旨在考察染料木素(genistein,GEN)、川芎嗪(tetramethylpyrazine,TMP)、染料木素-川芎嗪共晶(GEN-TMP)及染料木素与川芎嗪的物理混合物(GEN+TMP)在Caco-2细胞模型中的转运特征;建立Caco-2细胞模型,并以细胞跨膜电阻和标志物渗漏检查等...旨在考察染料木素(genistein,GEN)、川芎嗪(tetramethylpyrazine,TMP)、染料木素-川芎嗪共晶(GEN-TMP)及染料木素与川芎嗪的物理混合物(GEN+TMP)在Caco-2细胞模型中的转运特征;建立Caco-2细胞模型,并以细胞跨膜电阻和标志物渗漏检查等指标进行验证,采用高效液相色谱法,考察并计算安全浓度下药物的累积转运量、表观渗透系数和外排率,并探讨P糖蛋白(P-gp)抑制剂维拉帕米、乳腺癌耐药蛋白(breast cancer resistant protein,BCRP)抑制剂KO143和多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)抑制剂MK571对转运的影响。结果显示,Caco-2细胞模型完整性与功能性良好,GEN浓度为40μg/mL时,GEN、TMP、GEN-TMP以及GEN+TMP的细胞存活率分别为90.06%、84.21%、97.60%和89.37%;GEN、TMP、GEN-TMP和GEN+TMP的表观渗透系数(P_(app))大于1.0×10^(-6)cm/s,属于吸收良好药物;GEN-TMP中GEN的累积转运量和P_(app)值分别为(2.78±0.11)μg和(8.61±0.33)×10^(-6)cm/s,比GEN的(1.92±0.15)μg和(5.96±0.47)×10^(-6)cm/s提高了44.79%和44.46%;GEN+TMP中GEN的累积转运量和P_(app)值与GEN无显著性差异。GEN只受BCRP的外排作用,GEN-TMP中的GEN同时受到P-gp和BCRP的外排作用,TMP、GEN-TMP和GEN+TMP中的TMP均受到MRP2的外排作用。结果表明,相同浓度下,GEN-TMP的细胞存活率高于GEN+TMP。GEN-TMP的吸收强于GEN和GEN+TMP中的GEN,共晶受外排蛋白的作用区别于GEN和GEN+TMP中的GEN,研究工作为共晶的转运研究提供了借鉴和参考。展开更多
目的:探讨猕猴桃皮多酚(kiwifruit peel polyphenols,KPP)对脂多糖(lipopolysaccharide,LPS)应激Caco-2细胞抗氧化能力的影响。方法:采用CCK-8法测定不同处理组Caco-2的细胞活力,荧光分光光度计测定活性氧和线粒体膜电位,分光光度计测...目的:探讨猕猴桃皮多酚(kiwifruit peel polyphenols,KPP)对脂多糖(lipopolysaccharide,LPS)应激Caco-2细胞抗氧化能力的影响。方法:采用CCK-8法测定不同处理组Caco-2的细胞活力,荧光分光光度计测定活性氧和线粒体膜电位,分光光度计测定超氧化物歧化酶(superoxide dismutase,SOD)活力和谷胱甘肽(glutathione,GSH)、丙二醛(malonaldehyde,MDA)含量,实时荧光定量聚合酶链式反应测定核红细胞2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)、NAD(P)H:醌氧化还原酶1(NAD(P)H:quinone oxidoreductase 1,NQO1)、超氧化物歧化酶1(superoxide dismutase 1,S O D 1)、S O D 2基因的表达;蛋白印迹测定N r f 2、Ke a p 1及N Q O 1蛋白表达水平。结果:与L P S组相比,经50μg/mL KPP干预后细胞活力显著升高(P<0.05);活性氧水平和MDA含量分别显著下降至1.82±0.28、5.08 nmol/mg(P<0.05),线粒体膜电位显著升高至1.84±0.10(P<0.05),SOD活力和GSH含量分别显著升高至52.57 U/mg和69.46μmol/g(P<0.05);同时KPP干预能显著提高Nrf2、NQO1基因和蛋白表达(P<0.05),显著降低Keap1基因和蛋白表达(P<0.05)。结论:KPP能够通过Keap1/Nrf2/NQO1信号通路提高Caco-2的抗氧化水平,缓解LPS应激造成的细胞损伤。展开更多
采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转...采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.展开更多
Background:The ingestion of golden thread contaminated with heavy metals through the food chain leads to detrimental effects to human health.During digestion,not all of the heavy metals could be released to the gastro...Background:The ingestion of golden thread contaminated with heavy metals through the food chain leads to detrimental effects to human health.During digestion,not all of the heavy metals could be released to the gastrointestinal tract and readily to be absorbed by human body.Thus,bioaccessibility is an important issue in health risk assessments.Aims and Objectives:The aims and