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1.
鼠I型可溶性白细胞介素1受体基因在昆虫细胞的克隆和表达 总被引:1,自引:0,他引:1
白细胞介素1(IL-1)是一种重要的细胞因子,具有广泛的生物学活性。它通过与细胞表面的白细胞介素1受体(IL-1R)结合而起作用,以杆状病毒为载体在昆虫细胞中克隆表达了小鼠I型可溶性白细胞介素1受体基因,以NIH/3T3细胞RNA为模板,采用RT-PCR方法扩增得到小鼠sIL-1RI的cDNA,克隆至杆状病毒转移载体pAcGP67B,将转移重组质粒与野生病毒AcNPV DNA共转染昆虫细胞Sf9, 相似文献
2.
白细胞介素1(IL-1)是一种重要的细胞因子,具有广泛的生物学活性。它通过与细胞表面的白细胞介素 1受体(IL-1R)结合而起作用。以杆状病毒为载体在昆虫细胞中克隆表达了小鼠I型可溶性白细胞介素1受体(sIL-1 RI)基因。以NIH/3T3细胞RNA为模板,采用RT-PCR方法扩增得到小鼠sIL-IRI的cDNA,克隆至杆状病毒转移载体pAcGP67B,将转移重组质粒与野生病毒ACNPV DNA共转染昆虫细胞Sf9,经同源重组得到重组杆状病毒rACNPV。应用经纯化的rAcNPV感染昆虫细胞Sf9,表达获得重组的sIL-1RI。经对亲和层析样品的SDS-PAGE分析和对IL-1β生物活性阻断作用实验证实,表达产物能够与其配基结合,并且能够分泌至细胞培养上清中。 相似文献
3.
血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)是近年发现的高效、特异促血管内皮细胞有丝分裂 ,继而促进血管形成的一种糖蛋白[1 ] 。我们克隆了人VEGF1 6 5基因 ,构建了含人VEGF1 6 5cDNA的真核表达载体 ,并经肌肉直接注射该质粒对兔动脉损伤模型进行治疗 ,通过血管造影观察血管新生和侧支循环的建立情况。1 材料与方法1.1 VEGF1 6 5cDNA的克隆及序列分析取人肝癌细胞 (SMMC linM) ,细胞数约 2× 10 7,用RNA提取试剂盒 (Promega)抽提细胞总RN… 相似文献
4.
为建立鸭乙型肝炎病毒LJ-76的转染细胞系,将LJ-76病毒DNA插入到pUC19的EcoRⅠ位点上,分离得到含有双拷贝LJ-76DNA的重组质粒.通过磷酸钙沉淀方法,将经CsCl等密度离心纯化的LJ-76DNA双体导入到人肝癌细胞BEL7402中.收集转染细胞的培养液进行蔗糖密度梯度离心,所得沉淀经检测发现含有LJ-76DNA并具有特异性DHBV内源性DNA多聚酶活性;对上述样品通过DotEIA检测DHBV核心抗原及表面抗原结果为阳性.Southernblot分析表明转染细胞内存在病毒DNA复制中间体cccDNA、ssDNA和rcDNA,而cccDNA被认为是复制活动较为活跃的标志.电镜观察转染细胞的上清发现有病毒颗粒的存在. 相似文献
5.
鸡贫血病毒(CAV)是一种全球性的禽传染病的病原。为环状单链DNA,大小为2.3kb,是圆环病毒科的成员。CAV基因组包含了3个部分或完全重叠的基因。CAV可通过细胞凋亡使胸腺细胞及人工培养的转化型单核细胞产生致命的细胞病变(CPE)。在转化的(鸡)细胞中,VP3基因的编码产物凋亡素可以造成凋亡,而这种效应不依赖于p53,且不受凋亡抑制蛋白Bcl-2及CrmA的抑制。令人注目的是,凋亡素能使人肿瘤 相似文献
6.
杆状病毒早期基因启动子载体的构及报道基因的表达 总被引:1,自引:0,他引:1
以AcNPVp10基因为侧翼序列,用AcNPV的IEI基因启动子构建了杆状病毒早期基因启动子载体,并将新霉天抗性基因插入IE1基因启动子下游,得到转移载体pAcPIneo。将它和野生型AcNPV DNA共转染昆虫细胞Sf9,由于neo基因的表达,通过G418的选择和富集作用,得到重组病毒vAcPIneo的纯培养。 相似文献
7.
