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目的:研究齐多夫定对原代大鼠肝细胞的毒性。方法:采用二步灌流法分离Wistar大鼠肝细胞,经台盼蓝拒染试验测定细胞活力,活力>85%用于实验。实验分4组:药物组、阳性对照组、阴性对照组和空白对照组。肝细胞悬液(3×105个/ml)接种于各培养板:96孔板每孔200μl,24孔板每孔1ml,6孔板每孔2.5ml,各板置37℃、5%二氧化碳培养箱培养4h,弃上清液。药物组分别加入齐多夫定10、5、3.3、2、0.4、0.08mmol/L,阳性对照组分别加入四氯化碳10、2、0.5mmol/L,阴性对照组加入含1%二甲基亚砜(DMSO)的DMEM培养液,空白对照组加入DMEM培养液。96孔板于加样后6、12、24h进行MTT试验测定细胞活性,24孔板于加样后6、12h测定肝细胞AST、ALT及LDH的释放量,6孔板加样后12h将细胞苏木素-伊红(HE)染色,观察细胞形态。结果:齐多夫定浓度为3.3mmol/L、四氯化碳浓度为2mmol/L时,于6、12、24h的吸光度(OD)值分别为(1.20±0.17)、(0.99±0.08)和(0.89±0.09)与(1.20±0.13)、(1.01±0.09)和(0.88±0.13),同时间点阴性对照组OD值分别为(1.34±0.08)、(1.11±0.10)和(1.03±0.11)。与阴性对照组比较,齐多夫定组、四氯化碳组的细胞活力均显著降低,差异均有统计学意义(均P<0.05),但10mmol/L齐多夫定对细胞活性的影响远低于10mmol/L四氯化碳。加3.3mmol/L齐多夫定后6和12h,AST释放量为(18.53±2.02)卡门单位和(26.86±2.61)卡门单位,ALT的释放量为(15.16±2.18)卡门单位和(27.48±2.27)卡门单位,LDH释放量为(1681.00±193.98)U/L和(2708.55±78.73)U/L;阴性对照组同时间点AST释放量为(15.91±1.62)卡门单位和(37.71±2.54)卡门单位,ALT释放量为(19.66±0.74)卡门单位和(23.42±1.46)卡门单位,LDH释放量为(2036.39±134.56)U/L和(2870.21±87.73)U/L;与阴性对照组比较,齐多夫定对AST、ALT及LDH的释放量均有显著影响,差异均有统计学意义(均P<0.05)。齐多夫定浓度≥3.3mmol/L时,可引起细胞膜破裂、核浓缩,四氯化碳浓度为10mmol/L时,可使细胞变小、细胞膜破裂及核碎裂。结论:齐多夫定浓度≥3.3mmol/L时,具有肝细胞毒性作用,齐多夫定的细胞毒性低于四氯化碳。
Objective: To study the toxicity of zidovudine on primary rat hepatocytes. METHODS: Wistar rat hepatocytes were isolated by two-step perfusion method. Cell viability and viability> 85% were determined by trypan blue exclusion assay. The experiment was divided into 4 groups: drug group, positive control group, negative control group and blank control group. Hepatocyte suspension (3 × 10 5 cells / ml) was inoculated into each culture plate: 200 μl per well of a 96-well plate, 1 ml per well of a 24-well plate, 2.5 ml per well of a 6-well plate, Box cultured 4h, the supernatant was discarded. Drug groups were added zidovudine 10,5,3.3,2,0.4,0.08 mmol / L, the positive control group were added carbon tetrachloride 10,2,0.5mmol / L, the negative control group added with 1% dimethyl Sulfoxide (DMSO) in DMEM medium and blank control group were added DMEM medium. The cell viability was measured by MTT assay at 6, 12, and 24 h after the addition of 96-well plates. The release of AST, ALT and LDH of hepatocytes was measured at 24 and 12 h after the addition in the 24- Hematoxylin-eosin (HE) staining, observed cell morphology. Results: The absorbance (OD) values at 6, 12 and 24 h were (1.20 ± 0.17) and (0.99 ± 0.08) and (2) when the concentration of zidovudine was 3.3 mmol / L and the concentration of carbon tetrachloride was 2 mmol / (1.89 ± 0.09) and (1.20 ± 0.13), (1.01 ± 0.09) and (0.88 ± 0.13) respectively in the control group at the same time point.The OD values in the negative control group were (1.34 ± 0.08), (1.11 ± 0.10) and ). Compared with the negative control group, the cell viability of zidovudine group and carbon tetrachloride group were significantly decreased (all P <0.05), but the effect of 10 mmol / L zidovudine on cell activity Well below 10 mmol / L carbon tetrachloride. At 6 and 12 h after the administration of 3.3 mmol / L zidovudine, the release of AST was (18.53 ± 2.02) and (26.86 ± 2.61) and the release of ALT was (15.16 ± 2.18) and (27.48 ± 2.27), and the release of LDH was (1681.00 ± 193.98) U / L and (2708.55 ± 78.73) U / L respectively in the Carmen unit. The positive rate of AST release in the negative control group was (15.91 ± 1.62) (19.66 ± 0.74) Carmen units and (23.42 ± 1.46) Carmen units, respectively. The release of LDH was (2036.39 ± 134.56) U / L and (2870.21 ± 87.73) U / L, respectively. Compared with the negative control group , And zidovudine had significant effects on the release of AST, ALT and LDH (all P <0.05). Zidovudine concentration ≥ 3.3mmol / L, can cause cell membrane rupture, nuclear concentration, carbon tetrachloride concentration of 10mmol / L, can make cells smaller, cell membrane rupture and nuclear fragmentation. Conclusion: Zidovudine concentration ≥3.3mmol / L, with hepatotoxicity, zidovudine cytotoxicity lower than carbon tetrachloride.