[构建和验证用于筛选和评价抑制I型胶原蛋白α1链编码基因转录的抗肝纤维化药物的双荧光素酶报告基因系统]

[Construction and validation of a dual-luciferase reporter gene system for screening and evaluating anti-liver fibrosis drugs that inhibit transcription of the gene encoding collagen I, chain a1].

作者信息

Zhang Wei, Dai Xiaoming, Yu Hong, Wang Luyan, Sun Shihui, Li Junfeng, Zhou Yusen

机构信息

Department of Liver Surgery, Renji Hospital School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China.

出版信息

Zhonghua Gan Zang Bing Za Zhi. 2014 Oct;22(10):747-51. doi: 10.3760/cma.j.issn.1007-3418.2014.10.007.

DOI:10.3760/cma.j.issn.1007-3418.2014.10.007

PMID:25496864

Abstract

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 at1 (COL1A1).

METHODS

The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.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-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter 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 TGFbeta1-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 TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 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 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035).

CONCLUSION

The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.

摘要

目的

构建一种双报告基因系统，用作筛选和评估抑制I型胶原蛋白α1链（COL1A1）基因转录的治疗性药物化合物的工具。

方法

通过逆转录聚合酶链反应（RT-PCR）克隆转化生长因子β1（TGFβ1）的全长互补DNA（cDNA），并将其插入两个载体pcDNA3.1和pJW4303中，构建两个真核表达载体pcDNA3.1-TGFβ1和pJW4303-TGFβ1。接下来，以人类基因组DNA为模板，通过PCR克隆COL1A1的启动子区域，并将其插入载体pGL4.29中，构建报告基因载体pGL4.29-COL1A1启动子。通过限制性内切酶消化和DNA测序对所有三种重组载体进行验证。将pcDNA3.1-TGFβ1或pJW4303-TGFβ1载体与pGL4.29-COL1A1启动子载体或对照报告载体pRL-null共转染到LX-2人肝星状细胞中，建立转录激活双报告基因系统。该系统用作筛选抑制COL1A1转录的抗肝纤维化化合物的细胞模型。已知抑制COL1A1表达的模型药物地塞米松用作对照，以验证双报告基因系统。

结果

成功构建了两种表达TGFβ1的载体和含有COL1A1启动子区域的报告基因载体。双报告基因检测结果表明，TGFβ1共表达使COL1A1启动子活性增加200倍以上（t = 21.78，P = 0.0001），而在没有TGFβ1共表达的情况下，活性低于2倍（t = 3.396，P = 0.0274）。成功建立了转录激活双报告基因系统。模型药物地塞米松以剂量依赖性方式有效抑制COL1A1启动子的活性；10μmol/L地塞米松使活性降低29.6%（t = 4.140，P = 0.0144），100μmol/L时降低53.9%（t = 6.193，P = 0.0035）。