高通量筛选肿瘤预防药物技术平台的建立及中医药应用研究
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摘要
“应用天然或人工合成的化合物去阻断、逆转或预防侵袭性肿瘤的发生”,即肿瘤的化学预防(Chemoprevention)。国际癌症中心曾指出,在复杂多样的致癌因素中,80%～90%的人类肿瘤是由化学致癌物引起的。化学致癌物可损伤细胞DNA,使基因发生突变,从而引起肿瘤。肿瘤预防研究的目的之一就是寻找高效、低毒的药物。由于化学药品的毒副作用以及人们认识到“回归大自然”的重要性,天然药物在肿瘤预防中所起到的作用,日益受到重视。我国有着宝贵、丰富的中药资源,如何高通量地从中药中筛选出预防肿瘤的有效成分,已成为一个迫切需要解决的瓶颈问题。
目前,国内外应用于药物筛选的模型大致分为三类:整体动物水平模型、组织器官水平模型和细胞分子水平模型。前两种模型往往消耗样品量大,要使用大量的实验动物,劳动强度高,耗时耗力,并且一次试验筛选样品量有限,不适合成分复杂的中药有效活性成分的筛选。细胞分子水平的药物筛选模型具有材料用量少、省时高效、药物作用机制比较明确、可实现大规模的筛选等特点,但在分子和细胞水平上对化学预防剂的有效鉴别的研究还相当缺乏,通过建立转基因细胞模型对预防肿瘤中药进行高通量筛选的研究,国内外均未见报道。
本研究旨在建立一种简便易行、高度敏感和高效率的筛选肿瘤预防药物的细胞模型,并用于从现有中药中筛选预防肿瘤的有效成分。
1.应用重组PCR技术,以质粒pRL-TK为模板,获得重组TK启动子,并在其基本启动子上游引入Sac I酶切位点。将TK启动子片段克隆入pMD18-T载体中,构建载体pMD-TK。用Sal I和BamH I对质粒pMD-TK和pEGFP进行双酶切,将TK启动子连接到载体pEGFP中构建载体pTK-GFP。再以质粒pTK-GFP为模板扩增TK目的片段,用Vsp I和BamH I对扩增出的TK目的片段和载体pEGFP-N1进行酶切,将TK启动子连接到载体pEGFP-N1上,构建真核报告载体pTK-GFP/neo。人工合成的4个ARE序列,经退火磷酸化后逐个连接到载体pTK-GFP/neo上。首次在国内分别构建了真核报告载体pARE-TK- GFP/neo、p2ARE-TK-GFP/neo、p3ARE-TK-GFP/neo和p4ARE-TK-GFP/neo。
2.真核报告载体pTK-GFP/neo、pARE-TK-GFP/neo、p2ARE-TK-GFP/neo和p4ARE-TK-GFP/neo用脂质体转染HepG2细胞,24 h后在荧光显微镜下观察到4种细胞都发出绿色荧光,说明启动子的选择是较为合适的。接着用600μg/mL G418筛选出阳性细胞克隆。首次在国内构建了无ARE调控的细胞模型HepG2-TK-GFP和不同ARE重复序列调控的细胞模型HepG2-ARE-TK-GFP、HepG2-2ARE-TK-GFP、HepG2-3ARE-TK-GFP和HepG2-4ARE-TK-GFP。
3. HepG2-TK-GFP、HepG2-ARE-TK-GFP、HepG2-2ARE-TK-GFP和HepG2- 4ARE-TK-GFP细胞扩增后,经胰酶消化,接种入黑色96孔细胞培养板,每孔细胞数为5×104个,培养24 h后,除对照孔外,各孔加入终浓度为100μM的tBHQ,并设5个重复。24小时后用PBS洗涤,再加入100μg/mL EB 200μL室温下染色20 min,再用PBS洗涤1次后加入200μL PBS,用多功能荧光酶标仪检测细胞的GFP和EB荧光强度,求其相对发光度。结果HepG2-4ARE-TK-GFP细胞的GFP诱导表达水平最高,为HepG2-TK-GFP细胞的6倍,选用该细胞中对GFP具有最高表达水平和最低基础水平的克隆做后续实验。
4.将HepG2-TK-GFP和HepG2-4ARE-TK-GFP细胞扩增后,接种入黑色96孔细胞培养板,培养24 h后加入不同浓度(0(DMSO对照)、12.5、25、50、100、200μM)的阳性受试物PDTC和tBHQ。每浓度设5个重复,作用24 h后用PBS洗涤,加入EB染色,用多功能荧光酶标仪检测细胞的GFP和EB荧光强度。结果显示PDTC和tBHQ对HepG2-TK-GFP细胞的GFP荧光均无诱导作用,其荧光强度呈不规则变化,各浓度组之间差异不显著(P>0.05);PDTC诱导的HepG2-4ARE-TK-GFP细胞中的GFP荧光随着PDTC浓度的升高,荧光表达水平呈先上升后下降的趋势,具有一定的剂量效应关系。在浓度为50μM时诱导效果最好,与对照组比较差异有显著性(P<0.05);tBHQ诱导的HepG2-4ARE-TK-GFP细胞中的GFP荧光随着tBHQ浓度的升高,荧光表达水平持续上升,各组(12.5, 25, 50, 100, 200μM)与对照组比较差异都有显著性(P<0.05),且具有一定的剂量效应关系。从而证实本实验所构建的这种转基因细胞模型可用来筛选不同结构性质的化学预防剂。
