分析

目的 利用杆状病毒表达系统合成重组小鼠细小病毒(MVM)VP2蛋白，并对其免疫原性分析。方法 将优化后的MVM VP2序列克隆至表达载体pFastBac1,命名为pFastBac1-MVM VP2,再将其转化至DH10Bac感受态大肠杆菌中形成重组杆粒，提取重组杆粒经PCR鉴定正确后转染昆虫细胞Sf9,镜下观察到细胞明显病变后收获重组杆状病毒rBac-MVM VP2。Sf9细胞接重组病毒48和72 h后，用间接免疫荧光(IFA)和蛋白免疫印迹(Western blot)法验证重组蛋白的免疫原性。结果 构建的rBac-MVM VP2重组杆状病毒感染Sf9细胞后，可成功表达MVM VP2重组蛋白，经Western blot和IFA检测分析，重组蛋白具有较好的免疫原性且在病毒感染72 h时表达量较高。经蛋白纯化后可得到纯度较高的VP2重组蛋白。结论 本研究利用昆虫细胞-杆状病毒系统成功表达MVM VP2蛋白并具有良好的免疫原性，为VP2相关功能的研究和临床诊断方法的建立奠定基础。     

有机化学品分子片段定量结构与致癌活性研究

以分子连接性能和微扰分子轨道指数作结构、能量参数，从分析ＤＮＡ链互补碱基对２癌有机物分子片段发生共价交联的构效角度出发，首次得出产生碱基移码或置换型化学致癌的主要过程是化学品分子片段中活性原子与ＤＮＡ互补碱基对间氢键的共价结合，显示致癌性的充分必要条件是碱基Ｇ≡Ｃ间两个氢键与致癌片的特异交联。     

细小病毒抑瘤活性研究的历史与现状

细小病毒（PV）（包括自主性的及依存性的）有抗肿瘤活性。本文在介绍PV绵结构及理化性质、病毒复制后，阐述了PV与肿瘤的关系，即其对自发肿瘤及诱发肿瘤的抑制作用，特别虽本实验室多年来利用离体细胞培养系统广泛地测试了PVH－1及MVM抑制转化细胞及癌细胞活性得到的结果，对33株细胞及人癌（肝、肾）与癌周组织原代培养的细胞测得的抑瘤效应亦列于表内。PV抑瘤机理仍在分子水平上研讨之中，文中强调了非结构蛋白NS／rep的细胞毒作用，它可部分地阐明了PV的抗肿瘤机制。     

小鼠微小病毒( MVM) 荧光定量PCR 检测方法的建立及初步应用

摘要: 目的建立小鼠微小病毒( MVM) 荧光定量PCR 方法,并初步应用于实验小鼠中。方法根据NCBI 上发表的MVM( NC_001510) 基因组序列,设计引物和探针,建立MVM 荧光定量PCR 方法。考察引物探针的最佳线形范围,特异性及灵敏度,并使用该方法对178 份清洁级小鼠粪便DNA 样本进行检测。结果MVM 荧光定量PCR 方法最佳线性范围为109 ～ 104 拷贝/μL,标准曲线的线性关系良好,R2 值可达0. 99,灵敏度为101 拷贝/μL,特异性强,与大鼠细小病毒H-1 株和KRV 株、猪细小病毒均无交叉反应。应用荧光定量PCR 方法对178 份小鼠粪便DNA进行检测,结果均为阴性。结论建立的MVM 荧光定量PCR 方法具有特异、灵敏的特点,为MVM 的流行病学调查、检测提供了有效的技术支持。     

哪些药物可能致癌

随着医学的发展．某些致癌病因已逐步查明．其中就包括滥用药物导致癌症这一不容忽视的因素在内。     

供体内致癌试验用的转基因小鼠在美实现商品化

美国GenPharm Interna tional公司由科学顾问Anton Berns等领导的荷兰癌症研究所一研究组发现,导入癌基因Pim-1的转基因小鼠可用于高灵敏度的体内致癌试验。导入和人癌有关的基因的转基因小鼠,与一般的动物相比,对致癌物的灵敏度可提高25倍。     

两种细小病毒MVM宿主细胞的比较研究

ＭＶＭ属自主性细小病毒，对相当多种类的体内外生长的肿瘤细胞和转化细胞有抑制作用，本文对ＮＢＫ细胞的软琼脂克隆形成率和以体进行了分析，确认ＮＢＫ细胞为转化细胞，且对ＭＶＭ敏感。在此基础上，比较了两种细小病毒ＭＶＭ宿主细胞ＮＢＫ和Ａ９的细胞存活率、单位体积培养液中的细胞产量和病毒产量，认为Ａ９细胞更适合于作为细小病毒ＭＶＭ的宿主细胞进行病毒的制备和生产。     

Ha-ras和Ki-ras癌基因转染和细胞对小鼠细小病毒杀伤敏感性的相关性(英文)

