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1.
通过PCR扩增出猪传染性胃肠炎病毒(TGEV)聚合酶部分片段,反向插入逆转录病毒表达载体pLXSN中,用脂质体法将重组质粒plxas-pol转染PA317细胞,经抗生素G418筛选出稳定的产毒细胞克隆,分别扩大培养后,取其上清液感染小鼠成纤维细胞NIH3T3,细胞克隆产生的重组病毒效价达9.0×105CFU/mL。用高效价假病毒感染IBRS2细胞,再经抗生素G418筛选,提取细胞克隆总RNA,经RT-PCR证明plxas-pol整合入IBRS2细胞。以TGEV感染IBRS2细胞和具有抗性的IBRS2细胞所产生的细胞病变为指标,证明该反义RNA对病毒有明显抑制作用,抑制率约为80%。用2×103和4×102TCID50/mL剂量的TGEV感染时,引起细胞死亡的时间分别为21 h和27 h,与对照组相比,抗性细胞系可明显延迟因病毒感染引起细胞死亡的时间。 相似文献
2.
Mitsuaki Yoshida 《Proceedings of the Japan Academy. Series B, Physical and biological sciences》2010,86(2):117-130
Molecular biology of mouse and chicken retroviruses had identified oncogenes and provided a revolutionary concept in understanding of cancers. A human retrovirus was established during 1980–1982 in linkage with a unique human leukemia, concurrently in Japan and USA. This review covers our efforts on the discovery of new retrovirus, Human T-cell Leukemia Virus Type 1 (HTLV-1), first introducing to a new class of retroviruses with a unique regulatory factors, Tax and Rex. Then it is followed by analyses of molecular interaction of the vial Tax with cellular machineries involved in the pathogenesis of Adult T-cell Leukemia (ATL). And then a probable mechanism of pathogenesis of ATL is proposed including recent findings on HBZ after our efforts. 相似文献
3.
Jin Sung Park Da-Young Chang Ji-Hoi Kim Jin Hwa Jung JoonSeong Park Se-Hyuk Kim Young-Don Lee Sung-Soo Kim Haeyoung Suh-Kim 《Experimental & molecular medicine》2013,45(2):e10
Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles
to deliver therapeutic genes for ex-vivo therapy of diverse diseases;
this is, in part, because they have the capability to migrate into tumor or
lesion sites. Previously, we showed that MSCs could be utilized to deliver a
bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we
assessed whether transduction with a retroviral vector encoding CD gene
altered the stem cell property of MSCs. MSCs were transduced at passage 1 and
cultivated up to passage 11. We found that proliferation and differentiation
potentials, chromosomal stability and surface antigenicity of MSCs were not
altered by retroviral transduction. The results indicate that retroviral vectors
can be safely utilized for delivery of suicide genes to MSCs for
ex-vivo therapy. We also found that a single retroviral
transduction was sufficient for sustainable expression up to passage 10. The
persistent expression of the transduced gene indicates that transduced MSCs
provide a tractable and manageable approach for potential use in allogeneic
transplantation. 相似文献
4.
Jun Yang Ying Wu Ping Ma Yanhua Zhao Ying You Susan E. Kane Ning Mao Yangpei Zhang 《科学通报(英文版)》2000,45(15):1390-1394
A series of retro viral vectors encoding humanmdr1 gene alone as wetl as in combination with either humanmgmt gene or human mutantSer
31-dhfr gene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34+ cetls. It has been shown that expression of dual drug resistance genes in transduced cetls confers a broad range of resistance
to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs
in anin vivo model in which transduced hematopoietic cetls will acquire multiple protection against the cytotoxic side effects of combination
chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cetls more effectivety. 相似文献
5.
