Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells

Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.     

人endostatin基因重组逆转录病毒载体的构建及其在NIH3T3细胞中表达

为探讨转人血管内皮抑制基因（endostatin）在转染细胞中的表达，利用逆转录病毒载体构建人endostatin基因的重组质粒，通过脂质体（lipofectamine）将重组质粒导入包装细胞PA317,制备重组病毒液。用重组病毒液感染NIH3T3细胞，经G418筛选获得转入endostatin基因细胞株NIH3T3-endo。同法制备对照细胞株NIH3T3-pLncx.PCR检测NIH3T3-endo细胞基因组，在扩增产物中一份550bp人endostatin基因特异性片段，对照组为阴性。免疫组化测定示仅NIH3T3-endo细胞中有外源性endostatin蛋白的表达，说明人endostatin基因已被成功导入NIH3T3细胞，并获得稳定表达。     

Prevention of chemical carcinogenesis using glutathine S-transferase-pi (GST-pi)

In order to explore whether the protective function of GST-pi can prevent transformation in vitro, NIH3T3 cells and carcinogen glycidyl methacrylate (GMA) have been used in cell transformation study. NIH3T3 cells have been transfected with GST-pi cDNA inserted retrovirus vector, pXT1, and then G418 resistant clones have been analyzed by Southern and Northern analyses. NIH3T3/pXGST clones that stably express GST-pi and control cells, untransfected NIH3T3 and NIH3T3/pXT1, have been treated three times discontinuously with GMA. 1.287% of untransfected NIH3T3 and 1.197% of NIH3T3/pXT, cells obtained a transformation pheno-type, forming type Ⅲ transformed clones, which could grow in soft agar and form fibrosarcoma in nude mice. In comparison, the transformation rate is only 0.007% in NIH3T3/pXGST cells, which could not grow in soft agar and formed no tumor in vivo. The results showed that expression of exogenous GST-pi in NIH3T3 do protect NIH3T3 cells from GMA induced transformation in vitro, which provides an evidence that GST-pi may play a role in preventing chemical car-cinogenesis.     

Raf-1 protein kinase is required for growth of induced NIH/3T3 cells

Many growth factors regulate the cytoplasmic Raf-1 protein kinase, consistent with its having a central role in transduction of growth signals. The kinase is ubiquitously expressed and can promote proliferation, presumably in a manner dependent on growth-factor receptors and membrane-associated oncogenes. We have now examined the dependence of serum- and TPA (12-O-tetradecanoylphorbol-13-acetate)-regulated NIH/3T3 cell growth on RAF-1 kinase to determine whether Raf-1 is essential for receptor signalling. We inhibited Raf-1 function by expressing c-raf-1 antisense RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1 interferes with proliferation of normal NIH/3T3 cells and reverts raf-transformed cells. In revertant cells, DNA replication induced by serum or TPA was eliminated or reduced proportionately to the reduction in Raf protein levels. Expression of a kinase-defective Raf-1 mutant (craf301) or a regulatory domain fragment (HCR) inhibited serum-induced NIH/3T3-cell proliferation and raf transformation even more efficiently. Inhibition by antisense RNA or craf301 blocked proliferation and transformation by Ki- and Ha-ras oncogenes. We conclude that raf functions as an essential signal transducer downstream of serum growth factor receptors, protein kinase C and ras.     

Rapid senescence induced by overexpression of p53 in NIH3T3 cells

An NIH3T3 cell line which overexpresses temperature-sensitive p53Val135 was constructed by introduction of p53Val135 gene. It exhibited rapidly characteristic morphological and biochemical alterations related to repli-cative senescence when being cultured in 32℃. We suggested that the overexpression of p53 activated probably the onset of senescence in NIH3T3 cells, which induced a rapid cellular senescence.     

重组PAI—2基因在NIH3T3细胞中的活性表达

构建了人ＰＡＩ—２ｃＤＮＡ睥达质粒，以ＮＩＨ３Ｔ３为转染宿主细胞，ＮｏｒｔｈｅｒｎＢｌｏｔ，ＷｅｓｔｅｒｎＢｌｏｔ和ＰＡＩ活性扩散试验结果表明人ＰＡＩ—２ｃＤＮＡ可以在ＮＩＨ３Ｔ３细胞中正确表达出ＰＡＩ—２蛋白，且具有抑制ＰＡ的活性，同时未能检测到ＮＩＨ３Ｔ３细胞表达ＰＡＩ—２蛋白质．为探讨ＰＡ—ＰＡＩ—２系统的调控机制打下基础．     

N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

The L protein (241kD) of vesicular stomatitis virus (VSV) is the most important snbnnit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein (GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kept on the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in the perinuclear regions. And the fusion protein with 96—120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108—120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.     

