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肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用

贺芳, 邬力祥, 刘发益, 杨丽娟, 张琰, 张海福, 周炬, 黄柏胜, 邓小鹿

中南大学湘雅医学院生理学系.湖南,长沙 410008

摘要

该文旨在探讨肝细胞生长因子(hepatocyte growth factor, HGF)对神经元氧糖剥夺/再灌注损伤的影响。取原代培养12 d 的Sprague--Dawley大鼠大脑皮层神经元，无糖、缺氧(95% N_(2) + 5% CO_(2))孵育2 h后，换含25 mmol/L葡萄糖的培养液、常氧培养0---24 h，以MTT比色法检测细胞活力、乳酸脱氢酶(lactate dehydrogenase, LDH)漏出率作为细胞损伤指标，建立体外氧糖剥夺/再灌注损伤细胞模型；用流式细胞仪和Hoechst 33258染色分析细胞凋亡率；用RT--PCR和Western blot分别检测大鼠脑皮层神经元c--Met受体mRNA和蛋白的表达。于氧糖剥夺2 h/再灌注24 h处理前2 h，加入不同终浓度(5---120 ng/mL)的HGF，观察HGF对皮层神经元的影响。结果显示，c--Met受体表达于皮层神经元，氧糖剥夺2 h/再灌注24 h后，c--Met受体mRNA和蛋白表达均显著上调，神经元细胞活力明显降低，LDH漏出率和细胞凋亡率显著增高。HGF预处理能明显促进氧糖剥夺/再灌注损伤神经元的存活，降低 LDH漏出率，最大效应剂量为80 ng/mL。流式细胞术和Hoechst 33258 染色法结果均显示HGF (80 ng/mL)能显著降低氧糖剥夺/再灌注神经元的细胞凋亡率。此外，c--Met抑制剂SU11274 (5 #mu#mol/L)完全阻断HGF的神经保护作用。结果表明，HGF对皮层神经元氧糖剥夺/再灌注损伤具有直接的保护作用，呈一定的剂量依赖关系，并能有效对抗神经元凋亡。

关键词： 肝细胞生长因子; 神经元; 氧; 葡萄糖; 再灌注

Protection of hepatocyte growth factor on neurons subjected to oxygen--glucose deprivation/reperfusion

He Fang, Wu Lixiang, Liu Fayi, Yang Lijuan, Zhang Yan, Zhang Haifu, Zhou Ju, Huang Baisheng, Deng Xiaolu

Department of Physiology, Xiangya School of Medicine, Central South University.Changsha 410008,Hunan

Abstract

The present study was conducted to investigate the effect of hepatocyte growth factor (HGF) on cortical neurons exposedto oxygen-glucose deprivation/reperfusion (OGD/R). Primary cultured cerebral cortical neurons were prepared from Sprague-Dawleyrats. The cells were used for experiments after culture for 12 d in vitro. To initiate OGD/R, the culture medium was replaced by glucose-free medium, and cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N_(2) and 5% CO_(2) at 37 ℃for 2 h. Following this treatment, neurons were fed with glucose-supplemented (25 mmol/L) medium, and returned to the incubatorunder normoxic condition for 0-24 h. The cell viability was assessed by MTT assay, and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. The percentage of apoptotic cells was analyzed by flow cytometry and Hoechst 33258 staining.The expressions of c-Met mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. Oxygen-glucosedeprivation for 2 h decreased the cell viability and increased LDH leakage rate in cultured cerebral cortical neurons. The cell viabilitydeclined and LDH leakage rate increased with the reperfusion time going on (0-24 h). To explore the influence of HGF on neurons underoxygen-glucose deprivation for 2 h/reperfusion for 24 h (OGD_(2)/R_(24)) condition, the cultures were pretreated with HGF at differentconcentrations (5-120 ng/mL) 2 h prior to OGD_(2)/R_(24) The results showed that OGD_(2)/R_(24) treatment significantly decreased the cellviability, increased LDH leakage rate and the percentage of apopototic cells. Pretreatment with HGF at 5 ng/mL and 10 ng/mL did notaffect the decrease in cell viability resulting from OGD_(2)/R_(24) In the presence of 20 nedmL HGF,the increase in cell viability in corticalneurons exposed to OGD_(2)/R_(24) began to appear,and 80 ng/mL of HGF exhibited the maximal effect.HGF at 5,10 and 20 ne/mL did notaffect the increase in LDH leakage rate in cortical neurons exposed to OGD_(2)/R_(24) In the presence of 40 nedmL HGF,the decrease in LDH leakage rate in cortical neurons subjected to OGD_(2)/R_(24) began to appear,and 80 ng/mL of HGF displayed the maximal effect. In addition, HGF at 80 ng/mL significantly attenuated cell apoptosis resulting from OGD_(2)/R_(24). As detected by semi-quantitative RT-PCR and Western blot analysis.c-Met mRNA and protein were expressed in cerebral cortical neurons cultured for 12 d in vitro.c-Met mRNA and protein expressions in cortical neurons exposed to OGD_(2)/R_(24) were significantly upregulated and were not affected by pretreatmentof HGF at 80 ne/mL.Treatment with c-Met inhibitor SU11274 (5 #mu#mol/L)completely eliminated HGF-mediated protection of corticalneurons subjected to OGD_(2)/R_(24). The results suggest that HGF directly protects cortical neurons against OGD/R-induced cell injury in a dose-dependent manner,and HGD has a potent anti-apoptotic action on neurons exposed to OGD/R.

Key words: hepatocyte growth factor;Neurons;oxygen;glucose;reperfusion

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引用本文：

贺芳, 邬力祥, 刘发益, 杨丽娟, 张琰, 张海福, 周炬, 黄柏胜, 邓小鹿. 肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用[J]. 生理学报 2008; 60 (2): 235-242.

He Fang, Wu Lixiang, Liu Fayi, Yang Lijuan, Zhang Yan, Zhang Haifu, Zhou Ju, Huang Baisheng, Deng Xiaolu. Protection of hepatocyte growth factor on neurons subjected to oxygen--glucose deprivation/reperfusion. Acta Physiol Sin 2008; 60 (2): 235-242 (in Chinese with English abstract).