共查询到19条相似文献,搜索用时 125 毫秒
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将细胞表面粘附分子CD44S的cDNA反向插入到真核细胞表达载体pMAMneo-CAT和MMTV-LTR启动子下游,构成CD44S的反义RNA载体.将其用电击法导入CD44+的人黑色素瘤细胞系HMM239,转录出的反义RNA能不同程度地抑制HMM239表面CD44的表达.CD44的表达被抑制后,瘤细胞与透明质酸的结合力下降,细胞的体外生长速率不受影响.将其接种裸鼠皮下,发现其致瘤性明显降低 相似文献
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反义凝血酶受体基因的表达对人ASMC增殖的影响 总被引:3,自引:0,他引:3
介入治疗后再狭窄的发生严重降低了该治疗手段的最终疗效.为探讨再狭窄的发生机理、寻找有效的预防手段,利用反义RNA技术构建了含有部分反义凝血酶受体(ATR)cDNA片段的真核表达质粒pcDNA3/ATR,并观察了其对培养的人主动脉平滑肌细胞(ASMC)增殖的影响.结果表明pcDNA3/ATR的瞬时转染即能显著抑制人ASMC的3H-TdR参入量,且该作用与导入的DNA量呈剂量依赖性.说明部分反义凝血酶受体基因的表达可以抑制ASMC的增殖 相似文献
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AHF及NE、人参皂甙对不同年龄大鼠VSMC c-myc基因和HSP_(70)基因表达的影响 总被引:1,自引:0,他引:1
《中国应用生理学杂志》1998,(4)
用原位杂交法及激光光密度计灰度扫描半定量法,比较了老年组(24月龄)与青年组(6周龄)Wistar大鼠离体培养的主动脉VSMC(5-10代)c-myc与HSP70基因的表达及红细胞抗高血压因子(AHF)、去甲肾上腺素(NE)与人参皂甙对上述基因表达的影响。结果表明:1.增龄导致的VSMC增殖很可能与c-myc基因的过度表达有关,NE、AHF和人参皂甙可以通过调控c-myc基因的表达来影响VSMC增殖。2.增龄伴随的应激能力下降可能与HSP70基因表达的减少有关。3.HSP70基因也可能参与了VSMC增殖与分化的调控,NE、AHF与人参皂甙可能通过调控HSP70基因的表达来影响VSMC的增殖 相似文献
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报道了内皮素A型受体反义寡聚核苷酸(ODNs)对大鼠血管平滑肌细胞(VSMC)增殖及内皮素受体基因表达的影响.~3H-TdR参入结果显示,内皮素A型受体反义ODNs处理细胞可显著抑制内皮素诱导的VSMC的DNA合成,反转录-PCR及受体结合实验结果表明,ODNs的上述作用与降低VSMC内皮素A型受体基因表达活性有关. 相似文献
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天然及氧化修饰脂蛋白对人动脉平滑肌细胞原癌基因表达的影响 总被引:10,自引:0,他引:10
动脉平滑肌细胞(SMC)的增殖在动脉粥样硬化(AS)的形成过程中极其重要。我们在建立人主动脉SMC体外培养方法的基础上,观察了LDL,VLDL及HDL和相应的氧化修饰型脂蛋白对培养人SMCsis,jun,H-ras原癌基因及Rb抗癌基因转录表达的影响。结果表明:(1)HDL对SMCsis,jun,ras基因表达无影响;(2)LDL和VLDL有使这些基因表达增加的趋势;(3)ox-LDL,ox-VLDL和ox-HDL具有使SMCsis,jun,和ras基因表达显著增强的作用(P<0.01),且其作用较相应的天然脂蛋白大(P<0.01);(4)天然和氧化修饰型脂蛋白对Rb基因表达均无影响。据上述结果推测:LDL,VLDL,ox-LDL,ox-VLDL和ox-HDL的致AS作用可能与刺激SMCsis,jun和ras原癌基因表达增加有关。 相似文献
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利用RT-PCR方法,从小鼠肝脏组织总RNA中扩增出4.5SRNA的cDNA。该cDNA被克隆到pGEM3Zf(+)质粒上,酶切鉴定并测序。然后将该序列插入以Luc基因作为报道基因的表达载体pSVluc20的PvuⅡ位点,构建了含4.2SRNA逆转座子的表达载体pSVluc20-4.5S。脂质转染法将表达载体导入小鼠骨髓瘤细胞NS-1、SP2/0和人乳腺癌细胞Bca61。结果表明,小鼠4.5SRN 相似文献
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为研究反义RNA表达载体在细胞内的稳定性,构建了一个特异性针对β地中海贫血基因IVS-2-654C→T(β654)突变mRNA前体异常剪接位点的反义RNA表达载体pCMVA.pCMVA转染β654HeLa细胞后,通过RNA定量检测反义片段对β654mRNA异常剪接的纠正作用;再从转染后传代5次并冰冻保存1年的HeLa细胞中回收反义表达载体,转染另外的β654HeLa细胞,同样检测它对β654mRNA异常剪接的纠正作用.结果显示该载体在细胞传代前后均能阻断β654异常剪接,部分恢复其正常剪接途径:用回收的pCMVA转染β654HeLa细胞后,正常剪接的βmRNA水平[β/(β+β*)]由0.05上升到处理后15d的0.48,而2种对照质粒处理后对这一比值影响不大.表明pCMVA可在HeLa细胞中随着细胞传代而传递下去,并保持结构与功能完整 相似文献
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ras原癌基因的点突变是人胃癌发生发展的重要机理之一。利用能表达c-H-ras癌基因反义RNA的质粒,导入人胃癌细胞系BGC-823,研究了ras癌基因反义RNA对人胃癌细胞生长及恶性表型的作用,结果表明,c-H-ras反义RNA可引起BGC-823生长速率及形态的变化,在半固体培养基中细胞集落形成能力减弱,部分地抑制了BGC-823在裸鼠体内的致瘤性。c-H-ras反义RNA对其RNA的过量表达呈特异性抑制作用。 相似文献
