腺苷酸活化蛋白激酶在星形胶质细胞中的表达

Expression of Adenosine Monophosphate-activated Protein Kinase in Cultured Rat Astrocytes

目的通过检测腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)α1、α2、β1、β2、γ1及γ2亚基在培养大鼠星形胶质细胞中的表达,探讨AMPK在星形胶质细胞中以何种形式组成三聚体。方法取新生1 d的Wistar大鼠脑皮层进行星形胶质细胞培养,用胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和S100β免疫荧光染色鉴定星形胶质细胞。应用反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)法检测培养星形胶质细胞中AMPK各亚基的表达情况。结果 GFAP和S100β细胞免疫荧光染色显示,培养细胞95%以上为免疫荧光染色阳性细胞。RT-PCR实验结果显示培养的星形胶质细胞中有AMPKα1、α2、β1、γ1及γ2亚基的特异性目的条带出现,没有AMPKβ2亚基目的条带出现;在出现的5种特异性目的条带中,AMPKα2、β1及γ1亚基目的条带表达相对较多,而AMPKα1及γ2亚基只有微弱表达。结论星形胶质细胞中AMPK由α2、β1及γ1亚基组成三聚体,在星形胶质细胞能量代谢调节中发挥重要作用。

Objective To explore the way of adenosine monophosphate-activated protein kinase （AMPK） forming into heterotrimer in astrocytes through detecting the expression of AMPK α1, α2, β1, β2, γ1 and γ2 subunits in cultured rat astrocytes. Methods Primary cultured astrocytes derived from the cerebral cortex of one-day-old Wistar rats. The purity of cell culture was assessed by immunofluorescence staining for anti-glial fibrillary acidic protein （GFAP） and S100β antibody. The expression of AMPK subunits were detected through reverse transcription polymerase chain reaction （RT-PCR） in cultured astrocytes. Results Immunofluorescence staining revealed that over 95% of cells were GFAP and S100β-positive, and the monolayer cultures of astrocytes were presented as uniform shapes under the phase contrast microscopic. The results from RT-PCR showed that the five subunits including AMPK α1, α2,β1, γ1 and γ2 were found in the cultured astrocytes. The expression of AMPK α2, β1 and γ1 subunits were relatively high, and AMPK α1 and γ2 subunit expression were very weak. Conclusion AMPK heterotrimer is composed of α2, β1 and γ1 subunits in astrocytes, and plays a key role in the regulation of energy metabolism in astrocytes.