Glucose-Modulated Transgene Expression via Recombinant Adeno-Associated Virus
Glucose-Modulated Transgene Expression via Recombinant Adeno-Associated Virus
复制标题
通过重组腺相关病毒进行葡萄糖调节的转基因表达
作者：
Ya‐Wun Yang;Y. Hsieh;C. Chao
摘要目的。本研究的目的是通过重组腺相关病毒载体在体外和体内检测葡萄糖调节的报告基因的表达。
方法：研究方法。用携带荧光素酶基因的重组腺相关病毒(RAAV)载体，在大鼠胰岛素I基因启动子和巨细胞病毒即刻早期启动子驱动的增强型绿色荧光蛋白基因的控制下，转导HuH7人肝癌细胞。无论是否存在胰岛素促分泌剂，包括佛波醇-12-肉豆蔻酸酯-13-乙酸酯、二丁酰环-AMP和Forsklin，通过葡萄糖刺激来评估报告基因的表达。给链脲佐菌素诱导的糖尿病C57BL/6J小鼠肝脏注射rAAV，2周后测定小鼠血糖浓度和荧光素酶活性。
结果。在500倍感染复数时，约66-69%的细胞在转导后48小时表达增强的绿色荧光蛋白。在胰岛素基因启动子的驱动下，rAAV转导的肝癌细胞中的荧光素酶活性对毫米级葡萄糖做出反应。在1 mM或25 mM葡萄糖存在下，佛波醇-12-肉豆蔻酸酯-13-乙酸酯、二丁酰环AMP和Forsklin的加入增加了荧光素酶的表达。这些物质对荧光素酶活性的刺激作用可被100 nM的星形孢子素抑制。外源性胰岛素浓度增加到10-7M时，抑制了rAAV转导的HuH7细胞荧光素酶基因的表达。体内实验表明，链脲佐菌素诱导的糖尿病动物的荧光素酶活性与血糖水平具有良好的相关性。
结论。RAAV是一种很有前途的糖尿病肝脏基因治疗载体。葡萄糖和胰岛素促分泌剂调节rAAV转导的肝癌细胞中转基因表达，提示影响胰岛β细胞中胰岛素基因启动子功能的条件也影响有胰岛素基因启动子的人肝癌细胞中的转基因表达。体内实验结果表明，在rAAV治疗的糖尿病C57BL/6J小鼠中，可以获得葡萄糖调控的转基因表达。
AbstractPurpose. The objective of this study was to examine glucose-modulated reporter gene expression via recombinant adeno-associated viral vectors both in vitro and in vivo.
Methods. Huh7 human hepatoma cells were transduced by recombinant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluorescence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin secretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection.
Results. At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post-transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to millimolars of glucose. The addition of phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous insulin up to 10-7 M inhibited luciferase gene expression in rAAV-transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin-induced diabetic animals.
Conclusion. rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene expression in rAAV-transduced hepatoma cells, suggesting that conditions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells conferred with insulin gene promoter. Results obtained from in vivo experiments demonstrated that glucose modulated transgene expression can be obtained in rAAV-treated diabetic C57BL/6J mice.
DOI：
10.1073/pnas.91.19.8915
发表时间：
1994
影响因子：
11.1
作者：
Russell,DW;Miller,AD;Alexander,IE
通讯作者：
Alexander,IE
DOI：
10.1073/pnas.90.5.1781
发表时间：
1993-03-01
影响因子：
11.1
作者：
GERMAN, MS
通讯作者：
GERMAN, MS
糖尿病胰岛素替代的细胞工程和基因治疗策略。
影响因子：
7.7
作者：
Newgard,CB
通讯作者：
Newgard,CB