目的 介紹大鼠及小鼠 Pig-a基因突變試驗方法，并匯總國家藥物安全評價監(jiān)測中心 2015—2022年開展的基于免疫磁珠檢測法的大鼠及小鼠 Pig-a 基因突變試驗背景數(shù)據(jù)。方法 陰性物質(zhì)包括超純水和 0.5% 羧甲基纖維素鈉（CMCNa），雄性 C57BL/6J 小鼠間隔 24 h ig 0.5% CMC-Na，連續(xù) 7 d；雄性 SD 大鼠間隔 24 h ig 0.5% CMC-Na，連續(xù) 14 d；大鼠間隔24 h ig 超純水，連續(xù) 3 d。陽性對照為已知致細菌突變化合物，包括 N-乙基-N-亞硝基脲（ENU，10、40 mg·kg^-1）、鹽酸丙卡巴肼（PCZ，60、150 mg·kg^-1）、烏拉坦（EC，300、800 mg·kg^-1）、N- 亞硝基二甲胺（NDMA ，1.5 mg·kg^-1）、N- 亞硝基二乙胺（NDEA，15 mg·kg^-1）。小鼠間隔 24 h ig ENU 40 mg·kg^-1，連續(xù) 3 d；間隔 24 h ig NDMA 1.5 mg·kg^-1、NDEA 15 mg·kg^-1，連續(xù) 7 d。大鼠間隔 24 h ig PCZ 150 mg·kg^-1、EC 800 mg·kg^-1、ENU 40 mg·kg^-1，連續(xù) 3 d ；間隔 24 h ig PCZ 60 mg·kg^-1、EC300 mg·kg^-1、ENU 10 mg·kg^-1，連續(xù)28 d。分別于給予受試物前，首次給予后14、28 d采集外周血，用流式細胞術(shù)檢測大鼠紅細胞表面 CD59蛋白的結(jié)合情況，結(jié)合免疫磁性計數(shù)微球技術(shù)計算網(wǎng)織紅細胞（RETs）占總紅細胞的百分率（%RET）（作為外周血毒性考察指標）、總紅細胞中CD59表達為陰性細胞（RBC^CD59-，即突變的總紅細胞）發(fā)生率和RETs中CD59表達為陰性細胞（RET^CD59-，即突變的 RETs）發(fā)生率。結(jié)果 各試驗%RET數(shù)值均無大幅增加。SD大鼠和 C57BL/6J 小鼠的陰性對照組RBC^CD59-和 RET^CD59-突變率均低于 5×10-6，小鼠的背景值相對不穩(wěn)定。連續(xù) 3 d ig給予小鼠 40 mg·kg^-1的 ENU，RBC^CD59-和RET^CD59-發(fā)生率自給藥后2周開始均大幅增加（P＜0.05），給藥后4周進一步增加（P＜0.01、0.001）；給予小鼠NDMA后2、4周，RBC^CD59-發(fā)生率略有增加，但仍在陰性背景范圍內(nèi)，但RET^CD59-發(fā)生率在給藥后第2周大幅增加（P＜0.001），給藥后第4周則大幅回落；給予小鼠NDEA后2周，RBC^CD59-和RET^CD59-發(fā)生率均有所增加（P＜0.05、0.001），給藥后第 4 周則有所降低。連續(xù)3 d ig給予大鼠40 mg·kg^-1 ENU，或連續(xù)28 d ig給予大鼠10 mg·kg^-1 ENU，RBC^CD59-、RET^CD59-發(fā)生率自給藥后第2周開始均大幅增加（P＜0.001），給藥后第 4 周進一步增加（P＜0.001）；連續(xù) 3、28 d ig 給予大鼠不同劑量的 PCZ 或 EC 后，RBC^CD59-和RET^CD59-發(fā)生率的變化趨勢與 ENU類似，但 EC誘發(fā)的突變細胞率低于 ENU和 PCZ。結(jié)論 體內(nèi) Pig-a基因突變試驗可在首次給藥后4周內(nèi)有效檢出致細菌突變化合物ENU、PCZ、EC、NDMA、NDEA的致突變性。提供了大鼠和小鼠Pig-a基因突變試驗的背景值范圍，為標準化試驗方法的建立和研究結(jié)果的判定提供借鑒。