胰岛自身抗原GAD65片段和胰岛素B链基因共表达DNA疫苗的构建与表达

构建两种胰岛自身抗原谷氨酸脱羧酶（ glutamic acid decarboxylase, GAD） 65片段与胰岛素B链基因共表达DNA疫苗,并检测其在体外COS-7细胞中的表达。PCR方法从GAD65质粒中扩增出GAD190-385和GAD490-570两个片段的cDNA,overlap法再分别与信号肽基因进行拼接,将拼接后的融合基因SGAD190-515和SGAD490-570分别与胰岛素B链基因依次克隆入双启动子真核表达载体pBudCFA．1中。重组质粒经酶切和测序鉴定后,脂质体体外转染COS-7细胞,Western blot方法检测目的基因在该细胞中的表达。结果表明核酸序列测定克隆的融合基因和胰岛素B链基因序列与报告序列一致,开放阅读框正确,Western blot显示转染了DNA疫苗的COS-7细胞中均可检测两个目的基因的表达。两种GAD65片段与胰岛素B链基因共表达DNA疫苗均成功构建,为自身免疫糖尿病的免疫干预研究奠定了实验基础。     

人表皮生长因子融合蛋白在大肠杆菌表达的研究

在大肠杆菌中高效表达人表皮生长因子(hEGF)。根据大肠杆菌对密码的偏爱性,构建高效表达EGF融合蛋白的菌株,经离子交换层析和凝胶过滤层析两个步骤使产物得到纯化。融合蛋白表达产量为27%,EGF融合蛋白纯化产物纯度为95%以上,促3T3细胞增殖的活性测定为ED50为4.2ng/mL。在pET 3c:BL21(DE3)大肠杆菌表达系统中,以融合蛋白方式表达人表皮生长因子,表达效率高,纯化路线简单,产量较高,适合产业化生产。     

gD2 DNA疫苗诱导细胞免疫应答

用生殖器疱疹gD2 DNA疫苗pcDNA3-gD转染哺乳动物细胞COS-7,鉴定gD表达．再用其免疫BALB／c小鼠,测定脾细胞增殖、CTL细胞杀伤活性,以及CD4^ 和CD8^ 细胞群．结果表明：pcDNA3-gD能够在哺乳动物细胞COS-7中表达gD抗原,其免疫小鼠脾细胞增殖活性增加,CTL活性达37．69％,明显高于pcDNA3组和生理盐水组;CD4^ 占T细胞数量的33．38％,数量较pcDNA3组和生理盐水组显著增加．     

人白血病抑制因子真核表达载体的构建及在哺乳动物细胞中的表达

应用RT-PCR方法从人子宫内膜组织总RNA扩增出hLIF的全长基因,然后将其克隆至pcDNA3上,成功构建了重组真核表达载体pcDNA3/hLIF.利用脂质体介导将这一表达载体导入COS-7细胞和CHO-K1细胞,分别获得了hLIF的瞬时表达和稳定表达,为进一步进行hLIF生物学功能研究和hLIF在哺乳动物细胞中的高表达研究奠定了基础.     

降低聚腺苷二磷酸核糖聚合酶活性对外源癌基因诱导的NIH3T3转化的逆转作用研究

用ＰＡＲＰ酶抑制剂苯甲酰胺（ＢＡ）处理经ｃ－Ｈａ－Ｔ２４－ｒａｓ活化癌基因转染的ＮＩＨ３Ｔ３细胞得到转化细胞系，结果表明：经苯甲酸胺处理的细胞系，其细胞凝集、血清依赖性、形态特征等均发生了改变，呈现出正常细胞的特征．Ｓｏｕｔｈｅｒｎ杂交结果表明，已整合到细胞基因组中的外源Ｔ２４～ｒａｓ基因发生了丢失．     

2-TeCD对UVB致NIH3T3细胞损伤的保护作用

采用4,6 二脒基二苯基吲哚（DAPI）荧光染色法和流式细胞术考察谷胱甘肽过氧化物酶（GPx）活力的人工模拟物2-TeCD对UVB致NIH3T3细胞损伤的保护作用. 结果表明： 2-TeCD可影响UVB致NIH3T3细胞中DNA裂解率、 脂质过氧化水平、 活性氧体积分数、 细胞周期及 P53蛋白表达量等指标, 可通过清除自由基发挥对UVB损伤的防护作用.     

