荧光素酶标记转基因人胚胎干细胞的活体观察  

Viviperception of Transgenic Human Embryonic Stem Cells Labeled by Luciferase

作　　者：蔡柳洪[1] 周灿权[2] 张滨[1] 

机构地区：[1]中山大学附属第三医院不育与性医学科,广东广州510630 [2]中山大学附属第一医院生殖中心,广东广州510089

出　　处：《中山大学学报（医学科学版）》2009年第2期174-178,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广州市科技计划项目(2004Z1-E0101)

摘　　要：【目的】研究荧光素酶作为报告基因在活体定量和定位监测转基因人胚胎干细胞移植的动向的可行性。【方法】酶切pGL3 base质粒获得荧光素酶基因,酶切LTBR-uGIP获得载体骨架,连接荧光素酶基因和载体骨架构建慢病毒载体pULP,按照常规方法包装和扩增。ULP转导293T细胞以检测其荧光素酶的表达。人胚胎干细胞H9(P40)以W3R细胞为饲养层常规培养,人胚胎干细胞转导iDuet101-IDO(吲哚胺2,3双氧化酶,IDO)或iDuet101-CTLA4Ig(细胞毒性T细胞相关分子4免疫球蛋白,CTLA4Ig),经过筛选后再次转导ULP,即iDuet101-ULP(有荧光素酶表达)、iDuet101-CTLA4Ig-ULP(有荧光素酶和CTLA4Ig表达),iDuet101-IDO-ULP(有荧光素酶和IDO表达)。经过2次转导的3组人胚胎干细胞分别移植于5只BALB/C小鼠(实验组,共15只)和2只RAG-/-γC-/-小鼠(对照组,共6只),在注射后当日、3、5、7、14、21d和28d检测荧光素酶信号。【结果】在体外最少22000个两次转导外源基因的人胚胎干细胞可检测到荧光信号。异种移植只需要5×106的细胞即可检测到荧光信号,iDuet 101-CTLA4Ig-ULP组与iDuet101-IDO-ULP组RAG-/-γC-/-小鼠在移植后荧光信号逐渐增强,观察期间逐渐上升到3×107电子和1.5×107电子,iDuet101-ULP组两只RAG-/-γC-/-小鼠在移植后第7天因伤口感染退出实验。3组BALB/C小鼠在第7天荧光信号消失,直到移植后两个月信号没有恢复。【结论】构建慢病毒载体共表达抗生素抗性基因和目的基因,结合W3R细胞作为饲养层细胞以筛选细胞保证外源基因的表达。在细胞移植免疫缺陷小鼠后,荧光素酶基因可用于活体定量和定位监测转基因人胚胎干细胞移植的动向。[Objective] To study the feasibility of luciferase as a reporter gene in quantitative and located monitoring the trend of transgenic human embryonic stem cells after xenotransplantation in vivo. [Methods] Digested plasmid pGL3-base obtained luciferase gene, arid digested lentivirus vector LTBR-uGIP obtained vector backbone. Luciferase gene and veetor backbone were connected to construct lentivirus vector pULP packed and amplified using the conventional method. 293T cells were transduced with ULP to detect the expression of luciferase. Human embryonic stem cells H9 （P40） were cultured routinely on W3R cells as the feeder layers. H9 （P40） first transduced iDuet101-IDO gene （indoleamine 2, 3 dioxygenase, IDO） or iDuetl01-CTLA4Ig gene （cytotoxic T lymphocyte-associated molecule-4 immunoglobulin, CTLA4Ig）. After selection, ULP was transduced once again and three groups of human embryonic stem cells were obtained： iDuet101-ULP （with luciferase expression）, iDuet 101-CTLA4Ig-ULP （with lueiferase and CTLA4Ig expressions）, and iDuet101-IDO-ULP （with luciferase and IDO expressions）. After double transduction, the three groups of cells were transplanted in 15 BALB/C mice as experimeiatal group and 6 RAG^-/- γ/C^-/- mice as control group, respectively. Luciferase signals were detected on the 0, 3rd, 5th, 7th, 14th, and 21st day after the injection. [ Results] The minimum number for detecting fluorescence signal of human embryonic stem cells which transduced exogenous gene for two times in vitro was 22 000. Only 5 × 10^6 cells were enough for the detection of fluorescence signal in xenotransplantation in vivo. The signal kept enhancing in RAG^-/- γ/C^-/- mice after transplantation, and increased to 3 × 10^7 electrons and 1.5 × 10^7 electrons in iDuet 101-CTLA4Ig-ULP group and iDuet101- IDO-ULP group respectively. Two RAG^-/- γ/C^-/- mice in iDuet101-ULIP group dropped because of the infection in injection sites on the 7th d after transplantation. No signals could be detected in BALB/ C mice

关 键 词：荧光素酶 报告基因 人胚胎干细胞 异种移植 转基因 

分 类 号：R392.4[医药卫生—免疫学]