Liu Mingyue, Oyarzabal Esteban A, Yang Rui, Murphy Stephanie J, Hurn Patricia D
Department of Anesthesiology & Peri-Operative Medicine, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, UHS-2, Portland, OR, 97239-3098, USA.
J Neurosci Methods. 2008 Jun 30;171(2):214-7. doi: 10.1016/j.jneumeth.2008.03.002. Epub 2008 Mar 18.
Female astrocytes sustain less cell death from oxygen-glucose deprivation (OGD) than male astrocytes. Arimidex, an aromatase inhibitor, abolishes these sex differences. To verify sex-dependent differences in P450 aromatase function in astrocyte cell death following OGD, we developed a novel method that uses sex-specific and genotype-specific single pup primary astrocyte cultures from wild-type (WT) and aromatase-knockout (ArKO) mice. After determining sex by external and internal examination as well as PCR and genotype by PCR amplification of tail cDNA, we established cultures from 1-3-day-old male and female WT and ArKO mice pups and grew them to confluence in estrogen-free media. Cell death was measured by lactate dehydrogenase (LDH) assay. Our study shows that, while WT female astrocytes are more resistant to OGD than WT male cells, sex differences disappear in ArKO cells. Cell death is significantly increased in ArKO compared to WT in female astrocytes but not male cells. Therefore, P450 aromatase appears to be essential in endogenous neuroprotection in females, and this finding may have clinical implications. This innovative technique may also be applied to other in vitro studies of sex-related functional differences.
与雄性星形胶质细胞相比，雌性星形胶质细胞在氧糖剥夺（OGD）条件下的细胞死亡较少。芳香化酶抑制剂阿那曲唑可消除这些性别差异。为了验证在OGD后星形胶质细胞死亡过程中P450芳香化酶功能的性别依赖性差异，我们开发了一种新方法，该方法使用来自野生型（WT）和芳香化酶基因敲除（ArKO）小鼠的性别特异性和基因型特异性单只幼崽原代星形胶质细胞培养物。通过外部和内部检查以及PCR确定性别，并通过尾cDNA的PCR扩增确定基因型后，我们从1 - 3日龄的雄性和雌性WT及ArKO小鼠幼崽中建立培养物，并在无雌激素培养基中培养至汇合。通过乳酸脱氢酶（LDH）测定法测量细胞死亡。我们的研究表明，虽然WT雌性星形胶质细胞比WT雄性细胞对OGD更具抗性，但在ArKO细胞中性别差异消失。与WT相比，ArKO雌性星形胶质细胞的细胞死亡显著增加，而雄性细胞则不然。因此，P450芳香化酶似乎对雌性内源性神经保护至关重要，这一发现可能具有临床意义。这种创新技术也可能应用于其他与性别相关的功能差异的体外研究。