逆转录病毒载体介导的Anti-ras核酶对T24ras转化3T3细胞的抑制作用

突变ｒａｓ基因存在于多种人类肿瘤细胞中,本文将可特异性切割１２位点突变ｒａｓ（Ｔ２４ｒａｓ）之核酶Ｒ８通过逆转录病毒载体导入Ｔ２４ｒａｓ转化的ＮＩＨ３Ｔ３细胞,以期检测核酶在细胞内对其靶基因的作用．结果表明Ｒ８可特异性地切割突变ｒａｓｍＲＮＡ,导致转化细胞生物学特性如细胞形态、生长速度等在一定程度上发生逆转     

结核分枝杆菌mpt64基因真核表达载体的构建及表达

构建结核分枝杆菌分泌蛋白mpt64基因真核表达载体。通过PCR法从MTBH37Rv株基因组中扩增mpt64基因,插入pGEM-T-easy载体中,序列测定正确后,将其亚克隆到真核表达载体pcDNA3．1（-）,重组质粒酶切鉴定正确后,以Lipo-fectamine2000转染COS-7细胞后,分别以RT-PCR方法检测mRNA表达和间接免疫荧光技术检测目的蛋白的表达。构建了真核表达载体pcDNA-mpt64,RT-PCR结果证明mpt64基因可在COS-7细胞中转录,用间接免疫荧光检测,有表达mpt64蛋白的细胞着染。结果表明,构建结核分枝杆菌mpt64基因的真核表达载体pcDNA-mpt64成功,mpt64基因可以在COS-7细胞中表达。     

恶性疟原虫复合多表位基因的原核表达和多克隆抗体制备

原核表达恶性疟原虫多期多表位基因并制备其多克隆抗体。在前期构建恶性疟原虫复合多期多表位重组真核表达载体的基础上,将测序正确的AMAMEG基因克隆入原核表达载体pGEX4T—1并诱导表达。采用包涵体洗涤的方式纯化目的蛋白,以AMA—1抗体对纯化蛋白进行Western—blot分析。将纯化的融合蛋白免疫小鼠制备多克隆抗体。通过PCR成功获得长度为1 000 bp的恶性疟原虫AMA—1胞外域基因片段,获得了与多表位基因连接后的AMAMEG基因。通过诱导表达,显示在相对分子量约95 000处有预计大小的特异性条带,表明成功表达出融合蛋白。经此纯化的融合蛋白免疫的小鼠能产生特异性抗体,其滴度达到1∶105。纯化的融合蛋白和多克隆抗体为研究新型疟疾疫苗奠定一定的理论和实践基础。     

小鼠H-2D~b-BSP融合基因的构建与表达

目的:构建小鼠H-2Db-BSP融合基因,并诱导其在大肠杆菌中表达.方法:采用RT-PCR方法从C57BL/6小鼠淋巴细胞中扩增出H-2Db链的胞外段基因,经双酶切置换已构建的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使H-2Db与BirA酶底物肽(BSP)序列融合,构建H-2Db-BSP融合基因表达载体,转化大肠杆菌BL21(DE3)菌株,筛选重组子,并经IPTG诱导融合蛋白表达.结果:构建的H-2Db-BSP融合基因插入正确且序列与GenBank一致;经1mmol/L浓度的IPTG诱导后4h蛋白表达达到高峰,且融合蛋白以包涵体形式存在.结论:成功构建H-2Db-BSP融合基因,并诱导其在大肠杆菌得以表达,为进一步构建H-2Db四聚体,研究T细胞应答提供了物质基础.     

Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients

Kaposi's sarcoma (KS) is frequently associated with human immunodeficiency virus-1 (HIV-1) infection. Supernatants from HIV-1-infected T cells carrying the CD4 antigen promote the growth of cells derived from KS lesions of AIDS patients (AIDS-KS cells), and the HIV-1 tat gene, introduced into the germ line of mice, induces skin lesions closely resembling KS. Here we report that the tat gene product (Tat) is released from both HIV-1-acutely infected H9 cells and tat-transfected COS-1 cells. These Tat-containing supernatants specifically promote growth of AIDS-KS cells which are inhibited by anti-Tat antibodies; recombinant Tat has the same growth-promoting properties. Therefore a viral regulatory gene product can be released as a biologically active protein and directly act as a growth stimulator. These and previous data indicate that extracellular Tat could be involved in the development or progression, or both, of KS in HIV-1-infected individuals.     

重组白细胞介素-13及其受体对小鼠胚胎成纤维细胞增殖及表型转化的影响

将常规培养的NIH3T3细胞分为实验组和对照组,实验组加入80μg/L IL-13,作用24h后进行以下实验:使用RT-PCR技术检测NIH3T3细胞中IL-13Rα1、IL-13Rα2及α-SMA基因的表达;使用MTT法检测NIH3T3细胞的增殖率.研究结果表明:实验组NIH3T3细胞,胞体呈梭型,突起生长迅速,相互交织成网状,与对照组相比形态发生了明显的改变;MTT法测定显示,实验组NIH3T3细胞的增殖率与对照组相比显著增加,差异显著(P0.01);RT-PCR检测IL-13Rα2及α-SMA基因的表达量均显著增加,而IL-13Rα1表达量变化不大.本研究表明IL-13Rα2在成纤维细胞上的表达与其增殖和转型具有直接的相关性,IL-13可直接作用于成纤维细胞并可能以IL-13Rα2为起点发挥致肺纤维化作用.