5.以质粒pDsRed2-N1为模板扩增红色荧光蛋白及启动子片段克隆入载体pMD18-T上,构建载体pMD-DsRed。用Ade I和BspT I对载体pMD-DsRed和p4ARE-TK-GFP/neo进行双酶切,将目的红色荧光蛋白及启动子片段连接到载体p4ARE-TK-GFP/neo上,构建真核报告载体p4ARE-TK-GFP/DsRed/ neo。该载体用脂质体转染HepG2细胞,并用G418筛选出阳性细胞克隆。首次构建了以GFP为第一信号,以红色荧光蛋白为第二信号的细胞模型HepG2-4ARE-TK-GFP/DsRed。将该细胞接种入黑色96孔培养板,培养24 h后加入不同浓度(0(DMSO对照)、12.5、25、50、100、200μM)的阳性受试物PDTC和tBHQ。所得结果与HepG2-4ARE-TK-GFP细胞模型中的结果基本一致,证实对转基因细胞模型HepG2-4ARE-TK-GFP的完善是成功的,使筛选更简单、更方便、更准确。
6.首次利用HepG2-4ARE-TK-GFP细胞模型对22种中药单体、3种中药有效部位、复方六味地黄胶囊、四君子汤以及组成两方的各单味中药的提取物进行了筛选,结果中药单体中穿心莲内酯、黄芩苷、大黄酸、大黄素、槲皮素、大豆苷元、熊果酸、白藜芦醇显示结果为阳性,且都具有一定的剂量效应关系,提示8种单体能诱导Ⅱ相酶表达起到化学预防作用;三种有效部位中半枝莲总黄酮注射液和黄芪注射液显示结果为阳性,提示其阻断癌前病变和抗突变的作用可能是通过诱导Ⅱ相酶表达实现的;复方六味地黄胶囊和四君子汤的显示结果为阳性,单味中药提取物中熟地黄、人参、白术显示结果为阳性。其中复方六味地黄胶囊效果优于该方中的单味中药熟地黄,提示其诱导效果可能是因为复方产生了新的成分或是各成分的联合诱导了该模型中GFP的表达。
“The application of natural or synthetic compounds to block, reverse or prevent the occurrence of invasive tumors”, as known as chemoprevention, is an effective strategy. Among the diverse carcinogenic factors, the International Agency for Research on Cancer indicated that 80-90% of the development of human tumors are caused by chemical carcinogens. Chemical carcinogens can damage cell DNA to result in gene mutation, and then eventually lead to tumor development. One of the research objectives for chemoprevention is to discover effective drugs with low toxicity. Due to the toxic side effects of artificial chemical drugs, the importance of natural treatment and natural medicine in cancer prevention has gained more and more attention. China has valuable resources for traditional medicine. It has become an urgent need to investigate how to perform high-throughput screening for active preventive ingredients in Chinese herbal medicine.
