Sasaki K, Kitaguchi Y, Fukuda T, Aoyagi Y
Department of Animal Products, National Institute of Animal Industry, Tsukuba Norindanchi PO Box 5, Ibaraki 305-0901, Japan.
Int J Biochem Cell Biol. 2000 Sep;32(9):967-73. doi: 10.1016/s1357-2725(00)00043-1.
Chicken liver is lack of ascorbic acid biosynthesis system, different from mammals and highly evoluted birds. Chicken hepatocytes cultured without ascorbate was expected to have lower ascorbate amounts than physiological levels. Intracellular was decreased as compared with intact liver by cell preparation performed with in situ collagenase perfusion. We added ascorbate to a primary culture of chicken hepatocytes in order to restore the amount of ascorbate. Serum-free Leivobitz's L-15 medium which do not contain ascorbate was used for control medium. Cells were cultured with several concentrations of ascorbate for 24 or 48 h. After ascorbate supplementation for 24 to 48 h, cellular ascorbate concentration increased depending on the dose of medium ascorbate. Medium lactate dehydrogenase activity derived from hepatocytes, an index of cell injury, decreased upon 5-100 mg/l of ascorbate supplementation for 48 h. Tyrosine aminotransferase activity, an index of liver function, increased following culture with 50 and 100 mg/l ascorbate for 48 h. The activities, however, decreased by supplementation with 1000 mg/l of ascorbate. In conclusion hepatocytes lost intracellular ascorbate during preparation by in situ collagenase perfusion. Supplementation of ascorbate restored cellular ascorbate concentration, lowered cell injury and raised tyrosine aminotransferase activitv in primary cultured chicken hepatocytes. Ascorbate treatment for 48 h at 50 mg/l was the best combination in this study for primary culture of chicken hepatpcyte with non-serum L-15 medium
鸡肝缺乏抗坏血酸生物合成系统，这与哺乳动物和高度进化的鸟类不同。预计在无抗坏血酸条件下培养的鸡肝细胞的抗坏血酸含量低于生理水平。通过原位胶原酶灌注进行细胞制备后，与完整肝脏相比，细胞内抗坏血酸含量降低。我们向鸡肝细胞原代培养物中添加抗坏血酸以恢复抗坏血酸含量。不含抗坏血酸的无血清Leivobitz's L - 15培养基用作对照培养基。细胞用几种浓度的抗坏血酸培养24或48小时。在补充抗坏血酸24至48小时后，细胞内抗坏血酸浓度根据培养基中抗坏血酸的剂量而增加。作为细胞损伤指标的来自肝细胞的培养基乳酸脱氢酶活性，在补充5 - 100mg / l抗坏血酸48小时后降低。作为肝功能指标的酪氨酸转氨酶活性，在使用50和100mg / l抗坏血酸培养48小时后增加。然而，补充1000mg / l抗坏血酸后该活性降低。总之，在原位胶原酶灌注制备过程中，肝细胞失去了细胞内抗坏血酸。补充抗坏血酸可恢复细胞内抗坏血酸浓度，降低细胞损伤并提高鸡肝细胞原代培养物中的酪氨酸转氨酶活性。在本研究中，对于使用无血清L - 15培养基的鸡肝细胞原代培养，50mg / l抗坏血酸处理48小时是最佳组合。
