CAJ | 학술논문

目的观察大鼠大脑皮质神经元细胞缺氧时促红细胞生成素(erythropoietin,EPO)及其mRNA表达的变化,探讨内源性EPO对缺氧神经元细胞的保护作用.方法制备体外培养大鼠大脑皮质神经元细胞缺氧模型,分为正常对照组；缺氧12、24、48、72 h组.利用免疫组织化学、逆转录聚合酶链反应及蛋白质印迹观察细胞中EPO及其mRNA的表达,并在变化过程中观察培养液中乳酸脱氢酶来评估神经元细胞的活性.结果免疫组织化学、逆转录聚合酶链反应及蛋白印迹检测显示:缺氧组中EPO(20.79±2.98)及其mRNA(0.78±0.05)在12 h已有基本表达,与对照组(EPO:17.12±1.99；mRNA:0.39±0.05)比较,差异均有统计学意义(t=2.51,P＜0.05；t=13.51,P＜0.01)；且48 h达高峰(EPO:28.88±3.41,mRNA:1.45±0.07),与对照组比较,差异有统计学意义(t =7.29,P＜0.叭；=33.24,P＜0.01)；神经元细胞活性也最强.乳酸脱氢酶活性72 h后表达明显下降,各时间组之间比较差异同样具有统计学意义.结论神经元细胞缺氧后使细胞内EPO及EPO mRNA的表达增强,且可增强神经元细胞的活性.提示EPO参与了神经元细胞缺氧的发生发展过程,在神经元细胞缺氧的过程中起重要作用.
목적 관찰대서대뇌피질신경원세포결양시촉홍세포생성소(erythropoietin,EPO)급기mRNA표체적변화,탐토내원성EPO대결양신경원세포적보호작용.방법 제비체외배양대서대뇌피질신경원세포결양모형,분위정상대조조；결양12、24、48、72 h조.이용면역조직화학、역전록취합매련반응급단백질인적관찰세포중EPO급기mRNA적표체,병재변화과정중관찰배양액중유산탈경매래평고신경원세포적활성.결과 면역조직화학、역전록취합매련반응급단백인적검측현시:결양조중EPO(20.79±2.98)급기mRNA(0.78±0.05)재12 h이유기본표체,여대조조(EPO:17.12±1.99；mRNA:0.39±0.05)비교,차이균유통계학의의(t=2.51,P＜0.05；t=13.51,P＜0.01)；차48 h체고봉(EPO:28.88±3.41,mRNA:1.45±0.07),여대조조비교,차이유통계학의의(t =7.29,P＜0.팔；=33.24,P＜0.01)；신경원세포활성야최강.유산탈경매활성72 h후표체명현하강,각시간조지간비교차이동양구유통계학의의.결론 신경원세포결양후사세포내EPO급EPO mRNA적표체증강,차가증강신경원세포적활성.제시EPO삼여료신경원세포결양적발생발전과정,재신경원세포결양적과정중기중요작용.
Objective To observe erythropoietin (EPO) and its mRNA expression changes in rats cortical neurons when suffering hypoxia and investigate the endogenous EPO protective effect of hypoxia neuronal cells.Methods Cultured cortical neurons were prepared from hypoxia rats and divided into control,hypoxia 12,24,48,72 h group.Using immunohistochemistry,reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot,we observed EPO and its mRNA expression in cells,and also observe the culture medium containing lactate dehydrogenase to evaluate the activity of neurons in the whole process.Results Immunohistochemistry,RT-PCR and Western blot analysis showed:the EPO (20.79 ± 2.98) and its mRNA (0.78 ± 0.05) at 12 h had a basic expression in hypoxia group,compared with the control group (EPO:17.12 ± 1.99; mRNA:0.39 ± 0.05),and the difference was statistically significant (t =2.51,P ＜ 0.05 ; t =13.51,P ＜ 0.01) ; the strongest expression was observed at 48 h (EPO:28.88 ± 3.41,mRNA:1.45 ± 0.07),the difference was statistically significant (t =7.29,P ＜ 0.01 ; t =33.24,P ＜ 0.01) ; and neuronal activity was strongest.Lactate dehydrogenase activity was significantly decreased after hypoxia 72 h,and also a statistically significant difference was found between the groups at each point.Conclusions The EPO and EPO mRNA expression are increased after hypoxia in neuron cells,and may enhance the activity of neurons.Our study suggests that EPO might be involved in the development process of neuronal hypoxia and play an important role in neuronal hypoxia process.