World widely, drug screening models can be generally divided into three types: animal model, tissue and organ model, and cellular and molecular model. The first two models tend to consume a large amount of samples and to use a large number of laboratory animals. They are labour intensive and time consuming. Also, the number of samples being screened each time can be limited and these two models are not suitable for screening for complex active ingredients in Chinese medicine. The drug screening model at the cellular and molecular level has several properties: less sample consumption, high efficiency, understandable drug mechanism, and large sample size screening. However, there is limited research with regard to chemopreventive drug screening at the cellular and molecular level. To our knowledge, we are the first group to report on high-thoughput screening for chemopreventive Chinese medicine through transgenic cell model.
This paper aims to establish a simple, highly sensitive and efficient tumor preventive agents screening model to screen for active ingredients in traditional Chinese medicine for cancer prevention.
1. The TK promoter was amplified from plasmid pRL-TK by recombinant PCR and the Sac I enzyme restriction site was created. The TK promoter segments were cloned into pMD18-T vector to construct pMD-TK. The plasmids of pMD-TK and pEGFP were double digested with Sal I and BamH I. The TK promoter was connected to the correspondent restriction sites of the vector pEGFP to yield the vector pTK-GFP. The plasmid pTK-GFP was used as a template to amplify TK segments by PCR. The amplified TK segments and vector pEGFP-N1 were digested using Vsp I and BamH I. The TK promoter was ligated to the vector pEGFP-N1 to construct the eukaryotic reporter vector of pTK-GFP/neo. Four synthetic ARE sequences were inserted into the vector pTK-GFP/neo after annealing phosphorylation to regulate the expression of GFP. So the four eukaryotic vectors, pARE-TK-GFP/neo, p2ARE-TK-GFP/neo, p3ARE-TK-GFP / neo and p4ARE-TK- GFP/neo, were constructed respectively first in China.
2. The eukaryotic reporter vectors pTK-GFP/neo, pARE-TK-GFP/neo, p2ARE-TK- GFP/neo and p4ARE-TK-GFP/neo were transfected into HepG2 cells respectively. After 24 hours of incubation, the green fluorescence was obvious under the fluorescent microscope for the four types of cells, indicating that the choice of promoter was appropriate. First time in China, the clones resistant to 600μg/ml G418 were isolated to construct the ARE free cell model of HepG2-TK-GFP for the negative control and different ARE repeated sequence control cell models of HepG2-ARE-TK-GFP, HepG2-2ARE-TK-GFP, HepG2-3ARE-TK-GFP and HepG2-4ARE-TK-GFP.
3. After HepG2-TK-GFP, HepG2-ARE-TK-GFP, HepG2-2ARE-TK-GFP and HepG2- 4ARE-TK-GFP were amplified, they were digested by trypsin and seeded into wells of a black 96-well plate with 5×104 cells in each well. After the incubation for 24 hours, in addition to the control wells, each well was added tBHQ with a concentration of 100μM. Each type of cell was repeated for five times. After a 24-h exposure to the compounds, the medium was removed and 100μg / ml EB 200μl was added to stain the cells for 20 minutes at room temperature. The cells were then washed with PBS once and 200μl PBS was added into each well prior to measuring flurescence. Measurement of GFP or EB fluorescence was performed using a fluorescence microplate reader respectively. The induced GFP expression level was the highest in HepG2-4ARE-TK-GFP cells and it was about 6 times compared to HepG2-TK-GFP. The clone cells with the highest GFP expression level and lowest level were used to do the follow-up experiments.
4. After HepG2-TK-GFP and HepG2-4ARE-TK-GFP cell proliferation and inoculation into the black 96 well plates for 24 hours, different concentrations (0 (DMSO control), 12.5,25,50,100,200μM) of positive tested compounds PDTC and tBHQ were added in. Each concentration was repeated for five times. After 24 hours of incubation, the cells were washed with PBS and stained with EB, fluorescence intensity of GFP and EB was measured by the fluorescence microplate reader. The results indicated that the GFP fluorescence of HepG2-TK-GFP cells was not induced by PDTC and tBHQ. The fluorescence intensity were irregular and the differences between concentrations were not significant (P>0.05). As the GFP fluorescence intensity increased with PDTC concentration in PDTC-induced HepG2-4ARE-TK -GFP cells, the fluorescence expression was first increased and then decreased showing a dose-response relationship. At the concentration of 50μM, inducing effect had a high peak value and it was significantly different from the control (P<0.05). As the GFP fluorescence intensity increased with the tBHQ concentration in tBHQ-induced HepG2-4ARE-TK -GFP cells, the fluorescence expression continued to increase. There were significant differences between the experimental groups (12.5, 25, 50, 100, 200μM) and the control group (p<0.05) showing a dose-response relationship. Therefore, this transgenic cell model can be used to screen chemical compounds with different structural properties.
