缺氧时星形胶质细胞定量mRNA表达的内部参照标准研究

目的:观察缺氧对脑星形胶质细胞(AC)守家基因甘油醛-3-磷酸-脱氢酶(GAPDH)和β-肌动蛋白(actin)mRNA表达的影响,进一步观察内皮素转换酶(ECE)-2mRNA可否作为该条件下的内部参照标准。方法:从新生小鼠大脑获原代AC培养,分别在缺氧和正常氧条件下培养24h,提取mRNA,以溴化乙啶染色的28S及18SrRNA作内部参照,用Northernblot杂交技术定量测定GAPDH、β-actin和ECE-2mRNA水平。结果:缺氧24h,ACGAPDHmRNA的表达显著增加,β-actinmRNA表达明显下降;缺氧前后ECE-2的mRNA水平无明显差异。结论:在缺氧缺血条件下,GAPDH和β-actin不宜作为AC定量mRNA分析的内部参照标准,ECE-2可作为其替代。

Internal standards for mRNA quantitative expression in hypoxic astrocytes

AIM: To investigate the effect of hypoxia on glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin and endothelin-converting enzyme (ECE)-2 mRNA levels in cultured mouse brain astrocytes (AC). METHODS:AC from neonatal mouse brain was incubated for 24h in serum-free medium under hypoxic or normoxic conditions.The amount of transferred RNA was estimated using ethidium bromide stained 28SrRNA and 18S rRNA.The levels of tested mRNA were evaluated by Northern blot RNA hybridizat ion.RESULTS: The GAPDH mRNA was up-regulated to 503.0% of the normoxic controls in hypoxic AC (n=10. P<0.01), while the level of β-actin mRNA in hypoxic AC was down-regulated to 50.3% of control levels (n=6. P<0.01). In contrast, the ECE-2 mRNA level was not affected by hypoxia. CONCLUSION: The results indicate that GAPDH and β-actin are not suitable for the internal standards for quantitative mRNA expression analysis in AC after hypoxia or ischemia. ECE-2 may function as an alternative in such circumstance.