摘要
本研究构建1株能够稳定表达荧光素酶的人肺癌细胞，并进行生物学特性鉴定及体内成瘤情况验证。将携带有萤火虫荧光素酶基因的pGL4．50质粒转入A549细胞中，利用G418一直加压筛选出能够表达荧光素酶的阳性细胞克隆，并对阳性细胞克隆进行稳定性和体外活体成像检测。测定A549-luc细胞和A549细胞的生长曲线、迁移、侵袭及细胞周期，确定转染前后细胞生物学特性变化。为检测A549-luc细胞在体内成瘤和发光情况，建立裸鼠皮下荷瘤模型并应用小动物活体成像系统观察。结果表明，通过G418一直加压筛选和荧光素酶活性检测，筛选出2株荧光素酶活性高的克隆Clone20和Clone28，并连续传至40代，每5代检测1次荧光素酶活性，最终保留荧光素酶活性和稳定性最高的Clone28，Clone28通过体外活体成像检测显示生物发光值与细胞数目呈正相关的线性关系（R^2=0．9948）。A549-luc细胞和A549细胞具有相似的生长特性、迁移和侵袭能力以及细胞周期。成功建立了裸鼠皮下荷瘤模型，其发光强度与肿瘤体积呈正相关的线性关系（R^2=0．9715）。本研究稳定表达荧光素酶的人肺癌细胞的构建并成功建立裸鼠皮下荷瘤模型，可通过活体生物成像系统动态监测肿瘤的变化。
In this study,a kind of human lung cancer cell with stable expression of fluorescein was constructed,the biological characteristics and tumor formation in vivo were identified. Plasmid pGL 4.50 containing firefly luciferase gene was transfected into A549 cells by X-treme GENE HP, then the A549 cells expressing luciferase were screened with G418, and the stability and in vitro imaging of positive clones were detected. The determination of growth curve, migration, invasion and cell cycle of A549-1uc cells and A549 cells were measured to detect the changes of biological characteristics of cells before and after transfection. In order to detect the tumor formation and luminescence of A549-Luc cells in vivo ,a model of hypodermic tumor was established in nude mice and observed by small animal imaging system. Two strains of clone20 and clone28 with high luciferase activity were screened by G418 and luciferase activity detection; then they were passaged to 40th passage and luciferase activity were detected every five generations,ultimately retainied clone28 with the highest luciferase activity and stability, there was a positive correlation between the number of cells and bioluminescence value （R^2 =0. 994 8） ;A549-1uc cells and A549 cells had similar growth characteristics, ability of migration,invasion and cell cycle ; Subcutaneous tumor model was successfully established in nude mice,and there was a positive correlation between tumor volume and luminescence intensity （R^2 =0. 971 5）. In the research,the human lung cancer cells stably ex pressing luciferase and subcutaneous tumor model in nude mice were successfully established, which can be used to dynamically monitor the growth and progression of the tumor by bioluminescence imaging system.
作者
范园园
李铁
陈爽
李一权
荣凤君
李文杰
尹逊哲
韩继成
李善智
李敏
崔传信
李霄
金宁一
FAN Yuan-yuan;LI Tie;CHEN Shuang;LI Yi-quan;RONG Feng-jun;LI Wen-jie;YIN Xun- zhe;HAN Ji-cheng;LI Shan-zhi;LI Min;CUI Chuan-xin;LI Xiao;JIN Ning-yi(Institute of Veterinary Science, Academy of Military Medical Sciences, Changchun 130122, China;Changchun University of Chinese Medicine, Changchun 130012, China;Jilin Medical University ,Jilin City ,Jilin 132000 ,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第6期1177-1184,共8页
Chinese Journal of Veterinary Science
基金
新药创制科技重大专项资助项目(2014ZX09304314-002)
吉林省重点科技攻关资助项目(20150201002YY)
吉林省产业技术创新战略联盟资助项目(20140309006YY)
长春市重大科技攻关资助项目(16ss11)
关键词
A549细胞
荧光素酶
活体成像
裸鼠
肺癌肿瘤模型
A549 cells
luciferase
bioluminescence imaging
nude mice
tumor models of lung cancer