5. A red fluorescent protein and promoter fragment was cloned into the vector pMD18-T from pDSRed2-N1 to construct the vector pMD-DsRed. The vectors pMD-DsRed and p4ARE-TK-GFP/neo were digested by Ade I and BspT1. The red fluorescent protein and promoter fragments were cloned into the vector p4ARE-TK-GFP/neo to construct the eukaryotic reporter vector p4ARE-TK- GFP/DsRed / neo. This vector was transfected into HepG2 cells. Positive cell clones were screened by G418 to construct the cell model HepG2-4ARE-TK-GFP/DsRed using GFP as the first signal and red fluorescent protein as the second signal. The cells were inoculation into the black 96 well plates for 24 hours and different concentrations (0 (DMSO control), 12.5,25,50,100,200μM) of positive tested compounds PDTC and tBHQ were added in. The results were consistent with the conclusions made for the HepG2-4ARE-TK-GFP cell model. Therefore, the transgenic cell model of HepG2-4ARE-TK-GFP/DsRed was successfully established and screening will become easier, more convenient and more accurate.
6. The HepG2-4ARE-TK-GFP cell model was used to screen for 22 kinds of traditional Chinese medicine monomers, three types of active ingredients in Chinese medicine, Liu Wei Di Huang Capsule, Si Jun Zi Decoction, and ten single herb extracts. The results showed that andrographolide in Chinese medicine monomers, baicalin, rhein, emodin, quercetin, daidzein, ursolic acid, resveratrol were tested positive showing a dose-response relationship. Eight monomers could induce phase II enzymes expressions therefore played a chemopreventive role. Three effective parts of the total flavonoids in Scutellaria barbata and astragalus infection showed positive results, suggesting that their roles in blocking precancerous lesions and resistance to mutations may be achieved by the induction of phase II enzymes. The results for Liu Wei Di Huang capsule and Si Jun Zi Decoction were positive. Among single Chinese medicine extractions, the results were positive for Rehmannia glutinosa, ginseng, and Atractylodes. The effect of Liu Wei Di Huang capsule was better than the single herb Rehmannia glutinosa in the capsule, suggesting that the induced GFP expression in the model may be due to the new component produced or a combination of the compounds. This indicates that some natural chemopreventive agents was not less effective compared to some known synthetic chemical agents and more importantly, the side effect of these natural agents may be much smaller.
目前,国内外应用于药物筛选的模型大致分为三类:整体动物水平模型、组织器官水平模型和细胞分子水平模型。前两种模型往往消耗样品量大,要使用大量的实验动物,劳动强度高,耗时耗力,并且一次试验筛选样品量有限,不适合成分复杂的中药有效活性成分的筛选。细胞分子水平的药物筛选模型具有材料用量少、省时高效、药物作用机制比较明确、可实现大规模的筛选等特点,但在分子和细胞水平上对化学预防剂的有效鉴别的研究还相当缺乏,通过建立转基因细胞模型对预防肿瘤中药进行高通量筛选的研究,国内外均未见报道。
本研究旨在建立一种简便易行、高度敏感和高效率的筛选肿瘤预防药物的细胞模型,并用于从现有中药中筛选预防肿瘤的有效成分。