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动脉平滑肌细胞(SMC)是动脉粥样硬化(AS)斑块中的主要细胞,它的增殖在AS形成过程中极其重要。脂蛋白和氧化修饰型脂蛋白对SMC增殖的影响以及SMC增殖与原癌基因异常表达的关系是当前AS发病机制研究的热点之一。我们在建立人主动脉SMC体外培养方法的基础上,观察了LDL,VLDL及HDL和相应的氧化修饰型脂蛋白对培养人SMCfos,myc,erb-B原癌基因转录表达的影响。结果表明:①HDL对SMCfos,myc基因表达无影响;②LDL和VLDL有使这些基因表达增加的趋势,但与对照比较差异不显著(P>0.05);③OX-VLDL,OX-VLDL和OX-HDL有使SMCfos,myc基因表达显著增强的作用(P<0.01),且其作用较相应的天然脂蛋白大(P<0.01).上述结果说明:LDL,VLDL,OX-LDL,OX-VLDL和0X-HDL的致AS作用可能与刺激SMCfos和myc癌基因表达增加有关。 相似文献
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Antisense rescue defines specialized and generalized functional domains for c-Fos protein. 总被引:1,自引:0,他引:1
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J T Holt 《Molecular and cellular biology》1993,13(6):3821-3830
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反义封闭人多肽N-乙酰氨基半乳糖转移酶2 (pp-GalNAc-T2)的基因表达, 对胃癌细胞SGC7901中转化生长因子-β1(TGF-β1)与基质金属蛋白酶2 (MMP2)基因表达及细胞增殖有影响.在对几株肿瘤细胞的pp-GalNAc-T2基因表达水平进行分析后, 以高表达pp-GalNAc-T2的人胃癌细胞株SGC7901的总RNA为模板, 利用RT-PCR方法扩增两段不同长度pp-GalNAc-T2基因片段, 构建反义表达载体转染胃癌细胞SGC7901, 通过G418筛选, 建立一系列旨在封闭胃癌细胞SGC7901 ppGalNAc-T2基因表达的亚细胞克隆.通过流式细胞术、荧光显微镜、RT-PCR及Western印迹检测反义封闭pp-GalNAc-T2基因RNA表达后胃癌细胞SGC7901增殖以及TGF-β1、MMP2表达水平的变化. 反义封闭pp-GalNAc-T2基因表达后, 胃癌细胞SGC7901 pp-GalNAc-T2的表达水平明显降低, 细胞分裂增殖减慢, 表明反义封闭pp-GalNAc-T2基因表达对胃癌细胞SGC7901的生长增殖有影响.结果还显示, 反义封闭pp-GalNAc-T2基因表达可使TGF-β1、MMP2基因在mRNA与蛋白质表达水平均增加, 提示pp-GalNAc-T2基因表达可能对胃癌细胞SGC7901浸润转移产生影响.以上结果表明, pp-GalNAc-T2基因在肿瘤细胞中广泛表达, 并可能与肿瘤的增殖及浸润转移相关. 相似文献
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《中国科学:生命科学英文版》2015,(7)
Vascular smooth muscle cell(VSMC) proliferation and migration are pivotal for the pathogenesis of atherosclerosis and post-angioplasty restenosis. We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs-7(ADAMTS-7), a novel metalloproteinase, contributes directly to neointima formation by mediating VSMC migration. However, whether ADAMTS-7 affects VSMC proliferation remains unclear. In this study, we found that luminal adenoviral delivery of ADAMTS-7 aggravated intimal hyperplasia 7 d after injury, paralleled by an increased percentage of PCNA-positive cells in both intima and media. In contrast, perivascular administration of ADAMTS-7 si RNA, but not scrambled si RNA to injured arteries attenuated intimal thickening at day 7, paralleled with reduced intimal VSMC replication, without alteration of VSMC proliferation in the media. In accordance, [3H]-thymidine incorporation assay in primary cultured rat VSMCs revealed an enhanced replication rate(by 61%) upon ADAMTS-7 overexpression and retarded proliferation(by 23%) upon ADAMTS-7 si RNA administration. Our data demonstrates that ADAMTS-7 promotes VSMC proliferation both in vitro and in vivo. ADAMTS-7 may therefore serve as a novel therapeutic target for atherosclerosis and post-angioplasty restenosis. 相似文献