objectives of this study were to investigate the bioaccessibility of Cd in golden thread and assess the associated health risks based on the exposure to bioaccessible Cd.Materials and Methods:Inductively coupled plasma mass spectrometry(ICP-MS)has been applied to determine the Cd content in golden thread.Physiologically based extraction test(PBET)digestion was performed in the in vitro/Caco2 cell model to investigate the bioaccessibility of Cd in golden thread.Furthermore,the target hazard quotient(THQ)was used to assess the risks of the total and the bioaccessible content of Cd in golden thread.Results:The results revealed that the total Cd content in six batches of golden thread ranged from 3.203 to 5.723 mg/kg.After uptake by Caco2 cells,the bioaccessibility of Cd ranged from 42.36%to 59.73%.The results of the risk assessment indicated that prior to uptake by Caco2 cells,the THQ values of Cd for all batches of golden thread were greater than 1.However,after uptake by Caco2 cells,the THQ values of Cd in all samples were less than 1,thus suggesting that the risks were at a safe level.Conclusion:This study was the first to perform health risk assessment with bioaccessible heavy metals present in traditional Chinese medicine by PBET digestion using an in vitro/Caco2 cell model,thus enabling us to obtain more accurate and objective results while allowing us to avoid unnecessary government intervention and to establish more reasonable limit standards for heavy metals.展开更多
文摘旨在考察染料木素(genistein,GEN)、川芎嗪(tetramethylpyrazine,TMP)、染料木素-川芎嗪共晶(GEN-TMP)及染料木素与川芎嗪的物理混合物(GEN+TMP)在Caco-2细胞模型中的转运特征;建立Caco-2细胞模型,并以细胞跨膜电阻和标志物渗漏检查等指标进行验证,采用高效液相色谱法,考察并计算安全浓度下药物的累积转运量、表观渗透系数和外排率,并探讨P糖蛋白(P-gp)抑制剂维拉帕米、乳腺癌耐药蛋白(breast cancer resistant protein,BCRP)抑制剂KO143和多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)抑制剂MK571对转运的影响。结果显示,Caco-2细胞模型完整性与功能性良好,GEN浓度为40μg/mL时,GEN、TMP、GEN-TMP以及GEN+TMP的细胞存活率分别为90.06%、84.21%、97.60%和89.37%;GEN、TMP、GEN-TMP和GEN+TMP的表观渗透系数(P_(app))大于1.0×10^(-6)cm/s,属于吸收良好药物;GEN-TMP中GEN的累积转运量和P_(app)值分别为(2.78±0.11)μg和(8.61±0.33)×10^(-6)cm/s,比GEN的(1.92±0.15)μg和(5.96±0.47)×10^(-6)cm/s提高了44.79%和44.46%;GEN+TMP中GEN的累积转运量和P_(app)值与GEN无显著性差异。GEN只受BCRP的外排作用,GEN-TMP中的GEN同时受到P-gp和BCRP的外排作用,TMP、GEN-TMP和GEN+TMP中的TMP均受到MRP2的外排作用。结果表明,相同浓度下,GEN-TMP的细胞存活率高于GEN+TMP。GEN-TMP的吸收强于GEN和GEN+TMP中的GEN,共晶受外排蛋白的作用区别于GEN和GEN+TMP中的GEN,研究工作为共晶的转运研究提供了借鉴和参考。
