用于筛选和评价抑制I型胶原α1链基因表达的抗肝纤维化药物双报告基因系统的构建及其有效性鉴定  

Construction and validation of a dual-luciferase reporter gene system for greening and evaluating anti-fiver fibrosis drugs that inhibit transcription of the gene encoding collagen I, chain αl

作　　者：张伟 代晓朋[2] 于虹[2] 王鲁燕 孙世惠[2] 李军锋[2] 周育森[2] 

机构地区：[1]解放军八八医院肝病中心二区 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《中华肝脏病杂志》2014年第10期747-751,共5页Chinese Journal of Hepatology

摘　　要：目的构建一种双报告基因系统，用于筛选和评价抑制I型胶原α1链基因（COLIAl）表达的抗肝纤维化药物。方法RT-PCR克隆转化生长因子β1（TGFβ1）全长基因，酶切后插入载体pcDNA3．1和pJW4303，构建真核表达载体pcDNA3.1-TGFβ1和pJW4303-TGFβl；以基因组DNA为模板，克隆COLlAl启动子，插入报告基因载体pGL4.29，构建含COLlAl启动子的报告基因载体pGL4.29-COLlAl promoter。上述重组载体鉴定使用酶切和序列测定。将pcDNA3.1-TGFβ1或pJW4303-TGFβ1与pGL4-COLlAl promoter、内参报告基因载体pRL-null共转染入肝星形细胞系LX-2中，构建转录激活的双报告基因系统。使用地塞米松鉴定该双报告基因系统评价抗肝纤维化药物的有效性。组间比较用Studant t检验分析，P〈0.05为差异有统计学意义。结果成功构建TGFβ1的两种真核表达载体和含COLlAl启动子的报告基因载体。通过双报告基因检测两种方式表达TGFβ1对COLlAl启动子活性的影响，结果表明：分泌表达的TGFβ1使cOLlAl启动子活性提高200倍以上（t=21.78，P=0.0001），非分泌表达的TGFβ1仅使cOLlAl启动子活性提高了不到2倍t=3.396，P=0.0274）。成功构建了转录激活的双报告基因系统。用COLlAl表达抑制剂地塞米松作为模式药物来鉴定该系统的有效性，结果显示地塞米松对COLlAl启动子有抑制作用，且呈有剂量依赖性。结论成功构建了用于筛选和评价抑制COLlAl表达的抗肝纤维化药物的双榀告基因系统并答定该系统的有效件．Objective To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain αl （COL1A1）. Methods The full-length cDNA of transforming growth factor 131 （TGFβ1） was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFβl and pJW4303-TGFβ1. Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter. All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing. Either the pcDNA3.1-TGFβ1 or pJW4303- TGFβ1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vectorwere co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dual- reporter gene system. This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1. Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system. Results The two TGFβ1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed. The results of a dual-reporter gene assay showed that TGFβ1 co-expression increased the activity of the COL1A1 promoter by above 200-fold （t = 21.78, P = 0.0001）, whereas in the absence of TGFβ1 co-expression the activity was below 2-fold （t = 3.396, P = 0.0274）. The transcriptionactivated dual-reporter gene system was successfully established. The model drug, dexamethasone, effectively inhibited the activity of the COL 1A 1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 μmol/L dexamethasone （t = 4.140, P = 0.0144） and 53.9% with 100 μmol/L （t = 6.193, P = 0.0035）.

关 键 词：肝硬化 转化生长因子Β1 I型胶原α1链基因 

分 类 号：R575.2[医药卫生—消化系统]