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1.
目的 构建携带增强型绿色荧光蛋白基因(EGFP)标志针对大鼠诱导型一氧化氮合酶(iNOS)基因的shRNA重组腺病毒载体并在人胚肾-293细胞中扩增制备重组病毒.方法 利用AdMax包装系统,用先期构建的带有EGFP标记基因的穿梭质粒pDC316-iNOS-shRNA-EGFP,与骨架质粒pBHGlox_El,3Cre共转染293包装细胞,同源重组产生复制缺陷型重组腺病毒载体Ad5-inosshRNA-EGFP,反复感染293细胞扩增病毒后,离子交换法纯化病毒,并测定病毒颗粒数及滴度.结果 经聚合酶链反应(PCR)检测和EGFP表达证明已成功构建了携带iNOS-shRNA-EGFP的重组腺病毒载体;扩增纯化后,测得重组腺病毒颗粒数为3.6×1011 VP/ml,A260/A280值约为1.25,病毒活性为7.94×109IU/ml.结论 已成功构建重组腺病毒载体Ad5-inos-shRNA-EGFP,为利用基因激活技术治疗勃起功能障碍的研究奠定实验基础.Abstract: Objective To construct an adenovirus vector expressing short hairpin RNA (shRNA)of rat inducible oxide synthase (iNOS) carrying enhanced green fluorescent protein (EGFP) and amplify the adenovirus vector in HEK-293 cells. Methods The shuttle plasmid pDC316-inos-shRNA-EGFP that was constructed at an earlier date, was cotransfected with the adenovirus skeleton plasmid pBHGlox_E1,3Cre into 293 cells to obtain the produced replication defective recombinant adenovirus vector Ad5-inosshRNA-EGFP. The recombinant adenovirus was propagated by repeat infection of 293 cells and purified by ion exchange method, then the virus particles were counted and the purity and titer were determined.Results Recombinant adenoviral vector Ad-ACE-shRNA was constructed successfully, which was confirmed by polymerase chain reaction (PCR) and GFP expression. After amplification and purification, the virus particle count, A260/A280 and titer of recombinant adenovirus were 3.6 × 1011 VP/ml, 1.25 and 7.94 ×109 IU/ml,respectively. Conclusion Recombinant adenovirus vector Ad5-inos-shRNA-EGFP is successfully constructed, which laid a foundation for gene activation in erectile dysfunction treatment. 相似文献
2.
目的应用AdEasy复制缺陷型腺病毒载体系统构建小鼠FRNK基因重组腺病毒,并在293HEK细胞中扩增制备重组病毒。方法把小鼠FRNK基因克隆入腺病毒穿梭载体pAdTrack—CMV中,构建腺病毒穿梭质粒pAdTrack—CMV-FRNK,经PmeⅠ内切酶酶切成线性化后,采用电穿孔转化将其转化到含有腺病毒骨架载体pAdEasy-1的感受态大肠杆菌BJ5183中,通过卡那霉素筛选获得阳性腺病毒重组质粒。再将获得的腺病毒重组质粒转染到293HEK细胞进行包装,获得FRNK重组腺病毒pAdFRNK。荧光显微镜下观察绿荧光的表达。结果经酶切和绿荧光表达证明了小鼠FRNK基因的重组腺病毒载体pAdFRNK构建成功,并制备出高滴度的重组病毒。结论成功构建携带小鼠FRNK基因的重组腺病毒pAdFRNK,为研究FRNK的基因治疗奠定了基础。 相似文献
3.