为构建含绿色荧光蛋白(EGFP)和人胰岛素原基因的逆转录病毒表达载体,并检测其在人肝癌细胞HepG2中的表达,将IRES-EGFP片段克隆到含调控元件的人胰岛素原基因逆转录病毒表达载体(pLXSN-GI-Ins)中,构建得到表达质粒pLXSN-GI-Ins-EGFP.经脂质体介导转染HepG2细胞后,各孔分别加入含有30.0 mmol/L葡萄糖的培养液继续培养24 h,在荧光显微镜下观察EGFP基因的表达,检测细胞上清液中的胰岛素值.数据显示,成功构建逆转录病毒表达质粒pLXSN-GI-Ins-EGFP.转染HepG2细胞后48 h,表达EGFP基因的细胞数目占总细胞数目的比值为(38.0±5.0)%.结果表明,构建了含EGFP和调控元件的人胰岛素原基因逆转录病毒表达载体,并且能够在HepG2细胞中成功表达. 相似文献
6.
Astrocytes play an important role in the formation of glial scars. In order to investigate the effect of inhibitingGFAP gene expression on normal, reactive astrocytes and on glial scar formation, the efficiency of the recombinant antisenseGFAP retrovirus (PLBskG) on the growth, cell cycle, morphology andGFAP gene expression of astrocytesin vitro and on the formation of glial scarsin vivo has been studied by cell growth curves, flow cytometry, immunocytochemistry,in situ hybridization, RT-PCR and Southern blot. The results confirm the recombinant retrovirus (PLBskG) produced growth suppression
and G1 arrest of the normal and injured astrocytes. The infected cells become round or ellipoid. The cell processes become
fine or retracted. The intensity of staining ofGFAP is reduced. Expression ofGFAP mRNA is down regulated. However, in the control experiment, no obvious effects on the morphology or synthesis ofGFAP on cultured normal and scratched astrocytes infected by primary retrovirus vector (PLXSN) have been observed. The supernatant
of PLBskG has been injected into an injured site by microinjectionin vivo. The number and process lengths of GFAP positive cells are obviously reduced around the injured site. The formation of the
glial scar is inhibited, showing that the recombinant antisenseGFAP retrovirus can effectively inhibit the growth andGFAP expression of normal and injured astrocytesin vitro and the formation of glial scarin vivo. It is suggested thatGFAP plays an important role in glial scar formation. 相似文献
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8.
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。 相似文献
9.
检测FBXO6在多种肿瘤细胞中的表达和定位情况并构建稳定表达FBXO6的肿瘤细胞株。运用RT-PCR方法,在人胚肾293T细胞以及9种不同来源的肿瘤细胞中,检测了FBXO6的表达情况。以载体质粒pBabe-3Flag-FBXO6-puro/pBabe-3Flag-con、包装质粒pCMV-GAG-POL及包膜质粒pCMV-VSVG用Lipofectamine2000共转染包装细胞系293T,收集病毒颗粒感染A549细胞,经嘌呤霉素筛选稳定表达细胞株,Western blot鉴定FBXO6的表达。利用激光共聚焦荧光显微镜,采用间接免疫荧光方法,检测外源性FBXO6在A549中的亚细胞定位。在10种不同来源的细胞株中,FBXO6在A549细胞中的表达最高。成功筛选出嘌呤霉素抗性细胞系A549-Con与A549-3Flag-FBXO6。Western blot方法发现A549-3Flag-FBXO6细胞系稳定表达FBXO6蛋白。间接免疫荧光发现FBXO6与内质网tracker有共定位。FBXO6在肺癌A549细胞中高度表达,构建了稳定表达FBXO6的A549细胞系,部分FBXO6分布在内质网中。 相似文献
10.
单纯疱疾病毒胸苷激酶基因(HerpesSimplexVirus-ThymidineKinase,HSV-tk),配合核苷酸类似物(NAS)GCV或ACV的基因治疗是目前肿瘤基因治疗领域中一种较有希望的治疗方法.反转录病毒和腺病毒介导的HSV-tk/NAS肿瘤基因治疗系统的建立,并运用该系统进行脑胶质瘤、垂体瘤、黑色素瘤的基因治疗离体细胞和动物试验,取得了显著的肿瘤杀伤作用,特别是腺病毒介导的脑胶质瘤基因治疗动物试验效果很显著,能够完全消除肿瘤.同时,还建立了反转录病毒以及腺病毒HSV-tk基因治疗的安全性检测体系,为基因治疗的临床试验提供了安全性保证. 相似文献