基于报告基因检测的β3肾上腺素受体激动剂筛选模型的建立与应用

目的：建立基于双荧光素酶报告基因检测的β3肾上腺素受体（β3-AR）激动剂筛选模型,并用于β3-AR激动剂的筛选。方法：应用免疫细胞化学法鉴定β3-AR在β3-CHO细胞上的稳定表达,并将pCRE-luc质粒与pRL-TK质粒共同瞬时转染β3-CHO细胞,建立一种基于双荧光素酶报告基因检测的β3-AR激动剂筛选模型。通过检测报告基因萤火虫荧光素酶与海肾荧光素酶活性的比值,来间接反映配体对β3-AR的激动活性。并以β-AR激动剂异丙肾上腺素刺激对模型的有效性进行验证,在此基础上以此模型对20种新化合物的β3-AR激动活性进行筛选。结果：β3-CHO细胞经免疫细胞化学法鉴定有特异性受体蛋白表达。通过将两个报告基因质粒转染该细胞后,与加入溶剂对照相比,加入异丙肾上腺素可显著促进荧光素酶报告基因的表达,表明该模型有效、可靠。以此模型从20个化合物中初筛出8个活性较强的β3-AR激动剂。结论：本研究建立了基于双荧光素酶报告基因检测的β3-AR激动剂筛选模型,并以此模型筛选发现了几个活性较强的β3-AR激动剂。     

5-羟色胺1A受体激动剂高通量筛选模型的建立

目的为发现5-羟色胺1A(5-HT1A)受体的激动剂,通过报告基因活性检测的方法建立高通量筛选(HTS)细胞模型。方法将带有人源5-HT1A受体的真核表达质粒pcD-NA3.1(pcDNA3.1-h5-HT1AR)与带有报告基因pCRE-luc的pcDNA3.1质粒(pcDNA3.1-pCRE-luc)共转染到工具细胞中,通过对工具细胞选择、共转染质粒比例、化合物孵育时间及阳性化合物选择等条件进行探索和优化,建立了稳定表达人源5-HT1A受体并可用于该受体激动剂筛选的细胞模型(HEK293-h5-HT1AR)。结果①根据瞬转实验中信号值的高低,模型采用HEK293细胞作为工具细胞;②根据瞬转实验中的信噪比,发现pcDNA3.1-h5-HT1AR:pcDNA3.1-pCRE-luc的最佳比例为1∶3;③对化合物孵育时间进行优化,选择的最佳孵育时间为6 h;④阳性化合物的选择过程中,实验研究了5-HT.HCl,Flibanserin,8-OH-DPAT以及Lorcaser-in(APD356)4个已报道有5-HT1A受体激动活性的化合物,结果表明APD356在该体系中最适合作为阳性对照化合物。⑤该细胞株连续培养12代,信号稳定。结论通过对一系列实验条件进行选择和优化,建立了一个稳定表达人源5-HT1A受体的细胞模型,该模型可用于高通量5-HT1A激动剂的筛选。     

基于报告基因检测的PXR、FXR和LXRα激动剂高通量筛选模型的建立

目的建立基于报告基因法的高通量筛选细胞模型,用来发现PXR、FXR和LXRα受体激动剂。方法利用Real-time定量PCR方法比较HEK293、Hep G2和LS174T细胞中内源性核受体PXR、FXR和LXRα的表达量,将p SG5-h PXR和p GL3-XREM-CYP3A4、p EGFP-N3-h FXR和EcRETK-Luc、p CMX-FLAG-h LXRα和p GL3-XREM-CYP3A4等质粒分别共转染到工具细胞中,优化共转染比例,并考察阳性药与萤光素酶报告基因表达强度的量效关系、模型特异性和稳定性。结果 1根据Real-time定量PCR结果,模型选用低表达PXR、FXR和LXRα的HEK293细胞作为工具细胞;2根据不同共转染比例对报告基因活性的结果,PXR、FXR和LXRα报告基因药物筛选模型的报告基因和过表达质粒比例,最终分别选择1∶1、2∶1和2∶1;3模型中,报告基因活性均与相应阳性药物(PXR/Rif、FXR/CDCA和LXRα/T0901317)呈剂量依赖性增长;4仅PXR激动剂Rif、FXR激动剂CDCA和LXRα激动剂T0901317可分别明显增加相应筛选模型的报告基因活性,分别重复5次试验后,计算得Z'值分别为0.58、0.66和0.63。结论该研究建立的PXR、FXR和LXRα激动剂高通量筛选模型,具有良好的特异性和稳定性,适用于对PXR、FXR和LXRα受体激动剂的筛选,进而开发以核受体作为药物靶点的药物。     

基于报告基因和PPARγ信号通路的药物筛选模型的建立

目的　建立基于报告基因和PPARγ(peroxisomeprolif erator activatedreceptorγ)信号通路的药物筛选模型,用此模型筛选具有胰岛素增敏活性的小分子化合物。方法　五种细胞分别进行瞬时转染,将含有目的片段PPRE(peroxisomeproliferatorresponseelement)和报告基因荧光素酶(Luc)的质粒及表达PPARγ的质粒共转染到细胞中,通过测定荧光素酶活力来考察马来酸罗格列酮对PPARγ信号通路的影响,选取诱导表达倍数最高的细胞株建立模型。用其他类型核受体激动剂对此模型进行特异性考察。结果　293T细胞中,荧光素酶的表达受马来酸罗格列酮的诱导倍数最高,可达4 9倍,并呈现一定的剂量依赖关系,Z′因子为0 .72。而其他各类核受体激动剂的诱导表达率均在1倍左右。马来酸罗格列酮在转染剂量范围内无促进细胞增殖的作用。结论　马来酸罗格列酮对共转染报告基因质粒和表达PPARγ质粒的293T细胞Luc的表达具有较强的诱导作用,此模型具有较好的特异性和稳定性,适用于建立筛选PPARγ激动剂的高通量筛选模型。     

以GLP-1受体为靶点的药物筛选细胞模型的建立和应用

建立以GLP-1信号通路为靶点的药物筛选细胞模型,用于筛选GLP-1受体激动剂类的新型抗糖尿病药物。首先构建GLP-1受体信号通路调控的特异应答元件（RIP-CRE）多拷贝序列及报告基因E-GFP的重组载体。将该载体转染胰岛NIT-1细胞株,以检测转染细胞对GLP类似物的反应性和特异性,并通过稳定转染和单克隆培养,获得对GLP-1类似物特异应答的单克隆细胞株。该细胞模型在GLP-1类似物Exendin 4刺激下激活表达报告基因,激活作用可以被GLP-1受体阻断剂Exendin 9-39完全阻断,且激活途径非cAMP-PKA依赖,具有GLP-1受体特异性。构建该细胞模型,可以对肽类或非肽类GLP-1类似物进行高通量筛选。     

GPR120选择性剪切异构体2的高通量药物筛选细胞模型的构建

目的：建立GPR120选择性剪切异构体2的功能性筛选模型，筛选GPR120的激动剂和抑制剂，开发防治糖尿病等疾病的药物。方法：从健康人胎脑cDNA文库中得到缺失了第3外显子的GPR120选择性剪切异构体2（hGPR120-s）的cDNA，将其克隆到pcDNA3载体中，构建pcDNA3/hGPR120-s重组表达质粒，继而转染HEK293细胞，通过G418筛选，获得稳定表达该异构体的真核重组细胞系。利用特异性配体亚麻酸和GW9508探索和优化了筛选条件。并初步应用该模型研究各种天然脂肪酸的构效关系。结果：建立了稳定可靠的高通量筛选模型，Z’因子＝0.69。结论：该系统适合于GPR120激动剂的高通量筛选。     

基于COL1A1启动子的抗肝纤维化药物高通量筛选模型的建立及应用

为了有效筛选潜在的抗肝纤维化药物,本实验室建立了基于COL1A1启动子的高通量筛选细胞模型。该细胞模型在TGF-β1的刺激下,激活COL1A1启动子及荧光素酶报告基因的表达,而该激活作用可以被候选化合物抑制。作者首先构建COL1A1启动子和荧光素酶报告载体p GL4.17的重组质粒。采用双荧光素酶报告基因检测系统对该启动子的活性进行检测,发现重组质粒比对照组荧光素酶活性高15.98倍。该质粒转染到人肝星状细胞株LX2中,用无限稀释法得到单克隆细胞株,并利用活体成像仪筛选出稳定表达COL1A1启动子的单克隆细胞株LX2-COL。经TGF-β1诱导后检测该单克隆细胞株LX2-COL对候选化合物(S1~S7)的反应性,其中S7对启动子活性抑制作用最强。Real-time RT-PCR和Western blotting验证了经该细胞模型筛选得到的化合物S7能够抑制Ⅰ型胶原m RNA和蛋白水平的表达。结果表明,该细胞模型的建立能够实现对潜在的抗肝纤维化化合物的高通量筛选。     

稳定表达大麻受体2的CHO细胞株的构建

目的建立稳定高效表达人重组大麻受体2(CB2)的中国仓鼠卵巢(CHO-K1)细胞株,为体外高通量筛选CB2激动剂和拮抗剂奠定基础。方法通过脂质体介导的方法将构建的表达载体pcDNA3.1(+)-CB2转染入CHO-K1细胞中,然后用含G418的选择性培养液进行筛选,挑取耐药克隆;培养并收集耐药克隆细胞,用RT-PCR方法做进一步筛选;序列测定鉴定整合基因的序列;筛选的阳性克隆用放射性配体-受体结合实验进行进一步的鉴定和受体活性分析。结果转染细胞在含G418的选择性培养基中生长出28个耐药单克隆,用RT-PCR方法检测出17个CB2mRNA表达量较高的阳性克隆;RT-PCR扩增片段测定鉴定正确;挑选其中最优的克隆进行放射性配体-受体结合实验,结果显示,表达受体具有与CB2激动剂WIN55212-2特异结合的活性,其Kd和Bmax值分别(1.21±0.47)nmol.L-1和(3.12±0.7)nmol.g-1蛋白,这一结果与天然CB2的特性相似。结论建立了稳定高效表达人重组CB2的CHO-K1细胞株。     

以HIF2α为靶点的化合物筛选的细胞模型构建

目的 建立稳定表达血管内皮生长因子A（VEGFA）启动子区5×缺氧反应元件（5×HRE）和萤光素酶（Luc）报告基因的人肾透明细胞癌（786-O）单克隆细胞系,构建以缺氧诱导因子2α（HIF2α）为靶点的化合物筛选的细胞模型,并对其应用加以初步验证。方法 采用基因重组技术,构建含有VEGFA启动子区5×HRE以及Luc报告基因的慢病毒载体,并感染786-O细胞,经过嘌呤霉素筛选获得能稳定表达Luc活性的786-O-5×HRE-Luc细胞系;采用等浓度梯度稀释获得单克隆细胞系,运用HIF2α抑制剂PT2385和PT2399处理,检测Luc活性变化,筛选对PT2385和PT2399反应更灵敏的单克隆细胞系。结果 通过基因测序鉴定,VEGFA启动子区5×HRE和Luc报告基因慢病毒表达载体构建成功,经过嘌呤霉素（Puro）筛选获得稳定表达Luc的786-O-5×HRE-Luc细胞;PT2385和PT2399可剂量依赖性的降低细胞Luc的活性;得到对PT2385和PT2399反应灵敏的单克隆细胞系。结论 建立了786-O-5×HRE-Luc稳定表达5×HRE和Luc报告基因细胞系,为高通量筛选靶向HIF2α的药物提供一类新的细胞模型。     

A cell model for high-throughput screening GLP-1 receptor agonists北大核心CSCD

目的构建GLP-1受体激动剂高通量筛选细胞模型。方法构建pEGFP-GLP-1R-3C重组质粒,转染HEK293T细胞,用G418和流式细胞仪进行筛选。所构建的细胞系命名为HEK293T-GLP-1R-3C-eGFP细胞系。Western blot和激光共聚焦检测GLP-1R-3C-eGFP蛋白的表达。将环磷酸腺苷(cyclic adenosine monophosphate,cAMP)响应元件报告基因转染HEK293T-GLP-1R-3C-eGFP细胞,不同浓度的GLP-1刺激后,用一步法荧光素酶报告基因检测试剂盒检测发光值反映细胞cAMP水平,采用cAMP试剂盒(ELISA)进行验证。结果成功构建HEK293T-GLP-1R-3C-eGFP细胞系。不同浓度GLP-1刺激后,发光值变化趋势与细胞cAMP水平变化趋势相似。本实验中Z′因子的值为0.52。结论本研究建立了基于重组HEK293T细胞株,其可用于GLP-1受体激动剂高通量筛选。     

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

AIM: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library. METHODS: Synthetic responsive elements 6 cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. RESULTS: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC(50) values of SBG492 were 342.7 microg/mL for the D1 receptor and 31.7 microg/mL for the D5 receptor. CONCLUSION: We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.     

Anti-thyroid hormone activity of bisphenol A,tetrabromobisphenol A and tetrachlorobisphenol A in an improved reporter gene assay

Previously, we transiently transfected Gal4-fused thyroid hormone receptor (TR) expressing vector and the Gal4 response reporter structure pUAS-tk-luc into HepG2 cell, developed a TR beta-1 mediated reporter gene assay to screen for compounds that acted on the TR signaling pathway. In this study, we improved the test efficiency by changing the transfected cell line into CV-1 cell. Triiodothyronine (T3) and thyroxine (T4) induced higher luciferase expression, with the median effective concentration (EC₅₀) of 1.16 × 10^?8 and 1.36 × 10^?7 M, respectively. Bisphenol A (BPA) was selected as a positive antagonist, exhibiting weak anti-thyroid hormone activity with the median inhibitory concentration (IC₅₀) of 1.64 × 10^?5 M. The assay showed acceptable repeatability to T3 with inter coefficient of variability (CV) of 27.5% and intra CV of 18.6%. Two flame retardants, tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA), were tested for their agonist and antagonist activities. As a result, we found that both of them possessed TR antagonist activity and neither of them showed agonist activity. These results suggested that TBBPA and TCBPA could act as TH antagonists. This assay provided a useful tool for the assessment of environmental chemicals as thyroid system disruptors.     

Cloning, expression, and functional analysis of human dopamine D1 receptors

AIM: To construct an HEK293 cell line stably expressing human dopamine D1 receptor (D1R). METHODS: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D1R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D1R in HEK293 cells was monitored by the [3H]SCH23390 binding assay. The function of D1R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. RESULTS: An HEK293 cell line stably expressing human D1R was obtained. A saturation radioligand binding experiment with [3H]SCH23390 demonstrated that the Kd and Bmax values were 1.5+/-0.2 nmol/L and 2.94+/-0.15 nmol/g of protein, respectively. In the [3H]SCH23390 competition assay, D1R agonist SKF38393 displaced [3H]SCH23390 with an IC50 value of 2.0 (1.5-2.8) micromol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D1R expressed in HEK293 cells in a concentration-dependent manner with an EC50 value of 0.25 (0.12-0.53) micromol/L and 0.39 (0.27-0.57) micromol/L at 6 h/0.59 (0.22-1.58) micromol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC50 value of 27 (8.6-70) nmol/L. CONCLUSION: An HEK293 cell line stably expressing human D1R was obtained successfully. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research.     

Development and validation of a cell-based assay for the nuclear receptor retinoid-related orphan receptor gamma

The nuclear receptor retinoid-related orphan receptor gamma (RORγ) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for RORγ inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/RORγ ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-RORγ expression leads to increased reporter activity, which can be knocked down using RORγ ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for RORγ and observed varying levels of partial inverse agonist activity at μM concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.