体外研究汉滩病毒(HTNV)S基因及其5'端表达的意义,为核蛋 白T细胞表位的研究奠定基础。设计2套引物,用PCR方法从PBV220-S22原核质粒中扩增出S 基因全读码框(37-1326bp)及S基因5'端(37-501bp),用TA克隆将其克隆入pcDNA3.1/V5-His-TOPO载体中,成功构建pcDNA3.1-S及pcDNA3.1-S-N真核表达载体,并通过脂质体转 染至Vero细胞中,进行了瞬时表达。间接免疫荧光成功检测到pcDNA3.1-S及pcDNA3.1-S-N在Vero细胞中的表达。pcDNA3.1-S及pcDNA3.1-S-N真核表达载体有较高的转染效率,目 的基因能在宿主细胞中表达,有利于研究HTNV-S基因在T细胞表位研究中的意义。 相似文献
8.
三种类型辐射对质粒超螺旋DNA损伤的研究 总被引:7,自引:0,他引:7
用agarose电泳和图象处理技术比较了60Coγ射线、UV及低能N+离子处理pUC19DNA超螺旋结构的损伤效应及若干自由基清除剂的保护效应。结果表明:(1)γ射线和UV照射干燥DNA的损伤显著低于水溶液样品;(2)N+离子注入后超螺旋DNA的减少(SC%)与剂量呈良好线性关系,而γ射线和UV组的SC%随剂量升高呈指数下降;(3)干燥DNAγ辐照组的D37值为820Gy,SC完全消失的剂量LD为3814Gy,LD/D37=4.65;UV照射组的相应值分别为:1.65J/cm2,7.65J/cm2,4.64;N+离子组的相应值为:3.2×1015N+/cm2,5.0×1015N+/cm2和1.56。虽然上述三种辐射的剂量单位不同,不能直接比较其相对生物学效应,但从LD与D37的比值可反映DNASC破坏的程度和终点剂量(SC%=0)的大小。从而看出,N+离子(高LET辐射)比γ射线(低LET辐射)UV(非电离辐射)对DNA损伤作用更强;(4)乙醇、甘露醇等自由基清除剂对电离辐射损伤有很强的保护作用,但对UV损伤未见明显的保护效应 相似文献
9.
毛细血管扩张性共济失调突变蛋白在细胞凋亡过程中被Caspase┐3降解 总被引:1,自引:0,他引:1
新近的研究揭示:caspase蛋白酶在细胞凋亡中起着死亡执行者的重要功能.一些蛋白相继被证明在细胞凋亡中可被caspase特异切割,其中参与DNA损伤修复过程的聚ADP核糖聚合酶(PARP)以及DNA依赖的蛋白激酶(DNA-PK),在细胞凋亡过程中被caspase选择性切割具有特殊的功能意义.为探索与DNA-PK催化亚基有较高同源性,含有caspase切割位点,且功能上目前也被认为是感受DNA损伤和参与信号传导途径的ATM(Ataxiatelang-iectasiamutated)蛋白,是否在凋亡过程中也可被切割而降解?应用体外转录与翻译系统获得ATM蛋白的PI3K结构域,同时通过建立无细胞反应体系获得含caspase活性的细胞抽提液,将两者在体外共同保温.结果发现:ATM蛋白与caspase-3能免疫共沉淀,ATM蛋白的PI3K结构域可被caspase-3特异切割,并观察到辐射诱发细胞调亡中ATM蛋白的降解.从而进一步证实了DNA损伤修复的抑制,促进细胞凋亡的发生. 相似文献
10.
利用HSV—TK基因治疗肝癌的离体研究 总被引:2,自引:1,他引:1
利用DNA重组技术,将HSV-TK基因克隆至逆转录病毒载体(XM-6/TK)。经PA317细胞包装后,转染人肝癌细胞HepG2。结果显示,XM-6/TK转染HepG2细胞与野生型相比,其生长特点和细胞形态未有改变。给予抗病毒药物-环氧鸟苷(Ganciclovir,GCV)后,转染细胞生长受到严重抑制,细胞数量明显降低。细胞学检查证实,GCV处理后,转染细胞的死亡率显著增加。本结果提示,应用HSV-TK基因治疗肝癌可能为一种治疗肿瘤新的方法 相似文献
11.
MicroRNAs, the DNA damage response and cancer 总被引:1,自引:0,他引:1
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13.
Nicola Cogan Duncan M. Baird Ryan Phillips Lucy A. Crompton Maeve A. Caldwell Miguel A. Rubio Roger Newson Fiona Lyng C. Patrick Case 《Mutation research》2010,683(1-2):1-8
The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these ‘nearest neighbour’ cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells. 相似文献
14.