骨形成蛋白7重组腺病毒的构建及转染骨髓间充质干细胞的研究 总被引:5,自引:1,他引:4
目的 构建骨形成蛋白(BMP)7重组腺病毒,使其转染骨髓间充质干细胞并在细胞内得到表达。方法 将BMP7基因克隆到转移载体pAdTrack—CMV中,在细菌BJ5183中与pAdEasy腺病毒基因组进行同源重组,得到BMP7重组腺病毒基因组,通过转染HEK293细胞包装出重组腺病毒。利用BMP7重组腺病毒转染分离纯化后的骨髓间充质干细胞,鉴定BMP7在细胞内的表达。结果 经过多聚酶链反应(PCR)及酶切鉴定证明获得了BMP7转移质粒padTrack—BMP7和BMP7重组腺病毒基因组,并包装出重组腺病毒。逆转录PCR和免疫组织化学证明BMP7在重组腺病毒转染后的骨髓间充质干细胞中得到了表达。结论 BMP7重组腺病毒的构建和在骨髓间充质干细胞中的表达,为BMP7基因治疗的研究奠定了基础。 相似文献
4.
目的 构建携带EGFP标志人白细胞介素10(hIL-10)基因腺病毒载体.方法 以pSNAV2.0-hIL-10重组质粒为模板.PCR扩增hIL-10基因,获得hIL-10 cDNA片段,并酶切连接到带有含强绿色荧光蛋白基因(EGFP)标记的pDC316-IRES-EGFP-lacZalpha载体质粒上,Pmel线性化重组质粒pDC316-hIL-10-IRES-EGFP,与腺病毒包装系统AdMax转染293包装细胞,包装产生复制缺陷型重组腺病毒,经反复感染293细胞扩增病毒后,进行离子交换法纯化病毒,并测定病毒颗粒数、滴度.结果 经PCR鉴定、限制性酶切分析及序列测定,证明已正确构建重组穿梭质粒pDC316-hIL-10-IRES-EGFP和重组腺病毒质粒Ad-hIL-10-IRES-EGFP.扩增纯化后.测得重组腺病毒颗粒数为3.2×1011VP/ml,OD260/OD280值约为2.0,滴度为1.1×1010TCID50/ml.结论 成功构建了重组腺病毒质粒Ad-hIL-10-IRES-EGFP,为进一步研究hIL-10基因奠定实验基础. 相似文献
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目的 构建携带酪氨酸蛋白激酶受体2-小干扰RNA(Tie2-SiRNA)慢病毒载体,观察其对恶性黑色素瘤细胞的干扰作用.方法 将pSilencer 1.0-U6启动子-酪氨酸蛋白激酶受体2-小干扰RNA(pSilencer 1.0-U6-Tie2-siRNA)重组质粒经Xba Ⅰ酶切电泳鉴定后,与经Xba Ⅰ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒(pNL-EGFP)载体连接,产生加强绿色荧光蛋白的转移质粒-u6启动子-酪氨酸蛋白激酶受体2-Ⅰ(pNL-EGFP-U6-Tie2-Ⅰ)、pNL-EGFP-U6-Tie2-Ⅱ慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒,分别与水疱性口炎病毒G蛋白(pVSVG)包膜质粒和pHelper包装质粒组成慢病毒三质粒转染系统,再共转染293T细胞,产生pNL-EGFP-U6-Tie2-Ⅰ、pNL-EGFP-U6-Tie2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Tie2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Tie2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.8×103/ml.实时荧光定量RT-PCR结果显示:Tie2-SiRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Tie2基因的表达,其中较高者可达68.55%,并且2种慢病毒载体之间差异无统计学意义(t=0.362,P>0.05).结论 成功构建了Tie2-SiRNA慢病毒载体,体外研究显示其能抑制Tie2 mRNA的表达,为下一步进行抑制肿瘤生成的动物实验研究奠定了基础.Abstract: Objective To construct lentivector carrying Tie2-Small interfering RNA(SiRNA),so as to study its influence on malignant melanoma cells.Methods Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with Xbal.ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-Ⅰ or pNL-EGFP-U6-Tie2-Ⅱ,and then the electrophoresis clones was sequenced.Plasmids of pNL-EGFP-U6-Tie2-1 and pNL-EGFP-U6-Tie2-Ⅱ were constructed and combined with pVSVG and pHelper,respcectively,to constitute lentiviral vector system of three plasmids.The Lentiviral vector svstem was transfected into 293T cell to produce pNL-EGFP-U6-Tie2-Ⅰ and pNL-EGFP-U6-Tie2-Ⅱ lentivirus.Then the supernatant was collected tO determine the titer.Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. Results The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing.And the titer of lentiviral vector was 8.8×103/ml,which was determined by 293T cell.The results of Reahime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells(P<0.01).There was no significant difference in the expression level (P>0.05)between the two lentiviral vectors of Tie2-RNAi.Conclusions Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly.The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo. 相似文献
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目的 构建过表达大鼠阴茎神经源性一氧化氮合酶(PnNOS)基因大鼠脂肪源性干细胞(ADSCs),为基因修饰ADSCs移植治疗勃起功能障碍(ED)大鼠模型提供种子细胞.方法 获取大鼠PnNOS基因,并与线性化的腺病毒真核表达载体pDC315-EGFP定向克隆连接.构建正确的pDC315-PnNOS-EGFP穿梭质粒转染293 T细胞,采用Western Blot检测PnNOS基因在293 T细胞中的表达情况.利用AdMax腺病毒包装系统包装产生重组腺病毒,包装后扩增纯化并测定病毒滴度.PnNOS基因重组腺病毒转染大鼠ADSCs,观察GFP表达情况和Western Blot检测PnNOS基因表达情况.结果 经PCR鉴定、限制性酶切分析、测序鉴定和目的质粒Western Blot表达检测鉴定,证实pDC315-PnNOS-EGFP重组腺病毒载体构建成功.重组腺病毒包装成功且病毒滴度为5.0×109 PFU/ml.Western Blot于大鼠ADSCs中检测到约161 KDa大小条带,其与PnNOS蛋白分子量大小基本相一致.结论 大鼠PnNOS基因修饰的ADSCs构建成功,从而为基因修饰ADSCs移植治疗ED提供了良好的基因工程细胞. 相似文献
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目的 构建含人β1转化生长因子(TGF-β1)短发夹RNA(short hairpin RNA,shRNA)的复制缺陷型腺病毒载体.方法 通过聚合酶链式反应(PCR)法将带有TGF-β1 shRNA 序列构建至U6启动子(U6sh TGF-β1)下游,与T载体连接,将质粒转化人大肠杆菌.将U6sh TGF-β1连到穿梭质粒pDC316上,与腺病毒基因组质粒pBHGlox△E1、3Cre共转染293T细胞,得到重组腺病毒载体(rAd TGF-β1).经PCR鉴定正确后,进行扩增、纯化、滴度测定及感染性鉴定.结果 PCR法得到目的 片段U6sh TGF-β1,经测序,序列与设计方案完全一致.rAd TGF-β1具有良好的感染性,腺病毒滴度可达107.3TCID50/ml.双引物PCR法鉴定Ad TGF-β1可扩增出腺病毒E2b区片段及特异性片段U6sh TGFβ1.结论 成功构建了能表达TGF-β1 shRNA 的重组腺病毒载体rAd TGF-β1(A)及对照载体rAdTGF-β1(B),可能为临床应用RNA干涉--新的基因技术防治瘢痕疙瘩提供高效的方法. 相似文献
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目的:观察淋巴细胞特异性蛋白酪氨酸激酶(Lck)在肾小管上皮细胞中的表达及Lck反义RNA对其表达的影响.方法:采用RT-PCR方法从肾小管上皮细胞中获得Lck目的基因片段,将目的基因片段反向插入pDC 316质粒中,构建Lck反义RNA穿梭质粒(pDC 316-antisenseLck),经PCR、酶切及测序鉴定正确后,将此穿梭质粒与辅助质粒pBHGloxΔE1,3Cre 共转染HEK 293细胞,重组产生Lck基因反义RNA腺病毒载体(Ad-antisenseLck).用此重组腺病毒感染体外培养的肾小管上皮细胞,Western blotting方法观测此重组腺病毒载体对IL-2诱导的Lck蛋白表达的影响.结果:构建出Lck反义RNA重组腺病毒载体,此重组腺病毒载体感染培养的肾小管上皮细胞后可明显抑制IL-2诱导的Lck蛋白表达(P<0.05),抑制率可达47%.结论:成功构建出Lck反义RNA重组腺病毒载体,此载体可在肾小管上皮细胞内发挥生物学作用,显著阻抑肾小管上皮细胞的Lck蛋白表达. 相似文献