17.
Shaw S Wang X Redd H Alexander GD Isales CM Marrero MB 《The Journal of biological chemistry》2003,278(33):30634-30641
Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2. 相似文献
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Co-packaging of sense and antisense RNAs: a novel strategy for blocking HIV-1 replication.
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Retroviral vectors were engineered to express either sense (MoTiN-TRPsie+) or sense and antisense (MoTN-TRPsie+/-) RNAs containing the human immunodeficiency virus type-1 (HIV-1) trans -activation response (TAR) element and the extended packaging (Psie) signal. The Psie signal includes the dimer linkage structure (DLS) and the Rev response element (RRE). Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid (MT4) cell line. Stable transductants were then tested for sense and antisense RNA production and susceptibility to HIV-1 infection. HIV-1 production was significantly decreased in cells transduced with MoTiN-TRPsie+ and MoTN-TRPsie+/-vectors. Efficient packaging of sense and most remarkably of antisense RNA was observed within the virus progeny. Infectivity of this virus was significantly decreased in both cases, suggesting that the interfering RNAs were co-packaged with HIV-1 RNA. Vector transduction was not expected to occur and was not observed. Inhibition of HIV-1 replication was also demonstrated in human peripheral blood lymphocytes transduced with retroviral vectors expressing antisense RNA. These results suggest that (i) both sense and antisense RNAs were co-packaged with HIV-1 RNA, (ii) the co-packaged sense and antisense RNAs inhibited virus infectivity and (iii) the co-packaged sense and antisense RNAs were not transduced. Sense and antisense RNA-based strategies may also be used to co-package other interfering RNAs (e.g. ribozymes) to cleave HIV-1 virion RNA. 相似文献
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Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline‐inducible vectors
Here, we report on the construction of doxycycline (tetracycline analogue)‐inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl‐β‐D‐galactopyranoside‐inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline‐inducible promoter with the T7 promoter‐T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline‐inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications.