基于Ⅱ相代谢酶的转基因细胞模型探索半枝莲黄酮类化合物防治肿瘤作用及机理

Phase Ⅱ metabolic enzyme can catalyze the binding reaction of exogegous compound, producing more easily eliminated metabolic products, and thus can promote the metabolism of carcinogens, prevent cell mutation, block or even reverse tumorigenesis.In our previous research, the ARE control cell model of GFP was constructed first time in China. It was found that the Scutellaria barbatae D.DON total flavonoids could induce the expression of phase Ⅱ metabolic enzymes. we got seven flavonoids of different polarity parts which were A-G by extraction and separation of scutellaria barbatae D.DON. Based on our previous research, we are planning to utilize the gene targeting technology to effectively combine the phase Ⅱ metabolic enzyme gene regulation mechanism and the light-emitting mechanism of red, green fluorescent protein in order to build a double signalling transgenic cell model which performs the high-throughput screening on ingredients that increase the level of phase Ⅱ metabolic enzymes. It will be investigated that what the inducing active ingredients are and if there is a dose-response relationship between active ingredients and target molecules of signalling pathways. We also will detect and assess the induction mechanism of active ingredients in Scutellaria barbatae D.DON flavonoids on phase Ⅱ metabolic enzymes through gene transduction, protein expression and enzyme function. Our research will build a new technology platform on research on screening cancer preventive ingredients of traditional Chinese medicine and studying its mechanism.It can also provide new theoretical basis for developing new traditional Chinese medicine of cancer prevention and treatment。

Ⅱ相代谢酶在体内可催化外来化合物的结合反应，形成易于排出的代谢产物，促进致癌物或促癌物的体内代谢，防止细胞突变，阻滞和逆转肿瘤增生。我们前期成功构建了ARE调控的GFP转基因细胞，并发现半枝莲总黄酮能诱导Ⅱ相代谢酶的表达，进而对半枝莲提取与分离得到7个不同极性部位的黄酮类化合物。在此基础上拟将Ⅱ相代谢酶基因调控机制与红、绿色荧光蛋白的发光机制巧妙地结合起来，同时利用基因打靶技术，构建一种能高通量从中药里筛选出专使Ⅱ相代谢酶升高成分的双信号转基因细胞模型，进一步探讨半枝莲黄酮类化合物对Ⅱ相代谢酶发挥诱导作用的有效成分是什么？其有效成分和信号通路靶分子之间是否存在量效关系？并从基因转录、蛋白质表达及酶功能水平上，检测和评价半枝莲黄酮类化合物中防治肿瘤有效成分对Ⅱ相酶的诱导机理。期冀为简便、高效筛选防治肿瘤中药及探索其活性成分作用机制创建新的技术平台，也可为防治肿瘤新药研制开发提供新的理论依据。

Ⅱ相代谢酶在体内可催化外来化合物的结合反应，形成易于排出的代谢产物，促进致癌物或促癌物的体内代谢，防止细胞突变，阻滞和逆转肿瘤增生。我们前期成功构建了ARE调控的GFP转基因细胞，并发现半枝莲总黄酮能诱导Ⅱ相代谢酶的表达，进而对半枝莲提取与分离得到7个不同极性部位的黄酮类化合物。在此基础上我们将Ⅱ相代谢酶基因调控机制与红、绿色荧光蛋白的发光机制巧妙地结合起来，同时利用基因打靶技术，成功构建了一种能高通量从中药里筛选出专使Ⅱ相代谢酶升高成分的双信号转基因细胞模型，并通过阳性受试物证实了本实验所构建的这种转基因细胞模型可用来专一筛选Ⅱ相代谢酶单相诱导物，且具有一定的剂量效应关系。进一步通过该模型对半枝莲黄酮类化合物进行了筛选研究，发现7个不同极性部位的半枝莲黄酮类化合物中化合物G（FGSB）对细胞中GFP荧光有诱导作用。7个不同极性部位的半枝莲黄酮类化合物的32种组合中，虽然有多组组合对GFP荧光有明显诱导作用，但并未发现7个不同极性部位的半枝莲黄酮类化合物之间存在协同作用。通过对FGSB的研究，我们发现FGSB对Ⅱ相酶GCLC、GST-P1均有诱导作用，FGSB可激活Nrf2从胞浆转移到核内，证实FGSB通过Keap1-Nrf2-ARE信号系统调控Ⅱ相酶基因的表达。总之，本项目为简便、高效筛选防治肿瘤中药及探索其活性成分作用机制创建了新的技术平台，也为防治肿瘤新药研制开发提供了新的理论依据。