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目的利用AdEasy腺病毒载体系统构建血管内皮生长因子165(VEGF165)重组腺病毒,并在293T细胞中扩增。方法将PCR获取的VEGF165基因酶切并插入到pAdTrack-CMV中,构建成腺病毒穿梭质粒pAdTrack-VEGF165,经PmeI酶切线性化后,采用电穿孔转化到事先电转化腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌感受态细胞中,挑选同源重组菌落。提取质粒并用Pac I酶切鉴定,线性化重组质粒pAdEasy-VEGF165转染293T细胞,包装成重组腺病毒颗粒,荧光显微镜下观察绿色荧光表达,并扩增收集重组腺病毒,测定病毒滴度。结果经限制性内切酶检测、基因测序及和绿色荧光观察证实成功构建了携带VEGF165基因的重组腺病毒,并扩增出10^9pfu/mL的高滴度重组腺病毒。结论成功构建了携带VEGF165基因的重组腺病毒载体,为研究骨组织工程血管化局部基因治疗奠定了基础。 相似文献
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Construction of recombinant adenoviral vector Ad-CMV-hTGFbeta1 for reversion of intervertebral disc degeneration by gene transfer. 总被引:1,自引:0,他引:1
OBJECTIVE: To provide a highly efficient adenoviral vector Ad-CMV-hTGFbeta1 for the study of gene therapy for reversion of the intervertebral disc degeneration. METHODS: A newly developed recombinant adenoviral vector construction system was used in the study. The cDNA of hTGFbeta1 was first subcloned into a shuttle plasmid pShuttle-CMV. The resultant plasmid was linearized by digesting with restriction endonuclease PmeI, and subsequently transformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected into 293 cells. Recombinant adenoviruses were generated within 2 weeks. RESULTS: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8% agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The expression of hTGFbeta1 was verified by immunohistochemical staining. CONCLUSIONS: The successful generation of the adenoviral vector Ad-CMV-hTGFbeta1 and the confirmation of the interest gene expression make it possible for the experimental study of the reversion of the intervertebral disc degeneration by gene therapy. 相似文献
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目的:探讨持续活化Akt且带有HA标签(myr-HA-Akt)基因的重组腺病毒在肝硬化大鼠肝脏中的表达特性。方法:酶切正向插入目的基因的真核表达载体pcDNA3.1-myr-HA-Akt,获得myr-HA-Akt cDNA后,将其定向克隆到穿梭质粒pDC316中,然后将重组质粒与病毒骨架质粒pBHGloxΔE1,3Cre共转染293 细胞,获得复制缺陷型重组腺病毒Ad-myr-HA-Akt,并进行扩增、纯化。通过观察腺病毒感染293细胞后是否出现细胞病变效应;PCR和基因测序方法对所构建病毒进行鉴定,并采用TCID50法检测病毒滴度。自尾静脉注射重组腺病毒Ad-myr-HA-Akt感染肝硬化模型大鼠。免疫印迹法检测大鼠肝组织内Akt 和p-Akt蛋白的表达。结果:感染的293 细胞出现明显的细胞病变效应,PCR产物电泳证实重组腺病毒的存在,基因测序证实myr-HA-Akt的cDNA正确插入穿梭质粒且与pBHGloxΔE1,3Cre正确同源重组;病毒滴度为5.5×1011 vp/mL。从蛋白水平证实感染病毒的肝硬化模型大鼠有外源性Akt基因的表达。结论:构建的重组腺病毒Ad-myr-HA-Akt能有效地感染肝硬化模型大鼠,并可在模型大鼠中正确转录和翻译,为进一步研究腺病毒介导的Akt基因对肝硬化的治疗奠定了实验基础。 相似文献