为研究Ha－ras和Ki－ras癌基因转染和细胞对小鼠细小病毒（MVM）杀伤敏感性间的关系，两个细胞株，即Ki－ras癌基因转化细胞株DT和Ha－ras癌基因转化细胞株REF4-3，被用来作为研究材料．体外细胞集落形成率和细胞在裸小鼠体内的成瘤能力测定显示，和对照细胞NIH／3T3相比，REF4－3和DT对MVM的杀伤作用更敏感．同时，DNA杂交和蛋白免疫沉淀实验结果也显示，在REF-3和DT细胞中，无论是MVM的DNA增殖能力还是其NS－1蛋白的表达能力，均较在其对照组NIH／3T3细胞中高很多．FACA测定也显示，在感染MVM30h后，S期细胞的比例在REF4－3和DT细胞中都有增加，而在NIH／3T3细胞中却略微减少．通过PCR方法也可发现，在受到MVM抑制的REF-3肿瘤中仍可检测到MVM的DNA．     

Tumour maintenance is mediated by eNOS

Tumour cells become addicted to the expression of initiating oncogenes like Ras, such that loss of oncogene expression in established tumours leads to tumour regression. HRas, NRas or KRas are mutated to remain in the active GTP-bound oncogenic state in many cancers. Although Ras activates several proteins to initiate human tumour growth, only PI3K, through activation of protein kinase B (PKB; also known as AKT), must remain activated by oncogenic Ras to maintain this growth. Here we show that blocking phosphorylation of the AKT substrate, endothelial nitric oxide synthase (eNOS or NOS3), inhibits tumour initiation and maintenance. Moreover, eNOS enhances the nitrosylation and activation of endogenous wild-type Ras proteins, which are required throughout tumorigenesis. We suggest that activation of the PI3K-AKT-eNOS-(wild-type) Ras pathway by oncogenic Ras in cancer cells is required to initiate and maintain tumour growth.     

The ras oncogene product p21 is not a regulatory component of adenylate cyclase

Harvey (Ha-MSV) and Kirsten (Ki-MSV) murine sarcoma viruses induce tumours in animals and transform various cells in culture because of the expression of the ras oncogene product, p21 (ref. 1). Proto-oncogenes homologous with these genes are highly conserved evolutionarily and activated ras oncogenes have been detected in many human cancers. Whether c-ras oncogenes are directly responsible for human carcinogenesis is uncertain; however, it is clear that p21 mediates virus-induced transformation, although by an unknown mechanism. Epithelial and fibroblast cell lines transformed with Ha-MSV and Ki-MSV express p21 (ref. 8) and exhibit reduced adenylate cyclase activity. Like the guanine nucleotide regulatory proteins, Ns and Ni, which mediate stimulation and inhibition, respectively, of adenylate cyclase, p21 is a membrane-associated GTP binding protein, which exhibits GTPase activity. These similarities suggest that p21 and the adenylate cyclase regulatory proteins are related in cellular function, and that p21 depresses adenylate cyclase by inhibiting the activity of Ns or acting as Ni. We have therefore now examined the structural and functional similarities between p21 and Ns and Ni and find no evidence that p21 regulates adenylate cyclase activity by acting as one of these regulatory proteins.     

Glucocorticoid-dependent oncogenic transformation by type 16 but not type 11 human papilloma virus DNA

Squamous cell carcinoma of the uterine cervix is one of the most common cancers among women. Correlation between human papilloma virus (HPV) infection of the uterine cervix and the development of cervical neoplasia has been established. More recent studies have shown the presence and expression of integrated HPV types 16 and 18 DNA sequences in 70-80% of cervical tumours and tumour cell lines. It has been suggested that, in addition to HPVs, other agents such as hormones and tobacco products act as cofactors in cervical neoplasia (for review see ref. 15). The presence and expression of a glucocorticoid-responsive element in HPV-16 has been reported. Here we provide evidence for the oncogenic transformation of primary cells with a combination of HPV-16 DNA, but not HPV-11 DNA, and the activated form of the human Ha-ras oncogene only in the presence of the glucocorticoid hormone dexamethasone.     

Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein

The proteins encoded by the ras oncogene are thought to trigger expression of the transformed phenotype in some types of cancer cells. In human cells, the ras protein family consists of several members including normal (proto-oncogene) and mutant (oncogene) forms. In general, the proto-oncogene forms are thought to be involved in the normal growth control of cells, while the mutant forms (which apparently result from somatic mutation of the normal ras genes) appear to be responsible, in part, for the loss of normal growth control. On microinjection into living normal cells, the purified ras oncogene protein (p21) induces a characteristic loss of growth control in cells within several hours. The mutant forms of the different ras proteins typically contain a single amino-acid change, usually at position 12 or less frequently at position 61. Here we report that microinjection of antibodies specific for amino acid 12 of the oncogenic v-Ki-ras protein into cells transformed by this protein causes a transient reversion of the cells to a normal phenotype. The fact that this antibody inhibits binding of GTP to the v-Ki-ras protein supports the notion that GTP binding is essential to the transforming function of this oncogene product.