【摘要】：本研究采用HepaRG細(xì)胞建立類器官(organoids)3D培養(yǎng)模型,并結(jié)合高內(nèi)涵成像技術(shù)建立藥物肝毒性體外評(píng)價(jià)方法。通過氫化可的松和二甲基亞砜(DMSO)誘導(dǎo),使HepaRG細(xì)胞分化為明顯的肝細(xì)胞形態(tài)和膽管樣結(jié)構(gòu),并誘導(dǎo)藥物代謝酶、藥物轉(zhuǎn)運(yùn)體、核受體及肝細(xì)胞特異標(biāo)志性分子白蛋白(ALB)等相關(guān)基因的表達(dá),形成穩(wěn)定的模擬肝臟功能的類器官體外評(píng)價(jià)模型;采用高內(nèi)涵成像技術(shù),通過熒光染料進(jìn)行特異性、多靶點(diǎn)染色,從類器官球體表型、活/死細(xì)胞數(shù)量、線粒體膜電位(MMP)和細(xì)胞內(nèi)活性氧(ROS)評(píng)價(jià)藥物肝毒性。結(jié)果表明,采用HepaRG細(xì)胞建立的類器官體外評(píng)價(jià)模型可以準(zhǔn)確地評(píng)價(jià)肝毒性陽性對(duì)照藥胺碘酮(amiodarone,AMD)、環(huán)孢霉素(cyclosporin,CSP)及陰性對(duì)照藥阿司匹林(aspirin,ASP)的毒性差異:其中AMD和CSP均濃度依賴地導(dǎo)致類器官球體的總細(xì)胞數(shù)和活細(xì)胞數(shù)下降,并且AMD濃度超過50μmol·L~(-1)時(shí),還導(dǎo)致類器官球體中死細(xì)胞數(shù)急劇增多,而ASP對(duì)類器官無明顯損傷作用;AMD和CSP均濃度依賴地導(dǎo)致MMP下降和ROS水平增高,且AMD的作用強(qiáng)度大于CSP,而ASP對(duì)類器官無明顯損傷作用。綜上,HepaRG細(xì)胞建立的類器官高內(nèi)涵成像評(píng)價(jià)方法可用于藥物肝毒性的體外評(píng)價(jià),并且具有多靶標(biāo)高通量、結(jié)果直觀且可定量等優(yōu)勢。
[Abstract]:In this study, HepaRG cells were used to establish (organoids) 3D culture model and high intension imaging technique was used to establish in vitro evaluation method of drug hepatotoxicity. Induced by hydrocortisone and dimethyl sulfoxide (DMSO), HepaRG cells differentiated into hepatocyte morphology and bile duct like structure, and induced drug metabolizing enzymes and drug transporters. The expression of related genes such as nuclear receptor and hepatocyte specific iconic molecule albumin (ALB) forms a stable in vitro evaluation model of liver function, and uses high intension imaging technology to carry out specific, multi-target staining with fluorescent dyes. Hepatic toxicity was evaluated by organoid globular phenotype, number of living / dead cells, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS). The results show that The in vitro evaluation model established by HepaRG cells can accurately evaluate the toxicity of amiodarone (amiodarone,AMD), cyclosporine (cyclosporin,CSP) and aspirin (aspirin,ASP), in which both AMD and CSP are concentration-dependent on the conductance of hepatotoxic drugs, such as amiodarone (amiodarone,AMD), cyclosporine (cyclosporin,CSP) and aspirin (aspirin,ASP). The total number of cells and the number of living cells in the globules of organs decreased. When the concentration of AMD was more than 50 渭 mol L ~ (-1), the number of dead cells in the organoid spheres increased sharply, while ASP had no obvious damage to the organs. Both ASP and CSP caused the decrease of MMP and the increase of ROS levels in a concentration-dependent manner. The action intensity of AMD was higher than that of CSP, but ASP had no obvious damage to organ like organs. The high intension imaging method developed by HepaRG cells can be used to evaluate the hepatotoxicity of drugs in vitro and has the advantages of multiple targets and high throughput. The results are intuitive and quantifiable.
【作者單位】： 解放軍302醫(yī)院全軍中醫(yī)藥研究所;中國醫(yī)學(xué)科學(xué)院腫瘤醫(yī)院;河北北方學(xué)院;北京輸血醫(yī)學(xué)研究所;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(81503350) 北京市自然科學(xué)基金資助項(xiàng)目(7152142) 北京市科技新星計(jì)劃項(xiàng)目(Z16111000490000) 中國博士后科學(xué)基金資助項(xiàng)目(2016M590065)
【分類號(hào)】：R96

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