AANAT基因转染成肌细胞及其微囊化研究  

The experimental study on L6 cell microencapsule transfected with AANAT gene

作　　者：黄美贤[1] 廖华[1] 

机构地区：[1]南方医科大学解剖学教研室,广东省组织构建与检测重点实验室,广州510515

出　　处：《中国临床解剖学杂志》2009年第2期208-211,共4页Chinese Journal of Clinical Anatomy

基　　金：广东省科技计划攻关项目(2002C31306)

摘　　要：目的:构建pTARGETTM-AANAT真核表达载体并转化L6成肌细胞,检测目的基因的表达;制备pTARGETTMAANAT转基因细胞微囊,初步探讨AANAT转基因细胞微囊的生物活性。方法:RT-PCR技术扩增大鼠AANATcDNA,酶切连接pTARGETTM载体,脂质体法转染L6细胞并检测pTARGETTM-AANAT的基因表达产物;将高表达活性的L6细胞进行APA微囊化处理,倒置显微镜观察微囊形态,Western-blot检测囊内细胞AANAT蛋白表达活性。结果:酶切、DNA测序及凝胶电泳检测结果证实,pTARGETTM-AANAT真核表达载体构建成功。转染后的L6细胞表达AANATmRNA,胞浆中能检测到AANAT蛋白的表达。转染细胞微囊体外存活良好,微囊外形完整无粘连,大小较均一,囊内细胞清晰可见,体外培养两周后破囊检测证实囊内细胞具有AANAT蛋白表达活性。结论:pTARGETTM-AANAT真核表达载体在L6细胞中成功表达,转染细胞微囊体外存活良好,囊内细胞具备AANAT蛋白表达活性。Objective： To construct a eukaryotic expression vector system of rat NNNAT gene, transfect it into L6 myoblast, and entrap engineered cells into APA microcapsule for preparing artificial pineal tissue. Methods： The full-length eDNA fragment of rat AANAT gene was amplified by RT-PCR method. After retrieving the PCR products, ligating it with pTARGETTM vecter, transformating ligation reaction to JM 109 huge efficiency competent cells and identifying the recombinant plasmid, the recombinant eukaryotic expression vector pTARGET^TM-AANAT was transfected into rat L6 myoblast with lipofectamine. Accordingly, engineered cells selected by antibiotic G418 were enveloped into APA microcapsules, while the survival and activity of entrapped L6 cells was detected initially. Rosults： It was revealed that, amplified AANAT cDNA confirmed by agarose gel electrophoresis could ligate with pTARGETTM vector and subclone into JM109 cells. L6 cells transfected with pTARGET^TM-AANAT survived well after G418 selection and expressed AANAT protein regardless whether they were entrapped in capsules or not. Conclusions： The results suggest that we have prepared rat AANAT stable eukaryotic expression system successfully although it was just a primary result. This system can be used for the transfection of L6 myoblast which may be the seed cells of artificial pineal tissue after entrapping them into a microencapsule.

关 键 词：AANAT L6细胞 pTARGETTM载体 APA微囊 

分 类 号：R318.1[医药卫生—生物医学工程]