基于3D肝细胞球模型评价何首乌醇提物毒性研究  

Toxicity evaluation of alcohol extract of Polygonum multiflorum based on 3D hepatocyte ball model

作　　者：苏华龙 姚香草 陈佳敏 岑柏宏 王萍 陈宗正 许重远[1,2] SU Hua-long;YAO Xiang-cao;CHEN Jia-min;CEN Bo-hong;WANG Ping;CHEN Zong-zheng;XU Zhong-yuan(Clinical Pharmacy Center,Nanfang Hospital,Southern Medical University,Guangzhou 510515,Guangdong Province,China;National Medical Products Administration Key Laboratory for Research and Evaluation of Drug Metabolism&Guangdong Provincial Key Laboratory of New Drug Screening,Schoolof Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,Guangdong Province,China;The First Afiliated Hospital of Shenzhen University,Shenzhen 518035,Guangdong Province,China)

机构地区：[1]南方医科大学南方医院临床药学中心,广东广州510515 [2]南方医科大学药学院国家药品监督管理局药物代谢研究与评价重点实验室&广东省新药筛选重点实验室,广东广州510515 [3]深圳大学第一附属医院,广东深圳518035

出　　处：《中国临床药理学杂志》2024年第9期1272-1276,共5页The Chinese Journal of Clinical Pharmacology

基　　金：广东省基础与应用基础研究基金企业联合基金资助项目(2022A1515220136);广东省研究生教育创新计划基金资助项目(2022ANLK_023);青年基金-南方医科大学南方医院院长基金资助(2022B043);南方医院临床研究专项基金资助项目(2019CR015)。

摘　　要：目的探究何首乌醇提物对3D肝细胞球的毒性。方法设置不同的条件以及细胞比例,测量细胞球直径、致密度、圆度,探索最佳培养条件。3D肝细胞球分为对照组和低、中、高剂量实验组,低、中、高剂量实验组给予0.25、1.00和2.50 mg·mL^(-1)何首乌醇提物,对照组给予等量的培养基。用CellTiter-Glo试剂检测细胞球活性,用荧光定量聚合酶链反应(RT-qRCR)检测肝功能相关基因表达水平,用活死细胞双荧光染色试剂检测细胞球的毒性作用。结果细胞球理想接种条件为每微孔500个细胞,细胞比例为HepG2-Huvec-LX-2=8∶1∶1,圆度为0.91±0.07,坚实度为0.91±0.02,长径比为1.12±0.14,直径为(170.97±14.79)μm;第3、7、10和14天细胞球白蛋白(ALB)mRNA相对表达水平分别为1.00±0.02、0.96±0.02、0.54±0.07和0.52±0.07,细胞色素P4501A2(CYP1A2)mRNA相对表达水平分别为1.00±0.10、2.15±0.16、2.45±0.33和1.30±0.03。对照组和低、中、高剂量实验组多药耐药蛋白2(MRP2)mRNA相对表达水平分别为1.00±0.31、1.38±0.24、1.48±0.06和1.90±0.08,细胞球存活率分别为(98.19±0.49)%、(88.53±0.90)%、(71.60±2.91)%和(56.65±5.41)%。低、中、高剂量实验组上述指标与对照组比较,在统计学上差异均有统计学意义(均P<0.05)。结论建立的肝细胞球共培养模型中Ⅰ/Ⅱ相药物代谢酶、转运体、及肝细胞特异标志性分子白蛋白均有不同程度的表达,可用于中药何首乌的毒性评价,为何首乌的临床应用提供了参考。Objective To explore the toxicity ofPolygonum multiflorum alcohol extract on 3D hepatospheres.Methods Variations in culture conditions and cell ratios were implemented,followed by the assessment of cell sphere diameter,density,and roundness,aiming to explore the optimal culture conditions.The 3D hepatocyte spheres were divided into control group and experimental-L,-M,-H groups.The experimental-L,-M,-H groups were treated with 0.25,1.00 and 2.50 mg·mL^(-1)Polygounm multiforumalcohol extract,and the control group was given the same amount of culture medium.The cell viability of the cell spheroids was tested by CellTiter-Glo reagent,the expression level of liver function related genes was detected by fluorescent quantitative polymerase chain reaction(RT-qRCR).The toxicity of cell spheres was detected by double fluorescent staining of living and dead cells.Results The ideal culture condition of cell sphere was 500 cells per micropore,and the cell ratio was HepG2-Huvec-LX-2=8∶1∶1.It displayed the values of 0.91±0.07 for circularity,0.91±0.02 for firmness,1.12±0.14 for aspect ratio,and(170.97±14.79)μm for diameter.On the 3rd,7th,10th and 14th days,the expression levels of albumin(ALB)mRNA were 1.00±0.02,0.96±0.02,0.54±0.07,0.52±0.07,and the expression levels of cytochrome P4501A2(CYP1A2)mRNA were 1.00±0.10,2.15±0.16,2.45±0.33,1.30±0.03,respectively.The expression levels of multidrug resistance protein 2(MPR2)in the control group and the experimental-L,-M,-H groups were 1.00±0.31,1.38±0.24,1.48±0.06 and 1.90±0.08,respectively;spheroid viability were(98.19±0.49)%,(88.53±0.90)%,(71.60±2.91)%and(56.65±5.41)%.There were statistically significant differences in the above indexes between the experimental-L,-M,-H groups and the control group(all P<0.05).Conclusion The established hepatocyte sphere co-culture model showed varying degrees of expression of phaseⅠ/Ⅱdrug metabolism enzymes,transporters,and liver cell specific marker molecule albumin and can be used to evaluate the toxicity of multiflor

关 键 词：何首乌 肝毒性模型 共培养 3D细胞球 

分 类 号：R28[医药卫生—中药学]