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应用细菌重组系统构建小鼠Flt3配体的重组腺病毒及其在肝癌细胞中的表达 总被引:5,自引:0,他引:5
目的 构建携带小鼠Flt3配体(murine flt3 ligand,mFL)的重组腺病毒载体并检测它的感染后的小鼠肝癌细胞中的表达。为肝癌基因治疗提供实验基础。方法 PCR扩增含有mFL的质粒,应用高效细菌内同源重组系统构建携带mFL的重组腺病毒,感染小鼠肝癌细胞株Hepal-6,一步法提取细胞总RNA,RT-PCR检测mFL的表达。结果 构建了携带mFL的重组腺病毒,并在感染后的Hepal-6细胞中检测到了mFL的mRNA。结论 应用该细菌重组系统成功地构建了携带mFL的重组腺病毒载体。为下一步的基因治疗研究奠定了基础。 相似文献
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目的构建携带大鼠血管生成素.1(angiopoietin-1,ANG.1)基因的重组腺病毒载体,并检测其转染对大鼠骨髓间充质干细胞(bone marrow mesenchymal stemcells,BMSCs)活力的影响。方法RT-PCR法获取大鼠ANG-1基因并亚克隆至穿梭质粒pAdTrack-CMV,经测序无误后与骨架质粒pAdEasy-1在BJ5183中同源重组。重组质粒pAdEasy-1-ANG-1经鉴定后转染293细胞进行病毒包装扩增。体外转染BMSCs,检测转染后BMSCs中ANG-1的表达。MTT法评估常氧及缺氧环境下ANG-1对BMSCs的影响。结果重组腺病毒载体pAdEasy-1-ANG-1经测序及酶切鉴定正确;BMSCs经转染ANG-1基因后表达ANG-1。MTT法检测提示常氧及缺氧条件下转染前后BMSCs活性的差异均无统计学意义(缺氧组与缺氧下转染组相比,P〉0-05;常氧组与常氧下转染组相比,P〉0-05)。结论成功构建大鼠ANG-1基因重组腺病毒载体,且其转染在体外不影响BMSCs的活性。 相似文献
14.
目的 利用AdMax腺病毒载体系统构建人内皮型一氧化氮合成酶基因(heNOS)重组腺病毒并检测其在体外培养人血管平滑肌细胞中表达.方法 将质粒PMSCV-heNOS通过聚合酶链反应(PCR)法扩增出heNOS全长基因,插入PSUCMV中构建成腺病毒穿梭质粒PSUCMV-heNOS,转化DH5α大肠杆菌,挑选细菌单克隆进行DNA测序,酶切鉴定正确;通过PSUCMV-heNOS与质粒pBHGE3在293细胞中同源重组,得到携带heNOS基因的复制缺陷型重组腺病毒载体(AdCMV-he-NOS).转染体外培养的人血管平滑肌细胞,MTT检测细胞增殖,用免疫组织化学和Western blot等方法检测heNOS蛋白的表达.结果 (1)PCR产物电泳结果,酶切鉴定和测序鉴定结果证实构建人eNOS重组腺病毒载体成功,病毒滴度达3.5×l010PFU/ml;(2)根据体外培养的人血管平滑肌细胞(HVSMCs)形态学变化,免疫组织化学进行平滑肌细胞表型标志物α-actin染色,证实为HVSMCs;(3)转染120 h,AdCMV-heNOS病毒感染复数(MOI)分别为50、150、250、300、450、A570值分别为1.410±0.081、1.357±0.150、1.303±0.311、0.995±0.248、0.731±0.101,其中感染复数300明显稳定抑制血管平滑肌细胞的增殖,差异有统计学意义(P<0.01).免疫组织化学和Western blot检测显示转染细胞heNOS蛋白明显表达.结论 heNOS重组腺病毒的构建及其表达为血管内膜增生等疾病的基因治疗提供可能.Abstract: Objective To construct recombinant adenovirus vector carrying the human endothelial nitric oxide synthase gene (heNOS) and detect its expression in human vascular smooth muscle cells in vitro. Methods heNOS was cloned by polymerase chain reaction (PCR) using plasmid PMSCV-heNOS,heNOS cDNA was inserted into adenovirus shuttle plasmid-PSUCMV to generate a recombinant plasmid PSUCMV-heNOS, transfected into E. coli DH5α, and positive clones were correctly constructed and confirmed by DNA