1.应用重组PCR技术,以质粒pRL-TK为模板,获得重组TK启动子,并在其基本启动子上游引入Sac I酶切位点。将TK启动子片段克隆入pMD18-T载体中,构建载体pMD-TK。用Sal I和BamH I对质粒pMD-TK和pEGFP进行双酶切,将TK启动子连接到载体pEGFP中构建载体pTK-GFP。再以质粒pTK-GFP为模板扩增TK目的片段,用Vsp I和BamH I对扩增出的TK目的片段和载体pEGFP-N1进行酶切,将TK启动子连接到载体pEGFP-N1上,构建真核报告载体pTK-GFP/neo。人工合成的4个ARE序列,经退火磷酸化后逐个连接到载体pTK-GFP/neo上。首次在国内分别构建了真核报告载体pARE-TK- GFP/neo、p2ARE-TK-GFP/neo、p3ARE-TK-GFP/neo和p4ARE-TK-GFP/neo。
2.真核报告载体pTK-GFP/neo、pARE-TK-GFP/neo、p2ARE-TK-GFP/neo和p4ARE-TK-GFP/neo用脂质体转染HepG2细胞,24 h后在荧光显微镜下观察到4种细胞都发出绿色荧光,说明启动子的选择是较为合适的。接着用600μg/mL G418筛选出阳性细胞克隆。首次在国内构建了无ARE调控的细胞模型HepG2-TK-GFP和不同ARE重复序列调控的细胞模型HepG2-ARE-TK-GFP、HepG2-2ARE-TK-GFP、HepG2-3ARE-TK-GFP和HepG2-4ARE-TK-GFP。
3. HepG2-TK-GFP、HepG2-ARE-TK-GFP、HepG2-2ARE-TK-GFP和HepG2- 4ARE-TK-GFP细胞扩增后,经胰酶消化,接种入黑色96孔细胞培养板,每孔细胞数为5×104个,培养24 h后,除对照孔外,各孔加入终浓度为100μM的tBHQ,并设5个重复。24小时后用PBS洗涤,再加入100μg/mL EB 200μL室温下染色20 min,再用PBS洗涤1次后加入200μL PBS,用多功能荧光酶标仪检测细胞的GFP和EB荧光强度,求其相对发光度。结果HepG2-4ARE-TK-GFP细胞的GFP诱导表达水平最高,为HepG2-TK-GFP细胞的6倍,选用该细胞中对GFP具有最高表达水平和最低基础水平的克隆做后续实验。
4.将HepG2-TK-GFP和HepG2-4ARE-TK-GFP细胞扩增后,接种入黑色96孔细胞培养板,培养24 h后加入不同浓度(0(DMSO对照)、12.5、25、50、100、200μM)的阳性受试物PDTC和tBHQ。每浓度设5个重复,作用24 h后用PBS洗涤,加入EB染色,用多功能荧光酶标仪检测细胞的GFP和EB荧光强度。结果显示PDTC和tBHQ对HepG2-TK-GFP细胞的GFP荧光均无诱导作用,其荧光强度呈不规则变化,各浓度组之间差异不显著(P>0.05);PDTC诱导的HepG2-4ARE-TK-GFP细胞中的GFP荧光随着PDTC浓度的升高,荧光表达水平呈先上升后下降的趋势,具有一定的剂量效应关系。在浓度为50μM时诱导效果最好,与对照组比较差异有显著性(P<0.05);tBHQ诱导的HepG2-4ARE-TK-GFP细胞中的GFP荧光随着tBHQ浓度的升高,荧光表达水平持续上升,各组(12.5, 25, 50, 100, 200μM)与对照组比较差异都有显著性(P<0.05),且具有一定的剂量效应关系。从而证实本实验所构建的这种转基因细胞模型可用来筛选不同结构性质的化学预防剂。
5.以质粒pDsRed2-N1为模板扩增红色荧光蛋白及启动子片段克隆入载体pMD18-T上,构建载体pMD-DsRed。用Ade I和BspT I对载体pMD-DsRed和p4ARE-TK-GFP/neo进行双酶切,将目的红色荧光蛋白及启动子片段连接到载体p4ARE-TK-GFP/neo上,构建真核报告载体p4ARE-TK-GFP/DsRed/ neo。该载体用脂质体转染HepG2细胞,并用G418筛选出阳性细胞克隆。首次构建了以GFP为第一信号,以红色荧光蛋白为第二信号的细胞模型HepG2-4ARE-TK-GFP/DsRed。将该细胞接种入黑色96孔培养板,培养24 h后加入不同浓度(0(DMSO对照)、12.5、25、50、100、200μM)的阳性受试物PDTC和tBHQ。所得结果与HepG2-4ARE-TK-GFP细胞模型中的结果基本一致,证实对转基因细胞模型HepG2-4ARE-TK-GFP的完善是成功的,使筛选更简单、更方便、更准确。
6.首次利用HepG2-4ARE-TK-GFP细胞模型对22种中药单体、3种中药有效部位、复方六味地黄胶囊、四君子汤以及组成两方的各单味中药的提取物进行了筛选,结果中药单体中穿心莲内酯、黄芩苷、大黄酸、大黄素、槲皮素、大豆苷元、熊果酸、白藜芦醇显示结果为阳性,且都具有一定的剂量效应关系,提示8种单体能诱导Ⅱ相酶表达起到化学预防作用;三种有效部位中半枝莲总黄酮注射液和黄芪注射液显示结果为阳性,提示其阻断癌前病变和抗突变的作用可能是通过诱导Ⅱ相酶表达实现的;复方六味地黄胶囊和四君子汤的显示结果为阳性,单味中药提取物中熟地黄、人参、白术显示结果为阳性。其中复方六味地黄胶囊效果优于该方中的单味中药熟地黄,提示其诱导效果可能是因为复方产生了新的成分或是各成分的联合诱导了该模型中GFP的表达。
“The application of natural or synthetic compounds to block, reverse or prevent the occurrence of invasive tumors”, as known as chemoprevention, is an effective strategy. Among the diverse carcinogenic factors, the International Agency for Research on Cancer indicated that 80-90% of the development of human tumors are caused by chemical carcinogens. Chemical carcinogens can damage cell DNA to result in gene mutation, and then eventually lead to tumor development. One of the research objectives for chemoprevention is to discover effective drugs with low toxicity. Due to the toxic side effects of artificial chemical drugs, the importance of natural treatment and natural medicine in cancer prevention has gained more and more attention. China has valuable resources for traditional medicine. It has become an urgent need to investigate how to perform high-throughput screening for active preventive ingredients in Chinese herbal medicine.
