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目的建立具有高活力、高功能特性的原代小鼠肝细胞模型,并通过该体外模型评价受试物苯乙烯和氧化苯乙烯的急性毒性。方法以BABL/C小鼠作为肝细胞供者,在经典两步胶原酶消化法基础上进行优化,通过逆向灌流、间断灌注、限制消化时间及Percoll液离心纯化的方式获取小鼠肝细胞,并进行体外单层和夹层培养;通过细胞形态、细胞活力、胞内糖原颗粒以及上清中各指标即白蛋白(ALB)、乳酸脱氢酶(LDH)、丙氨酸氨基转移酶(ALT)及尿素氮(BUN)变化综合评价原代培养的肝细胞模型;以苯乙烯和氧化苯乙烯为受试物,用浓度分别为0.2、1、5、10和25μmol/L的受试物作用于夹层培养3 d的肝细胞,于染毒3、6、12、24及48 h后,采用CCK-8法和LDH法测定细胞存活率。结果改良法分离获取的小鼠肝细胞活力为(90.3±5.2)%,纯度为(95.3±4.2)%,产量达(2.4±0.9)×107;夹层培养7 d内90%以上细胞呈典型的肝细胞形态特征,培养第3 d胞浆内可见大量糖原颗粒。ALB分泌、LDH和ALT漏出、BUN合成、以及细胞活力夹层培养在8 d内、单层培养在6 d内呈波动性变化,且夹层培养法各指标明显优于单层培养法;夹层培养第3 d ALB分泌量[(1.42±0.20)g/L]和BUN合成量[(1.97±0.22)mmol/L]及细胞活力均达峰值,而LDH漏出量[(7.30±2.33)U/L]和ALT漏出量[(6.51±1.86)U/L]降到谷值,且3~7 d各指标变化相对稳定。苯乙烯和氧化苯乙烯在染毒6 h内,对肝细胞显示了较低的细胞毒性,细胞存活率在90%以上,且CCK-8和LDH两种毒性测定方法之间无显著差异;随着染毒时间的进一步延长,CCK-8法检测出的细胞存活率更低(85%以下),与LDH法相比差异有统计学意义。用不同浓度受试物处理肝细胞24 h后,从5μmol/L开始观察到相对较高的细胞毒性,用CCK-8法检测出细胞存活率约为85%,但与LDH法相比无显著差异;随着染毒浓度的继续增加,CCK-8法检测出的细胞存活率更低(80%以下),与LDH法相比差异有统计学意义。结论改良的胶原酶消化法结合夹层培养法可使肝细胞在较长时间(7 d)内维持良好的形态和功能,且用夹层培养3~7 d的肝细胞模型可以较准确地评价苯乙烯和氧化苯乙烯的毒性效应,结合毒性检测指标推测受试物主要影响肝细胞内线粒体亚细胞器的功能,对肝细胞膜的损伤程度影响较弱。
OBJECTIVE: To establish a primary mouse hepatocyte model with high activity and high functional properties and evaluate the acute toxicity of styrene and styrene oxide by the in vitro model. Methods BABL / C mice were used as donors of hepatocytes and optimized by the classic two-step collagenase digestion method. Mouse hepatocytes were obtained by reverse perfusion, intermittent perfusion, restriction digestion and Percoll centrifugation. In vitro monolayers and mesenchymal cultures were cultured. By means of cell morphology, cell viability, intracellular glycogen granules and various indicators of supernatant, such as albumin (ALB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) And urea nitrogen (BUN) were used to evaluate the primary cultured hepatocytes model. Styrene and styrene oxide were used as the test substance, and the test substances at concentrations of 0.2, 1, 5, 10 and 25 μmol / L Membranes were cultured for 3 days. After 3, 6, 12, 24 and 48 hours of exposure, cell viability was determined by CCK-8 assay and LDH assay. Results The results showed that the viability of hepatocytes was (90.3 ± 5.2)%, the purity was (95.3 ± 4.2)% and the yield was (2.4 ± 0.9) × 107. The more than 90% Hepatocyte morphological features, a large number of glycogen particles can be seen within 3 days after culture. ALB secretion, leakage of LDH and ALT, BUN synthesis, and cell viability cultured in 8 d, monolayer culture fluctuated within 6 d, and the indicators of sandwich culture were significantly better than those of monolayer culture; (1.42 ± 0.20) g / L], and the amount of BUN [(1.97 ± 0.22) mmol / L] and cell viability reached its peak at 3 d, while the leakage of LDH [(7.30 ± 2.33) U / L] And ALT leakage [(6.51 ± 1.86) U / L] dropped to the bottom, and the change of every index from 3 to 7 days was relatively stable. Styrene and styrene oxide showed less cytotoxicity on hepatocytes within 6 h after exposure, with cell viability above 90%. There was no significant difference between CCK-8 and LDH assays With the further extension of the exposure time, the cell survival rate detected by the CCK-8 method was lower (85% or less), which was significantly different from that of the LDH method. After treating hepatocytes with different concentration of test substance for 24 h, relatively high cytotoxicity was observed from 5 μmol / L, and cell viability was about 85% with CCK-8 assay, but no significant difference compared with LDH assay ; With the continuous increase of exposure concentration, the cell survival rate detected by CCK-8 method was lower (less than 80%), which was significantly different from LDH method. Conclusions Modified collagenase digestion combined with sandwich culture method can maintain good morphology and function of hepatocytes for a long time (7 days), and the hepatic cell model cultured for 3-7 days with sandwich can accurately evaluate the effect of styrene And the toxicity of styrene oxide toxicity, combined with toxicity test speculated that the test substance mainly affects the function of mitochondrial subcellular organelles in hepatocytes, the degree of damage to the liver cell membrane less affected.