sequencing analysis and restriction endonucleas analysis. The plasmid PSUCMV-heNOS was co-transfected with pBHGE3 in 293 cells to generate replication-defective recombinant adenovirus vector carrying heNOS gene. Human vascular smooth muscle cells were transfected by Ad-heNOS. The effect on the proliferation of human vascular smooth muscle cells was investigated by MTT assay. The expression of heNOS was detected by immunohistochemistry staining and Western blotting. Results ( 1 ) PCR, restrictive digestion, and sequencing revealed the successful construction of the recombinant adenovirus vector carrying heNOS gene and its titer was 3.5 × 1010 PFU/ml; (2) It was confirmed that the human vascular smooth muscle cells cultivated in vitro were characterized by morphology and immunohistochemistry strain of α-actin; (3) At 120 h, the A570 values in multiplicity of infection (MOI) 50, 150,250,300,450 were respectively 1. 410 ±0. 081, 1. 357 ±0. 150, 1.303 ±0. 311,0. 995 ±0. 248 and 0. 731 ±0. 101. The proliferation of human vascular smooth muscle cells was significantly and stably inhibited with MOI 300 of AdCMVheNOS ( P < 0. 01 ). Immunostaining and Western blotting with heNOS in engineered cells were positive.Conclusion The successful construction of AdCMV-heNOS and expression may provide an opportunity for the gene therapy of vascular intimal hyperplasia. 相似文献
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目的探讨人磷酸二脂酶5(PDE5)基因反义RNA重组腺病毒载体的构建方法。方法根据基因库确定具有完整PDE5A1启动子活性的cDNA基因序列,并设计反义链,两'端加内切酶修饰;通过转载体克隆方法构建转入载体(pENTR)并测序;借助Gateway腺病毒表达载体系统进行重组反应构建重组腺病毒表达载体pAd/CMV/V5/antisense PDE5A1;PacⅠ酶切后转染293A细胞系进行包装、扩增;病毒质粒酶切后电泳鉴定及PCR检测;CsCl梯度离心法纯化病毒,病毒空斑实验进行滴度测定。结果pENTR载体测序证实目的基因序列及插入方向正确;pAd/CMV/V5/antisense PDE5A1酶切后电泳鉴定,可见145bp的片段;PCR检测证明实验设计的目的反义基因成功插入腺病毒载体中;重组腺病毒滴度达108~1010/μl。结论借助Gateway腺病毒表达载体系统可成功地将人工合成的反义基因装入到腺病毒载体中;纯化病毒液量和滴度符合体内基因转染实验的要求。 相似文献
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腺病毒载体介导的Polo样激酶1短发夹环RNA对胶质瘤体内外增殖的抑制作用 总被引:1,自引:0,他引:1
