人护骨素转基因抑制破骨细胞功能的研究(英文)

Inhibition of osteoclast function by osteoprotegerin

背景:转基因治疗骨科系统疾病一直是骨科治疗的难点,构建携带有效基因的病毒是目前治疗因破骨细胞功能异常亢进的骨骼系统疾病的研究方向。目的:构建携带人护骨素(humanosteoprotegerin,hOPG)基因的重组腺病毒及其在大鼠体内共同表达。设计:单一样本研究。单位:解放军第三军医大学大坪医院全军战创中心骨创科。材料:实验在解放军第三军医大学烧伤研究所移植免疫实验室完成。材料:质粒PUC19-hOPG;穿梭质粒pAdTrack-CMV及腺病毒基因组;pAdeasy-1,293细胞株;限制性内切酶EcoRI,EcoRⅤ,BamHI,KpNI,PmeI,PacI,PCR引物。方法:将hOPG基因片段插入到含有GFP基因的穿梭质粒pAd-Track-CMV上,与骨架质粒在大肠杆菌BJ5183胞内进行同源重组,经293细胞包装、扩增后得到携带hOPG和GFP基因的重组腺病毒(Adeasy-Adtrack-CMV-hOPG,Ad-hOPG)。经大鼠尾静脉注射109pfu的Ad-hOPG,在不同时相处死大鼠取其心、肝、肾、脾等组织观察重组腺病毒在体内表达特点。主要结局观察:①重组质粒转染293细胞后,观察是否有绿色荧光蛋白的表达。②所获得的重组腺病毒基因组是否含有hOPG基因。③重组腺病毒Ad-hOPG所携带的基因是否能在大鼠体内表达蛋白,及表达时象特点。结果:成功地构建了高滴度的携带hOPG基因的重组腺病毒,并能在体内大量表?

BACKGROUND:Transgene therapy has been a difficulty for orthopaedic diseases.To construct a novel virus carrying effective gene becomes the focus of researches on the treatment of disease of skeletal system due to dysfunction of osteoclasts. OBJECTIVE:To construct recombinant adenovirus carrying human osteoprotegerin(hOPG) gene and observe its expression in rats in vivo. DESIGN:A single sampling study. SETTING:Department of Orthopaedic and Traumatic Surgery,Center for Military War Trauma of Daping Hospital,Third Military Medical University. MATERIALS:This study was completed in the Laboratory of Transplantation Immunity,Institute of Burns,Third Military Medical University.The materials were PUC19 hOPG,shuttle plasmid pAdTrack CMV;pAdeasy 1,293 cell strain;restriction endonuclease including EcoRI,EcoRV,BamHI,KpNI,PmeI and PacI,PCR primer. METHODS:The fragment of hOPG gene encoding region was cloned into the shuttle plasmid pAdTrack CMV,homologously recombined with the backbone plasmid pAdEasy 1 in the E.Coli BJ5183,and then recombinant adenovirus carrying hOPG and GFP gene could be achieved after packaged and amplified in the 293 cells(Adeasy Adtrack CMV hOPG,Ad hOPG). Then,expressive characteristics of recombinant adenovirus at different phases were observed by injecting 109 pfu Ad hOPG into caudal vein of SD mice after the tissues such as heart,liver,kidney,spleen,etc.were taken out of the scarified mice. MAIN OUTCOME MEASURES:①The expression of GFP of recombinant plasmid in 293 cells.②If the recombinant adenoviral genome contained hOPG gene or not.③The protein expression in vivo of gene carried by recombinant adenovirus Ad hOPG in rabbits and its time dependant expressing character. RESULTS:Recombinant adenovirus vector carrying hOPG gene was successfully constructed in higher titers and expressed in vivo for 24 days. CONCLUSION:The recombinant adenovirus vector carrying hOPG gene is successfully constructed in higher titers and the expressive characters of Ad hOPG in vivo are obtained initially.