From in vivo to in vitro: Major metabolic alterations take place in hepatocytes during and following isolation.
From in vivo to in vitro: Major metabolic alterations take place in hepatocytes during and following isolation.
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DOI：
10.1371/journal.pone.0190366
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Bilodeau M
中科院分区：
文献类型：
--
作者：
Cassim S;Raymond VA;Lapierre P;Bilodeau M
肝脏在维持生理动态平衡方面起着关键作用，而肝细胞在很大程度上对此负责。利用分离的原代肝细胞已经成为研究营养生理学、异体代谢和一些肝脏病理的重要工具。由于肝细胞被从其正常环境中移除，原代肝细胞的分离程序和体外培养在一定程度上会引起不必要的代谢变化。我们的目标是使用最先进的技术在分离前、分离期间和分离后对原代细胞进行彻底的代谢谱分析。在使用两步胶原酶灌注法分离肝细胞的过程中，以及在长达48小时的体外细胞培养过程中，使用高效液相色谱法进行了广泛的代谢物测量。用细胞外流量分析评估分离的原代肝细胞的线粒体呼吸能力和与ATP相关的呼吸。与原位肝脏相比，原代肝细胞在分离过程中显示出与抗氧化相关的代谢产物(NADPH、NADP、GSH和GSSG)的急剧下降(P<；0.001)。平行的柠檬酸循环活性评估显示，与原位肝脏相比，乙酰辅酶A、异柠檬酸/柠檬酸比值、琥珀酸、富马酸和苹果酸的含量降低了95%(P<；0.001)。虽然在分离过程和细胞培养过程中，几种细胞能量代谢产物如腺苷、腺苷、腺苷二磷酸和三磷酸腺苷的水平逐渐下降(P<；0.001)，但体外培养的原代细胞的三磷酸腺苷/腺苷二磷酸比值和能量电荷水平高于原位肝细胞(P<；0.05)。此外，培养24小时后，呼吸量显著下降。有趣的是，这与三磷酸腺苷相关呼吸的显著改变无关。总而言之，主要的代谢变化在肝细胞从肝脏中移除后立即发生。这些变化在体外培养过程中持续或增加。当使用原代肝细胞研究新陈代谢或肝脏生理病理时，需要考虑这些观察。
The liver plays a key role in maintaining physiological homeostasis and hepatocytes are largely responsible for this. The use of isolated primary hepatocytes has become an essential tool for the study of nutrient physiology, xenobiotic metabolism and several liver pathologies. Since hepatocytes are removed from their normal environment, the isolation procedure and in vitro culture of primary hepatocytes is partially known to induce undesired metabolic changes. We aimed to perform a thorough metabolic profiling of primary cells before, during and after isolation using state-of-the-art techniques. Extensive metabolite measurements using HPLC were performed in situ in the liver, during hepatocyte isolation using the two-step collagenase perfusion method and during in vitro cell culture for up to 48 hours. Assessment of mitochondrial respiratory capacity and ATP-linked respiration of isolated primary hepatocytes was performed using extracellular flux analysis. Primary hepatocytes displayed a drastic decrease in antioxidative-related metabolites (NADPH, NADP, GSH and GSSG) during the isolation procedure when compared to the in situ liver (P<0.001). Parallel assessment of citric acid cycle activity showed a significant decrease of up to 95% in Acetyl-CoA, Isocitrate/Citrate ratio, Succinate, Fumarate and Malate in comparison to the in situ liver (P<0.001). While the levels of several cellular energetic metabolites such as Adenosine, AMP, ADP and ATP were found to be progressively reduced during the isolation procedure and cell culture (P<0.001), higher ATP/ADP ratio and energy charge level were observed when primary cells were cultured in vitro compared to the in situ liver (P<0.05). In addition, a significant decrease in the respiratory capacity occurred after 24 hours in culture. Interestingly, this was not associated with a significant modification of ATP-linked respiration. In conclusion, major metabolic alterations occur immediately after hepatocytes are removed from the liver. These changes persist or increase during in vitro culture. These observations need to be taken into account when using primary hepatocytes for the study of metabolism or liver physiopathology.
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