目的探讨重组腺病毒载体介导的Polo样激酶1(PLK1)短发夹环RNA(shRNA)对人胶质瘤细胞株U251在体内外增殖的抑制作用。方法设计并合成针对PLK1的shRNA,并克隆至真核表达载体pEGFP—H1上,再将H1启动子和shRNA亚克隆至腺病毒穿梭质粒pAdTrack上,通过在携带有pAdeasy-1质粒的BJ5183细菌内同源重组,生成针对PLK1的shRNA重组腺病毒载体pAD-H1/PLK1,经293细胞包装产生重组腺病毒AD-H1/PLK1,感染U251细胞。应用逆转录-聚合酶链反应(RT—PCR)的方法检测U251细胞PLK1mRNA表达,流式细胞仪检测细胞凋亡以及细胞G2/M期转变情况,以噻唑蓝(MTT)比色法检测各组细胞的增殖活性。同时制备裸鼠U251细胞移植瘤模型并注射重组腺病毒,观察肿瘤生长情况。结果成功构建针对PLK1基因的RNA干扰腺病毒表达载体,与AD-H1组比较AD-H1/PLK1组在转染24h和48hU251细胞PLK1mRNA表达水平分别降低50.4%(P〈0.01)和71.9%(P〈0.01)。且感染AD-H1/PLK1病毒48h后的U251细胞凋亡率从8.3%升至65.6%(P〈0.01),而G2/M期的细胞从21.5%增至51.4%(P〈0.01),细胞的增殖能力则下降50%(P〈0.01)。体内实验表明重组腺病毒载体介导的PLK1shRNA也明显抑制肿瘤生长(P〈0.01)。结论腺病毒介导的RNA干扰技术可有效降低PLK1在胶质瘤细胞中的表达水平,抑制肿瘤细胞在体内外的增殖活性。 相似文献
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目的:探讨构建携带大鼠血管内皮细胞生长因子(VEGF)的重组腺病毒载体方法,为后续的基因转染研究作准备。方法:采用RT-PCR扩增的方法获取鼠源性的VEGF,并克隆入穿梭质粒pDC316。构建的质粒pDC316-VEGF经酶切及测序鉴定正确后,通过lipofectamine2000的介导与腺病毒包装质粒pBHGE3共转染至人胚肾细胞HEK293,经同源重组后获得携带鼠VEGF的重组腺病毒VDC316-VEGF。应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒。以50%组织培养感染剂量法(TCID50)测定病毒滴度。结果:PCR鉴定证实重组腺病毒含有鼠VEGF,病毒滴度为3×109pfu/mL。结论:成功构建的携带大鼠VEGF的重组腺病毒载体能在HEK293细胞内扩增获得足够高的病毒滴度,可作为后续基因治疗研究工作中可靠的基因转染工具。 相似文献
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转化生长因子β3基因转染兔骨髓间充质干细胞的实验研究 总被引:2,自引:2,他引:2
目的构建转化生长因子β3(transforming growth factor β3,TGF-β3)的腺病毒载体,并通过腺病毒载体将TGF-β3基因转染骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs),使其在细胞内得到表达。方法将人TGF-β3质粒定向克隆进入穿梭质粒pAdTrack—CMV中,与pAdEasy-1共同转入细菌并重组,获得TGF-β3重组腺病毒质粒,用脂质体法导入包装细胞HEK293中,48~96h后荧光显微镜下观察绿色荧光蛋白的表达。取1月龄新西兰大白兔5只,取其胫骨骨髓。采用密度梯度离心法分离纯化MSCs,并进行传代。取传至第3代细胞采用TGF-β3腺病毒载体转染,10h后荧光显微镜观察绿色荧光表达,转染TGF-β3重组腺病毒4d后的MSCs用免疫细胞化学方法观察TGF-β3在细胞内的表达。结果pAdEasy—TGF-β3转染HEK293细胞48~96h后,荧光显微镜可见明显的绿色荧光表达。兔骨髓分离培养10d后,培养MSCs融合达70%~80%、贴壁紧密,细胞形态均一,似成纤维细胞;14d完成原代细胞培养。TGF-β3重组腺病毒转染MSCs17h后,荧光显微镜下出现少量绿色荧光,48~72h荧光加强,荧光与MSCs轮廓一致。免疫细胞化学观察,转染TGF-β3重组腺病毒MSCs胞浆内有棕黄色阳性颗粒表达。结论TGF-β3基因重组腺病毒载体的成功构建并在MSCs内的表达,为创伤愈合的基因治疗提供了研究基础。 相似文献
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目的设计、构建并筛选出转染至人骨肉瘤细胞系OS-9901,对c-myc沉默效果最佳的复制缺陷型重组腺病毒质粒pAd-c-myc.短发夹RNA(short hairpinRNA,shRNA)包装出表达c-myc-shRNA的重组腺病毒,并测定其滴度。方法设计有shRNA结构的3对单链寡核苷酸(ss oligos),经变性退火为双链寡核苷酸(dsoligos),插入穿梭质粒pENTR/U6载体,测序正确后,经Lipofectamine^TM2000阳离子脂质体介导入人骨肉瘤细胞系OS-9901,采用RT-PCR筛选对c-myc沉默效果最佳的穿梭质粒pENTR/U6-shRNA,然后与腺病毒骨架质粒行同源重组,筛选出正确重组子。采用293A细胞包装出表达c-myc-shRNA的复制缺陷型重组腺病毒,观察其细胞病变效应(cytopathiceffect,CPE),采用病毒颗粒法(viral particles,vP)和50%组织培养感染剂量法(50%tissue culture infective dose,TCID50)测定病毒滴度。结果电泳验证后的dsoligos插入穿梭质粒pENTR/U6载体,测序结果提示构建的pENTR/U6-shRNA质粒正确。从3对dsoligos通过RT-PCR方法筛选出对c-myc沉默效果最佳的穿梭质粒pENTR/U6-shRNA,经PacI酶切线性化后同源重组成功构建了介导c-myc-shRNA复制缺陷型重组腺病毒,CPE和空泡现象3d开始出现,6d更加明显。采用VP法测定的第1代腺病毒滴度为5.23×109VP/mL,经3~4代扩增后可达2.26×10^12VP/mL。TCID50证实病毒滴度为10^-3.8/0.1mL。结论通过RNA干扰技术,体外成功构建了介导shRNA-c-myc复制缺陷型重组腺病毒。 相似文献
