基于雌激素受体的全人源基因表达调控系统的构建与优化  

Construction and Optimization of Humanized Regulatory System for Gene Expression

作　　者：闫梦迪 陈颖 吴侠 肖啸 YAN Meng-di;CHEN Ying;WU Xia;XIAO Xiao(School of Pharmacy,East China University of Science and Technology,Shanghai 200237,China;State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock,Inner Mongolia University,Hohhot 010070,China)

机构地区：[1]华东理工大学药学院,上海200237 [2]内蒙古大学省部共建草原家畜生殖调控与繁育国家重点实验室,呼和浩特010070

出　　处：《内蒙古大学学报（自然科学版）》2020年第4期418-426,共9页Journal of Inner Mongolia University：Natural Science Edition

基　　金：国家自然基金地区科学基金项目(31660343)。

摘　　要：目前基因治疗已经成为精准医疗体系中重要组成部分。然而,靶标基因在体内表达量的不可控给基因治疗的安全性带来一定风险。本研究在雌激素受体诱导系统的基础上,优化构建了全人源基因调控系统,并通过绿色荧光(Green fluorescent protein,GFP)或荧光素酶(Gaussia luciferase,Gluc)跟踪诱导系统的活性。当使用外源诱导药物4-羟基他莫昔芬和内源诱导药物17β-雌二醇进行诱导时,前者可以高效诱导GFP和Gluc在HEK293细胞中的表达,并且表达强度与剂量呈正相关。通过水高压将载体注入小鼠体内,进行药物诱导后发现,4-羟基他莫昔芬更高效的诱导报告基因GFP的表达。成年小鼠尾静脉注射1×1013vg/kg的AAV2/8-pZFHD1-hER-GFP病毒实验结果显示,第一次给药后,能够明显观察到GFP在肝脏组织的表达,但是药物在小鼠体内被代谢后,GFP的表达消失;当再次给药后,GFP荧光又重新被诱导。综上所述,本研究构建的人源化雌激素受体介导的调控系统在体内外均可调控目的基因的表达及开关。Gene therapy has became an important part of precision medical system.However,it is important to finely control the gene expression level within human body.Quantity and cycle of exogenous genes in vivo affects cellular and physiological status,even induces cytotoxicity.Using the human regulatory elements,we constructed the whole humanized transcription regulatory system,and tracked activity of the regulatory system by green fluorescent protein(GFP)or luciferase(Gluc).The exogenous inducer 4-hydroxytamoxifen was more effective than 17β-estradiol in the induction of the expression of GFP and Gluc in HEK293 cells,and the expression intensity was positively correlated with the dose of the inducers.It was found that 4-hydroxytamoxifen induced the expression of GFP more efficiently.To verify function of regulatory system as a gene switch,we treated C57 BL/6 J mice with an intravenous injection of AAV2/8-pZFHD1-hER-GFP at 1×1013vg/kg doses.After the first drug induction,the expression of GFP in liver tissues was clearly observed.After the drug was metabolized in vivo,the expression of GFP disappeared.While re-induced by the drug,GFP expressed again.In summary,the humanized estrogen receptor-mediated regulatory system constructed in this study can efficiently regulate expression of the target gene as a switch both in vitro and in vivo.

关 键 词：人源化 诱导表达调控 基因治疗 基因开关 重组腺相关病毒 

分 类 号：R96[医药卫生—药理学]