World widely, drug screening models can be generally divided into three types: animal model, tissue and organ model, and cellular and molecular model. The first two models tend to consume a large amount of samples and to use a large number of laboratory animals. They are labour intensive and time consuming. Also, the number of samples being screened each time can be limited and these two models are not suitable for screening for complex active ingredients in Chinese medicine. The drug screening model at the cellular and molecular level has several properties: less sample consumption, high efficiency, understandable drug mechanism, and large sample size screening. However, there is limited research with regard to chemopreventive drug screening at the cellular and molecular level. To our knowledge, we are the first group to report on high-thoughput screening for chemopreventive Chinese medicine through transgenic cell model.
This paper aims to establish a simple, highly sensitive and efficient tumor preventive agents screening model to screen for active ingredients in traditional Chinese medicine for cancer prevention.
1. The TK promoter was amplified from plasmid pRL-TK by recombinant PCR and the Sac I enzyme restriction site was created. The TK promoter segments were cloned into pMD18-T vector to construct pMD-TK. The plasmids of pMD-TK and pEGFP were double digested with Sal I and BamH I. The TK promoter was connected to the correspondent restriction sites of the vector pEGFP to yield the vector pTK-GFP. The plasmid pTK-GFP was used as a template to amplify TK segments by PCR. The amplified TK segments and vector pEGFP-N1 were digested using Vsp I and BamH I. The TK promoter was ligated to the vector pEGFP-N1 to construct the eukaryotic reporter vector of pTK-GFP/neo. Four synthetic ARE sequences were inserted into the vector pTK-GFP/neo after annealing phosphorylation to regulate the expression of GFP. So the four eukaryotic vectors, pARE-TK-GFP/neo, p2ARE-TK-GFP/neo, p3ARE-TK-GFP / neo and p4ARE-TK- GFP/neo, were constructed respectively first in China.
2. The eukaryotic reporter vectors pTK-GFP/neo, pARE-TK-GFP/neo, p2ARE-TK- GFP/neo and p4ARE-TK-GFP/neo were transfected into HepG2 cells respectively. After 24 hours of incubation, the green fluorescence was obvious under the fluorescent microscope for the four types of cells, indicating that the choice of promoter was appropriate. First time in China, the clones resistant to 600μg/ml G418 were isolated to construct the ARE free cell model of HepG2-TK-GFP for the negative control and different ARE repeated sequence control cell models of HepG2-ARE-TK-GFP, HepG2-2ARE-TK-GFP, HepG2-3ARE-TK-GFP and HepG2-4ARE-TK-GFP.