文摘目的:探讨猕猴桃皮多酚(kiwifruit peel polyphenols,KPP)对脂多糖(lipopolysaccharide,LPS)应激Caco-2细胞抗氧化能力的影响。方法:采用CCK-8法测定不同处理组Caco-2的细胞活力,荧光分光光度计测定活性氧和线粒体膜电位,分光光度计测定超氧化物歧化酶(superoxide dismutase,SOD)活力和谷胱甘肽(glutathione,GSH)、丙二醛(malonaldehyde,MDA)含量,实时荧光定量聚合酶链式反应测定核红细胞2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)、NAD(P)H:醌氧化还原酶1(NAD(P)H:quinone oxidoreductase 1,NQO1)、超氧化物歧化酶1(superoxide dismutase 1,S O D 1)、S O D 2基因的表达;蛋白印迹测定N r f 2、Ke a p 1及N Q O 1蛋白表达水平。结果:与L P S组相比,经50μg/mL KPP干预后细胞活力显著升高(P<0.05);活性氧水平和MDA含量分别显著下降至1.82±0.28、5.08 nmol/mg(P<0.05),线粒体膜电位显著升高至1.84±0.10(P<0.05),SOD活力和GSH含量分别显著升高至52.57 U/mg和69.46μmol/g(P<0.05);同时KPP干预能显著提高Nrf2、NQO1基因和蛋白表达(P<0.05),显著降低Keap1基因和蛋白表达(P<0.05)。结论:KPP能够通过Keap1/Nrf2/NQO1信号通路提高Caco-2的抗氧化水平,缓解LPS应激造成的细胞损伤。
文摘采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.
基金financially supported by the 13th Five-Year National Significant New Drugs Creation Feature Subjects grant(2018ZX09735006)by the Project for Medicine and Medical Instruments Review and Approval System Reform grant(ZG2016-1)
文摘Background:The ingestion of golden thread contaminated with heavy metals through the food chain leads to detrimental effects to human health.During digestion,not all of the heavy metals could be released to the gastrointestinal tract and readily to be absorbed by human body.Thus,bioaccessibility is an important issue in health risk assessments.Aims and Objectives:The aims and objectives of this study were to investigate the bioaccessibility of Cd in golden thread and assess the associated health risks based on the exposure to bioaccessible Cd.Materials and Methods:Inductively coupled plasma mass spectrometry(ICP-MS)has been applied to determine the Cd content in golden thread.Physiologically based extraction test(PBET)digestion was performed in the in vitro/Caco2 cell model to investigate the bioaccessibility of Cd in golden thread.Furthermore,the target hazard quotient(THQ)was used to assess the risks of the total and the bioaccessible content of Cd in golden thread.Results:The results revealed that the total Cd content in six batches of golden thread ranged from 3.203 to 5.723 mg/kg.After uptake by Caco2 cells,the bioaccessibility of Cd ranged from 42.36%to 59.73%.The results of the risk assessment indicated that prior to uptake by Caco2 cells,the THQ values of Cd for all batches of golden thread were greater than 1.However,after uptake by Caco2 cells,the THQ values of Cd in all samples were less than 1,thus suggesting that the risks were at a safe level.Conclusion:This study was the first to perform health risk assessment with bioaccessible heavy metals present in traditional Chinese medicine by PBET digestion using an in vitro/Caco2 cell model,thus enabling us to obtain more accurate and objective results while allowing us to avoid unnecessary government intervention and to establish more reasonable limit standards for heavy metals.
