雷公藤甲素致L5178Y细胞及小鼠Pig-a基因突变风险评价  

Risk Assessment of Pig-a Gene Mutation Risk on L5178Y Cell and Mice Induced by Triptolide

作　　者：王亚楠 闫明 汪祺[1] 文海若 WANG Ya-nan;YAN Ming;WANG Qi;WEN Hai-ruo(National Institutes for Food and Drug Control,Beijing 100050,China;China Pharmaceutical University,Nanjing 210009,China)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]中国药科大学,江苏南京210009

出　　处：《中国现代中药》2020年第10期1630-1637,共8页Modern Chinese Medicine

基　　金：国家自然科学基金项目(81503347);国家十三五“重大新药创制”专项(2018ZX09201-017)。

摘　　要：目的:评价雷公藤甲素(TPL)的潜在致Pig-a基因突变风险。方法:采用小鼠淋巴瘤L5178Y细胞分别进行非S9(-S9)代谢和S9(+S9)代谢条件下体外实验。-S9代谢条件实验中,L5178Y细胞分为对照组(1%二甲基亚砜)、阳性对照甲基磺酸乙酯(EMS,500μg·mL-1)组和TPL各质量浓度(6.3、12.5、25.0、50.0、100.0 ng·mL^-1)组,受试物处理24 h后细胞计数,更换培养基培养8 d,表达结束后进行流式检测。+S9代谢条件实验中,L5178Y细胞分为对照组(1%二甲基亚砜)、阳性对照苯并芘[B(a)P,5μg·mL^-1]组和TPL各质量浓度(12.5、25.0、50.0、100.0、200.0 ng·mL^-1)组,受试物处理4 h后更换培养基,24 h后计数,培养8 d,表达结束后进行流式检测。体内Pig-a基因突变实验中,KM小鼠按体质量随机分为对照组(0.5%羧甲基纤维素钠)、阳性对照N-乙基-N-亚硝基脲(ENU,100 mg·kg^-1)组和TPL各剂量(50、100、200μg·kg^-1)组。ENU组仅于首次给药日给药1次,TPL各剂量组连续给药28 d,分别于给药前和首次给药后14、28、42、56 d于小鼠眼内眦采血,血样经缓冲液洗脱后,进行anti-CD24-PE和anti-CD61-PE标记,采用免疫磁性分离架进行柱前和柱后样品的收集,利用流式细胞仪对样本进行检测计数,对突变率进行统计分析。结果:体外Pig-a基因突变实验结果表明,在-S和+S 9代谢条件下,与对照组比较,TPL各剂量组Pig-a基因突变率差异无统计学意义,且无剂量依赖性。体内Pig-a基因突变实验结果显示,与对照组比较,TPL组小鼠红细胞(RBC)和网织红细胞(RTC)CD24缺失差异无统计学意义,且无剂量依赖性。结论:TPL质量浓度为200 ng·mL^-1及200μg·kg^-1以下时不会引起L5178Y细胞及KM小鼠发生Pig-a基因突变。Objective:To evaluate the potential genetic mutation risk of triptolide(TPL)by using in vitro/in vivo Pig-a gene mutation assay in L5178Y cells and KM mice,respectively.Methods:In vitro Pig-a gene mutation test:Metabolic conditions without S9:We set the control group(dimethyl sulfoxide,DMSO),the positive agent group(ethylmethylsulfone,EMS,500μg·mL^-1)and TPL group(6.3,12.5,25.0,50.0,100.0 ng·mL^-1).The cell was counted 24 hours after treatment,and the medium was changed for 8 days.The cell density was maintained at 1×10^6-2×10^6 cells/mL during the expression period,after that,testing was performed by flow cytometry.Metabolic conditions with S9:We set the control group(1% DMSO),positive agent group(benzo[a]pyrene,B(a)P,5μg·mL^-1),and TPL group(12.5,25.0,50.0,100.0,200.0 ng·mL^-1).The cell was counted 24 hours after treatment for 4 hours,and the medium was changed for 8 days.The cell density was maintained at 1×10^6-2×10^6 cells/mL during the expression period,after that,testing was performed by flow cytometry.In vivo Pig-a gene mutation test:We set the 0.5% CMC-Na group,the TPL group(50,100,200μg·kg^-1)and ENU group(100 mg·kg-1).ENU was administered only once on the first day of administration.The CMC-Na and TPL continuous administration for 28 days,respectively.Blood samples were collected on days 0,14,28,42 and 56 after the first administration in the eyes.Cell surface targets were labeled with the anti-CD24-PE and anti-CD61-PE and the pre-column and post-column samples were collected by flow cytometry.The number of Pig-a mutated cells in a million total and reticulocytes was counted and the mutation rate was analyzed.Results:In vitro Pig-a gene mutation test:There was no significant difference of TPL group in the frequency of Pig-a gene mutation compared with the vehicle control group with or without S9 metabolic activation,and no concentration gradient relationship.In vivo Pig-a gene mutation:In terms of lack of CD24 about red blood cell(RBC)and reticulocyte,there was no significant difference an

关 键 词：雷公藤甲素 PIG-A 基因突变 流式细胞术 KM小鼠 L5178Y细胞 

分 类 号：R285.5[医药卫生—中药学]