Neurobiology of Selenium, Institute for Experimental Endocrinology, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.
J Trace Elem Med Biol. 2010 Apr;24(2):130-7. doi: 10.1016/j.jtemb.2010.01.007. Epub 2010 Feb 2.
Serum-free media require a number of supplements in order to support long-term neuronal survival. Commercially available B27, in combination with Neurobasal medium, supports neuronal survival and suppresses glial proliferation. However, B27 contains many biological antioxidants as well as catalase and superoxide dismutase, eventually demanding the application of unphysiologically high peroxide concentrations in survival assays. Moreover, optimal amounts of selenium (Se) are included in "B27 supplement minus antioxidants", a commercially available supplement used for the study of the role of antioxidants. Hence, Se-dependent enzymes like glutathione peroxidase are maximally expressed when this supplement is used and Se-depletion studies are not possible without changing the medium composition. We have therefore developed a modified serum-free media supplement which allows for free variation of all constituents. Our supplement was comparable to B27 with regard to cell survival and expression of neurochemical markers. Reduction of Se content in the supplement reduced selenoprotein expression and made cortical neurons more sensitive towards challenges with peroxides. Withdrawal from the medium supplement of vitamin E alone did not alter the survival of neurons in response to peroxides, while simultaneous reduction of Se and vitamin E rendered neurons hypersensitive towards peroxide challenge. This finding implied that adequate Se supply of neurons is required to minimize lipid peroxidation. Our medium supplement is easily prepared, inexpensive, and should be applicable to the analysis of survival mechanisms beyond peroxide challenge.
无血清培养基需要添加多种补充剂才能支持神经元的长期存活。商品化的 B27 与 Neurobasal 培养基结合使用可支持神经元存活并抑制神经胶质细胞增殖。然而，B27 含有许多生物抗氧化剂以及过氧化氢酶和超氧化物歧化酶，最终需要在存活测定中应用不生理的高过氧化物浓度。此外，“B27 补充剂减去抗氧化剂”中包含了最佳量的硒 (Se)，这是一种用于研究抗氧化剂作用的市售补充剂。因此，当使用这种补充剂时，依赖 Se 的酶如谷胱甘肽过氧化物酶的表达达到最大值，而如果不改变培养基成分，则无法进行 Se 耗竭研究。因此，我们开发了一种改良的无血清培养基补充剂，可自由改变所有成分。我们的补充剂在细胞存活和神经化学标志物表达方面与 B27 相当。补充剂中 Se 含量的减少降低了硒蛋白的表达，使皮质神经元对过氧化物的挑战更加敏感。单独从培养基补充剂中去除维生素 E 不会改变神经元对过氧化物的存活反应，而同时减少 Se 和维生素 E 会使神经元对过氧化物的挑战更加敏感。这一发现意味着神经元需要充足的 Se 供应才能最大程度地减少脂质过氧化。我们的培养基补充剂易于制备、价格低廉，并且应该适用于分析过氧化物挑战以外的存活机制。