Objective The method of Pig-a gene mutation assay in rats and mice was introduced, and the background data based on immunomagnetic beads carried out by the National Center for Safety Evaluation of Drugs from 2015 to 2022 were summarized.Methods The negative substances included ultrapure water and 0.5% carboxymethyl cellulose sodium (CMC-Na). Male C57BL/6J mice received 0.5% CMC-Na at an interval of 24 h for seven consecutive days. Male SD rats were given 0.5% CMC-Na at an interval of 24 h for 14 consecutive days. Rats were given ultrapure water at an interval of 24 hours for three consecutive days.Positive controls were known bacterial mutagenic compounds, including N-ethyl-N-nitrosourea (ENU, 10 and 40 mg·kg^-1), procarbazide hydrochloride (PCZ, 60 and 150 mg·kg^-1), uratan (EC, 300 and 800 mg·kg^-1), N-nitrosodimethylamine (NDMA, 1.5 mg·kg^-1), and N-nitrosodiethylamine (NDEA, 15 mg·kg^-1). Mice were given ENU 40 mg·kg^-1 at an interval of 24 h for three consecutive days, and given NDMA 1.5 mg·kg^-1 and NDEA 15 mg·kg^-1 at an interval of 24 h for seven consecutive days. Rats were given PCZ 150 mg·kg^-1, EC 800 mg·kg^-1, and ENU 40 mg·kg^-1 at 24 hour intervals for three consecutive days, and given PCZ 60 mg·kg^-1, EC 300 mg·kg^-1, and ENU 10 mg·kg^-1 at 24 hour intervals for 28 consecutive days. Peripheral blood was collected before the administration of the test substance, 14 and 28 days after the first administration, and the binding of CD59 protein on the surface of rat red blood cells was detected by flow cytometry. The percentage of reticulocyte (RETs) in total red blood cells (%RET, as an indicator for peripheral blood toxicity), the incidence of CD59 expression in total red blood cells (RBC^CD59-, i.e. mutated total red blood cells) and the incidence of CD59 expression in RETs (RET^CD59- , i. e. mutated RETs) were calculated by using immunomagnetic counting microsphere technology.Results There was no significant increase in the % RET values for each experiment. The spontaneous RBC^CD59- and RET^CD59- mutation rates were lower than 5×10-6 in SD rats and C57BL/6J mice. The background values of mice are relatively unstable. After three consecutive days of ig administration of 40 mg·kg^-1 of ENU to mice, the incidence of RBC^CD59- and RET^CD59- increased significantly from two weeks after administration (P < 0.05), and further increased at four weeks after administration (P < 0.01 and 0.001). After two and four weeks of administration of NDMA to mice, the incidence of RBC^CD59- slightly increased, but remained within the negative background range. However, the incidence of RET^CD59- increased significantly (P < 0.001) in the second week after administration and decreased significantly in the fourth week after administration. The incidence of RBC^CD59- and RET^CD59- increased two weeks after administration of NDEA to mice (P < 0.05 and 0.001), but decreased four weeks after administration. The incidence of RBC^CD59- and RET^CD59- increased significantly (P < 0.001) after three consecutive days of ig administration of 40 mg·kg^-1 ENU to rats, or 28 consecutive days of ig administration of 10 mg·kg^-1 ENU to rats, starting from the 2nd week after administration and further increasing at the 4th week after administration. After continuous administration of different doses of PCZ or EC to rats for 3 and 28 days, the trend of changes in the incidence of RBC^CD59- and RET^CD59- was similar to that of ENU, but the mutation cell rate induced by EC was lower than that of ENU and PCZ.Conclusion In vivo Pig-a gene mutation assay can effectively detect the mutagenicity of bacterial mutant compounds ENU, PCZ, EC, NDMA, NDEA within four weeks after the first administration. This study provides the background value range of Pig-a gene mutation assay in rats and mice, which provides reference for the establishment of standardized test methods and the determination of research results.

R965.3

國家十三五“重大新藥創(chuàng)制”專項課題（2018ZX09201017）；國家自然科學基金資助項目（81503347）