用于包装腺相关病毒293细胞的培养(英文)

Culture of 293 cells for the package of adeno-associated viruses

背景:腺相关病毒作为一种主要的基因工程载体,已被广泛应用。但腺相关病毒是缺陷性病毒,需要包装细胞提供E1蛋白。腺相关病毒293细胞作为重要的腺相关病毒特异性包装细胞,能反式产生E1,但腺相关病毒293细胞娇嫩,培养困难,生物学特性易发生改变。因此,有必要建立一种腺相关病毒293细胞的培养方案以满足基因工程的需要。目的:建立一种体外培养腺相关病毒293细胞的方法。设计:开放性实验。单位:华中科技大学同济医学院附属同济医院神经内科。材料:腺相关病毒293细胞株购于Stratagene公司。高糖型DMEM粉(Gibco公司),AAV Helper-Free系统三质粒(Stratagene公司)。方法:实验于2006-10/2007-04在武汉同济医院神经内科实验室完成。在体外将腺相关病毒293细胞复苏,用高糖型DMEM生长培养基培养,当细胞单层融合度达到50%时传代和冻存,并倒置显微镜下观察细胞的生长状态,记录生长曲线。荧光倒置显微镜下观察,根据腺相关病毒293细胞在共转染腺相关病毒系统三质粒后以及被腺相关病毒上清感染后能否激发出绿色荧光,鉴定其包装腺相关病毒的生物学特性。主要观察指标:①腺相关病毒293细胞形态学观察。②生长曲线。③腺相关病毒包装。结果:①倒置显微镜下显示,腺相关病毒293细胞贴壁生长良好,呈现不规则的多角形,胞浆透亮,胞核隐约可见。②生长曲线显示,细胞传代后第1天为生长适应期,2～5d为生长活跃期,6d后细胞进入生长平台期。③荧光倒置显微镜下观察到,腺相关病毒293细胞激发绿色荧光,共转染腺相关病毒系统三质粒成功。腺相关病毒293细胞被腺相关病毒上清感染后激发出绿色荧光,腺相关病毒包装成功。结论:本实验的培养方法简单有用,培养的腺相关病毒293细胞可保持良好的腺相关病毒包装功能。

BACKGROUND： As a main gene engineering vector, adeno-associated virus （AAV） is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV- 293 cells to meet the need of gene engineering. OBJECTIVE： To establish a culture method of AAV- 293 cells in vitro DESIGN： An opening study SEI-FING： Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate DMEM （H-DMEM） powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company. METHODS： This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not （observed by fluorescence inverted microscope） after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed. MAIN OUTCOME MEASURES ： （1） Morphological observation of AAV-293 cells; （2） the growth curve; （（3） the package of AAV RESULTS ： （1） AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei appearances. （2） The growth curve showed that the growing adaptive phase was the first day after AAV-293 cells were passaged, the actively growing phase was from the second day to the fifth day, and the growing platform phase was after the sixth day. （3） AAV-293 cells with green fluorescence observed by fluorescence inverted microscope, and cotransfection of the there AAV system plasmids was successful. AAV-293 cells gave out green fluorescence after infected with AAV supernatant, and AAV package succeeded. CONCLUSION ： The culture method established by the authors in the experiment is simple and useful, and the cultured AAV-293 cells remain a good function of AAV package.