High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA‐damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1‐dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA–ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA‐damaging agents. Finally, we demonstrate that the RPA1‐binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA‐damaging therapeutic agents. 相似文献
15.
Mutant mammalian cells hypersensitive to DNA damaging agents are a useful tool for the characterization of DNA repair pathways, and can be used to isolate, by transfection, DNA repair genes and genes that confer basal resistance to cytotoxic agents. These genes may be important in mutagenesis, carcinogenesis and resistance of cancer cells to therapy. 相似文献
16.
Assay and analysis for anti- and pro-oxidative effects of ascorbic acid on DNA with the bulk acoustic wave impedance technique 总被引:1,自引:0,他引:1
A bulk acoustic wave (BAW) impedance sensor has been applied for in situ monitoring of the whole process of DNA oxidative damage induced by the vitamin C (Vc)-Fe (III) system, based on its real-time responses to the density-viscosity change of the tested solution due to the damages occurring on the DNA molecules. The results showed that Vc exhibited two conflicting effects, i.e., pro-oxidation and anti-oxidation on the DNA at different Vc concentrations in the damage system, and the "threshold" concentration of Vc for these two effects was estimated to be about 100 micromol/L. The end-point frequency change of the sensor (Deltaf(m)) was found to be linearly related to the initial concentration of the soybean DNA (C(DNA)) in the range of 40-1000 microg/mL, and the exponential relationship between the frequency change (Deltaf(0)) vs damaging time suggested that the Fe (III)-mediated DNA damage by Vc could be described as a first-order kinetics reaction. The effects of variations in concentrations of Vc and Fe3+ on the DNA oxidative damage were discussed, and based on investigations for the enhancing influence of H2O2 and inhibiting influence of HO* scavengers on the DNA damage, the nature and physiological toxicity of the damage in biological system were also examined. In addition, UV-vis spectra and electrophoresis analysis were also used, and the experimental observations were in good agreement with the above results. 相似文献
17.
《DNA Repair》2019
Genomic DNA is constantly assaulted by both endogenous and exogenous damaging agents. The resulting DNA damage, if left unrepaired, can interfere with DNA replication and be converted into mutations. Genomic DNA is packaged into a highly compact yet dynamic chromatin structure, in order to fit into the limited space available in the nucleus of eukaryotic cells. This hierarchical chromatin organization serves as both the target of DNA damaging agents and the context for DNA repair enzymes. Biochemical studies have suggested that both the formation and repair of DNA damage are significantly modulated by chromatin. Our understanding of the impact of chromatin on damage and repair has been significantly enhanced by recent studies. We focus on the nucleosome, the primary building block of chromatin, and discuss how the intrinsic structural properties of nucleosomes, and their associated epigenetic modifications, affect damage formation and DNA repair, as well as subsequent mutagenesis in cancer. 相似文献
18.
In this study, cytotoxic, membrane and DNA damaging effects of the essential oil from Origanum majorana and its oxygenated monoterpene component linalool were tested on parental and epirubicin-resistant (drug-resistant) human lung cancer cell lines (H1299). Essential oil’s and linalool’s cytotoxicities were examined and parental cells were found more sensitive to the essential oil’s and linalool’s cytotoxicities than drug-resistant cells. O. majorana essential oil had more effective membrane damaging effect than linalool on parental cells, while in drug-resistant H1299 cells, linalool had more effective membrane damaging effect than the essential oil. O. majorana essential oil possessed more effective DNA damaging effect than linalool on both parental and drug-resistant cells. The conclusions from this study suggest that O. majorana essential oil and linalool exhibit cytotoxic, membrane and DNA damaging effects. They thus need further investigation as potential therapeutic agents for human lung cancer. 相似文献
19.
Jing Ge Somsak Prasongtanakij David K. Wood David M. Weingeist Jessica Fessler Panida Navasummrit Mathuros Ruchirawat Bevin P. Engelward 《Journal of visualized experiments : JoVE》2014,(92)
DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The ‘CometChip’ is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays. 相似文献
20.
《Cell cycle (Georgetown, Tex.)》2013,12(17):2029-2035
Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment. However, apoptotic pathway deficiencies are a hallmark of cancer cells. We propose that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death. Here we show that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways. While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a variety of cancer cells. We found that β-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo. This occurs within minutes of exposure to β-lapachone, resulting in selective necrotic cell death. Inhibition of PAR blocked β-lapachone-induced necrosis. Furthermore, necrotic cell death induced by β-lapachone was significantly reduced in PARP1 knockout cell lines. Our data suggest that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy 相似文献