慢病毒携带增强型绿色荧光蛋白转染的人脐静脉内皮细胞  

Transfection of human umbilical vein endothelial cells with lentivirus containing enhanced green fluorescent protein

作　　者：丁强[1] 杨波[2] 王簕[3] 尹飚[2] 唐龙[2] 章波[2] 

机构地区：[1]广州医科大学附属第三医院骨外一区,广东省广州市510150 [2]广州医科大学附属第三医院骨科,广东省广州市510150 [3]广州医科大学荔湾医院,广东省广州市510150

出　　处：《中国组织工程研究》2014年第15期2351-2356,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家青年科学基金项目(31200726);2011年广州市科技计划项目(2011J4100052)~~

摘　　要：背景:人脐静脉内皮细胞转染慢病毒携带增强型绿色荧光蛋白方便后期示踪该细胞,掌握转染的最佳感染复数(MOI)和产生较强荧光强度的时间,可为后期观察人脐静脉内皮细胞在动物模型体内的示踪奠定基础。目的:观察慢病毒携带增强型绿色荧光蛋白在人脐静脉内皮细胞中的表达,找到稳定标记人脐静脉内皮细胞的方法。方法:采用胶原酶灌注消化分离人脐静脉内皮细胞,将其置于含有体积分数20%胎牛血清、内皮细胞生长因子的培养基内培养,倒置显微镜观察内皮细胞生长情况,消化离心重悬后进行计数,平均分成4组,每组细胞数5.0×105个,接种于24孔培养皿,空白组加入10μL的DMEM无血清培养基,剩余3组加入慢病毒携带增强型绿色荧光蛋白进行转染,MOI分别为2,3,4,每组3个皿。结果与结论:分离的人脐静脉内皮细胞在体外培养5-7 d可长成单层,光镜下胞体为单层鹅卵石状排列,第Ⅷ因子相关抗原的检测为阳性。转染后24 h可见绿色荧光,72 h荧光达到最强,细胞转染率与MOI值呈线性增长关系,至21 d时仍可见绿色荧光。转染后0,7,14,21 d,MOI=3组的人脐静脉内皮细胞数量均与空白组差异无显著性意义,表明转染后细胞增殖能力不受慢病毒携带增强型绿色荧光蛋白影响。提示慢病毒携带增强型绿色荧光蛋白转染人脐静脉内皮细胞效率高,绿色荧光蛋白基因可持续表达21 d,且标记后不影响人脐静脉内皮细胞的增殖活性。BACKGROUND：Human umbilical vein endothelial cells transfected with lentivirus containing enhanced green fluorescent protein can be easily traced. The optimal multiplicity of infection and time for producing strong fluorescence intensity can lay the foundation of tracing human umbilical vein endothelial cells in animal models. OBJECTIVE：To observe expression of lentivirus containing enhanced green fluorescent protein in human umbilical vein endothelial cells, and thereby to find a stable method to label human umbilical vein endothelial cells. METHODS：Using 0.1%col agenase perfusion digestion, we isolated human umbilical vein endothelial cells, which then were placed into a culture medium containing 20%fetal bovine serum and endothelial cellgrowth factor and observed under an inverted microscope. Fol owing digestion, centrifugation and suspension, the cells were counted and divided into four groups, 5.0×105 cells in each group. After cells were seededonto 24-wel plates, 10μL serum-free Dulbecco’s modified Eagle’s medium was added into the blank group, and lentiviruses containing enhanced green fluorescent protein were added into another three groups for celltransfection respectively at multiplicities of infection of 2, 3, 4. There were three dishes in each group. RESULTS AND CONCLUSION：After cultured for 5-7 days, isolated cells grew into a single layer and exhibited a cobblestone-like arrangement under a light microscope. In addition, factor VIII related antigen test was positive. A green fluorescence was visible at 24 hours of transfection, and peaked at 72 hours. Transfection efficiency was in a linear growth with the multiplicity of infection. Up to the 21st day of transfection, the green fluorescence was stil visible. After 0, 7, 14, 21 days of transfection, the number of human umbilical vein endothelial cells showed no difference between the transfection group with the multiplicity of infection=3 and blank group, suggesting the proliferative ability of cells has no changes after transfecti

关 键 词：组织构建 血管内皮细胞 慢病毒 增强型绿色荧光蛋白 人脐静脉内皮细胞 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]