特异性切割HIF1αmRNA的锤头状核酶细胞内外活性研究

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1、徐州医学院硕士学位论文特异性切割HIF-1αmRNA的锤头状核酶细胞内外活性研究姓名：李婷申请学位级别：硕士专业：神经生物学指导教师：吴永平20030401摘簧特异性切割HIF-1amRNA的锤头状核酶细胞内外活性研究骚究玺；攀薅导师：鼹永平教授摘要匿瓣：设计特异性切割HIF．1amRNA的锤头状核酶，褐建核酶、底物的体外及翼核表达载髂，疆究箕在嚣乡}菠绷蕤蠹翡凌裁嚣彀，麓獍HIF．18mRNA竣酶痤霉予黪瘥鹣鏊因治疗奠寇萋础。方法：利用软件分析并预测HIF．1nmRNA二级结构，选择合适的核酶作用靶点，设计并合成核酶及底物的cDNA序列，分别克隆到pSP72、pSUPER

2、、phRL-LUC表达载体上。体外失控转录出棱酶及底物，避行体外切割实验。利用脂质俸介导的必转染技术，将核黪窝疯豹袭遮载薅共转染爨293T缎蕤孛，逶过双焚炎豢告基因系统捻爨缀瘫内孩鹜戆滔憋。结果：测序鉴定克隆成功，体井及雏臆内爽黢结果显示，抗HIF-1amRNA棱酶簌体外及细胞内均能有效切割底物。结论；成功构建抗HIF．1amRNA核酶及底物的体外及真核表达栽体，体外及细胞内实验结果谖骥抗HIF-1amRNA棱酶将成为肿瘤蒸阂治疗的手段。关镳溺；锤头凌竣酶、簸畿滂导嚣子一1a、鏊嚣浚瘳、终乡}切割、襞染2摘婺Hammerheadribozymewhichspecifical

3、lycleaveHIF一1amRNA：invitroandinvivoactivityPostgraduate：LiTingDirectedby：Dr．WuYongping．Abstractoqective：ThehammerheadribozymeswhichspecificallycleaveHIF-1amP．NAweredesignedandclonedintoallinvitrotranscriptionalormammalianexpressionvector．Tostudytheinvitroandinvivaactivltyoftheribozymeswere

4、forthefutureuseintumorgenetherapy．Method：UsingthesofewareRNAstructure．thesecondarystrBGtBreoftheHIF-1amRNAwaspredictedandthetargetsiteswereseleeed。ThecDNAsedeodingtheribozymesandthesubstratesweresynthesizedandclonedintopsP72、pSUPER、phRL-LUCexpressionvectorrespectively。Ribozymesandsubstrate

5、sforinvitrocleavagereactionweregeneratedfromrun-offinvitrotranscription．Mammalianvectorsofribozymesandsubstratesw∽cotransfectedinto293Tcells．ThecleavageactivityinvivawasmeasuredbyDual-Lueiferasereporterassaysystem．Results：DNAsequencingconfirmedthecorrectinsertionofthevectors，弧eresultsshowe

6、dthatthehammerheadribozymescancleavethesubstratesefficientlybothinvitroandinvivo．Conclusion：Wesuccessfullyconstructedtheanti-HIF—lnmRNAhammerheadribozymesandsubstratesvectors．弛eresultsbothinvitroandinvivosuggestedthattheanti-HIF-lamRNAhammerheadribozymescouldbeapotentialtoolforJ【lAHlorgene

7、therapy．Keyward：hammerheadribozymeHypoxia-inducedfactor-18genetherapyinvitrocleavetransfection3薷害丽蠢貉黢质瘩楚中枢裤经系统戢常藏静瑟往瀚瘤。尽管近年来采取手术、敬疗和纯疗等综合滚疗手段，毽憨嚣疆爱仍苓理想。礤究发现瓣癯内都存在显著豹袋襞区域，瓣瘤缨稳逶痰绞载主要通过两靴方妓：1)膊瘤血管生成，其巾盘鸷内发生长因子(VEGF)理黪痰斑管形成调控方面起麓重要作媚。2)?／arburg效应，即肿瘤教缺氧甚楚有鼠状态下增加糖酵