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目的 构建携带P50基因的重组腺病毒载体,观察其感染宫颈癌Hela细胞后对p-AKT(Thr308)的影响.方法 以PcDNA3.1-p50PIK质粒为模板,构建带有p50PIK基因的重组腺病毒质粒Ad-p50PIK-GFP,酶切鉴定后经PacI线性化后转染HEK293包装细胞,观察细胞内绿色荧光蛋白(GFP)表达情况,并进行3轮扩增,收获带有目的 片段的腺病毒.将重组腺病毒Ad-p50PIK-GFP感染Hela细胞并通过Western blot技术检测其对p-AKT的影响.结果 将Ad-p50PIK-GFP经Xho I和Kpn I双酶切鉴定和琼脂糖电泳,在1400bp附近有目的 条带,送测序结果与GeneBank报道的序列完全一致,表明重组腺病毒Ad-p50PIK-GFP构建成功;然后将其转染HEK293细胞,绿色荧光表明Ad-p50PIK-GFP在HEK293包装细胞中成功表达;用SDS-PAGE电泳方法检测p50对Akt磷酸化的影响,结果表明高表达蛋白p50明显促进AKT的磷酸化(Thr308).结论 成功构建重组腺病毒Ad-p50PIK,p50在宫颈癌Hela细胞株中的过表达可使p-AKT表达升高.Abstract: Objective To construct recombinant adenovirus carrying p50PIK gene and examine the effect on cervix cancer Hela cell lines after transfection with Ad-p50PIK. Methods p50PIK cDNA was amplified by polymerase chain reaction (PCR), with the template PcDNA3. 1-p50PIK, then cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrackCMV-p50 was linearized by PmeI, followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183, then identified by enzyme digestion.After linearized by PacI and transfection into HEK293 cells, recombinant adenovirus Ad-p50PIK were obtained in HEK293 cells then amplified by 3 circles. The green fluorescence in HEK293 cells was observed.Ad-p50PIK was transfected into Hela cells. The phosphorylation of Akt was detected by Western blotting.Results After double restriction enzyme digestion and agarose gel electrophoresis of The Ad-p50PIK-GFP,the 1400 bp purpose band was sequenced, Results was exactly the same with the sequence GeneBank had reported,indicating that the recombinant adenovirus Ad-p50PIK-GFP were successfully constructed; Then transfected into HEK293 cells, green fluorescence shows that Ad-p50PIK-GFP in HEK293 packaging cells successfully expressed; by SDS-PAGE electrophoresis was used to detect p50 on Akt phosphorylation, the Results show that high expression of p50 protein significantly increased AKT's Phosphorylated (Thr308).Conclusion Recombinant adenovirus Ad-p50PIK had been successfully constructed. Overexpression of p50PIK in Hela cell lines can promote phosphorylation of AKT. 相似文献