3. After HepG2-TK-GFP, HepG2-ARE-TK-GFP, HepG2-2ARE-TK-GFP and HepG2- 4ARE-TK-GFP were amplified, they were digested by trypsin and seeded into wells of a black 96-well plate with 5×104 cells in each well. After the incubation for 24 hours, in addition to the control wells, each well was added tBHQ with a concentration of 100μM. Each type of cell was repeated for five times. After a 24-h exposure to the compounds, the medium was removed and 100μg / ml EB 200μl was added to stain the cells for 20 minutes at room temperature. The cells were then washed with PBS once and 200μl PBS was added into each well prior to measuring flurescence. Measurement of GFP or EB fluorescence was performed using a fluorescence microplate reader respectively. The induced GFP expression level was the highest in HepG2-4ARE-TK-GFP cells and it was about 6 times compared to HepG2-TK-GFP. The clone cells with the highest GFP expression level and lowest level were used to do the follow-up experiments.
4. After HepG2-TK-GFP and HepG2-4ARE-TK-GFP cell proliferation and inoculation into the black 96 well plates for 24 hours, different concentrations (0 (DMSO control), 12.5,25,50,100,200μM) of positive tested compounds PDTC and tBHQ were added in. Each concentration was repeated for five times. After 24 hours of incubation, the cells were washed with PBS and stained with EB, fluorescence intensity of GFP and EB was measured by the fluorescence microplate reader. The results indicated that the GFP fluorescence of HepG2-TK-GFP cells was not induced by PDTC and tBHQ. The fluorescence intensity were irregular and the differences between concentrations were not significant (P>0.05). As the GFP fluorescence intensity increased with PDTC concentration in PDTC-induced HepG2-4ARE-TK -GFP cells, the fluorescence expression was first increased and then decreased showing a dose-response relationship. At the concentration of 50μM, inducing effect had a high peak value and it was significantly different from the control (P<0.05). As the GFP fluorescence intensity increased with the tBHQ concentration in tBHQ-induced HepG2-4ARE-TK -GFP cells, the fluorescence expression continued to increase. There were significant differences between the experimental groups (12.5, 25, 50, 100, 200μM) and the control group (p<0.05) showing a dose-response relationship. Therefore, this transgenic cell model can be used to screen chemical compounds with different structural properties.
5. A red fluorescent protein and promoter fragment was cloned into the vector pMD18-T from pDSRed2-N1 to construct the vector pMD-DsRed. The vectors pMD-DsRed and p4ARE-TK-GFP/neo were digested by Ade I and BspT1. The red fluorescent protein and promoter fragments were cloned into the vector p4ARE-TK-GFP/neo to construct the eukaryotic reporter vector p4ARE-TK- GFP/DsRed / neo. This vector was transfected into HepG2 cells. Positive cell clones were screened by G418 to construct the cell model HepG2-4ARE-TK-GFP/DsRed using GFP as the first signal and red fluorescent protein as the second signal. The cells were inoculation into the black 96 well plates for 24 hours and different concentrations (0 (DMSO control), 12.5,25,50,100,200μM) of positive tested compounds PDTC and tBHQ were added in. The results were consistent with the conclusions made for the HepG2-4ARE-TK-GFP cell model. Therefore, the transgenic cell model of HepG2-4ARE-TK-GFP/DsRed was successfully established and screening will become easier, more convenient and more accurate.
6. The HepG2-4ARE-TK-GFP cell model was used to screen for 22 kinds of traditional Chinese medicine monomers, three types of active ingredients in Chinese medicine, Liu Wei Di Huang Capsule, Si Jun Zi Decoction, and ten single herb extracts. The results showed that andrographolide in Chinese medicine monomers, baicalin, rhein, emodin, quercetin, daidzein, ursolic acid, resveratrol were tested positive showing a dose-response relationship. Eight monomers could induce phase II enzymes expressions therefore played a chemopreventive role. Three effective parts of the total flavonoids in Scutellaria barbata and astragalus infection showed positive results, suggesting that their roles in blocking precancerous lesions and resistance to mutations may be achieved by the induction of phase II enzymes. The results for Liu Wei Di Huang capsule and Si Jun Zi Decoction were positive. Among single Chinese medicine extractions, the results were positive for Rehmannia glutinosa, ginseng, and Atractylodes. The effect of Liu Wei Di Huang capsule was better than the single herb Rehmannia glutinosa in the capsule, suggesting that the induced GFP expression in the model may be due to the new component produced or a combination of the compounds. This indicates that some natural chemopreventive agents was not less effective compared to some known synthetic chemical agents and more importantly, the side effect of these natural agents may be much smaller.
引文
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