CAJ | 학술논문

本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、无氧(95%N2+5%CO2)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HGF受体c-Met 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表达于皮层神经元,氧糖剥夺2 h/再灌注24 h后,c-Met 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/mE.流式细胞术和Hoechst33258染色结果均显示,HGF(80 ng/mL)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 μmol/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.
본문지재탐토간세포생장인자(hepatocyte growth factor.HGF)대신경원양당박탈/재관주손상적영향.취원대배양12 d적Sprague-Dawley대서대뇌피층신경원,무당、무양(95%N2+5%CO2)부육2 h후,환함25 mmol/L포도당적배양액、상양배양0～24 h,이MTT비색법검측세포활력、유산탈경매(lactate dehydrogenase,LDH)루출솔작위세포손상지표,건입체외양당박탈/재관주손상세포모형;용류식세포의화Hoechst 33258염색분석세포조망솔;용RT-PCR화Western blot분별검측대서뇌피층신경원HGF수체c-Met mRNA화단백적표체.우양당박탈2 h/재관주24 h처리전2 h,가입불동종농도(5～120 ng/mL)적HGF,관찰HGF대피층신경원적영향.결과현시,c-Met표체우피층신경원,양당박탈2 h/재관주24 h후,c-Met mRNA화단백표체균현저상조,신경원세포활력명현강저,LDH루출솔화세포조망솔현저증고.HGF예처리명현촉진양당박탈/재관주손상신경원적존활,강저LDH루출솔,최대효응제량위80 ng/mE.류식세포술화Hoechst33258염색결과균현시,HGF(80 ng/mL)현저강저양당박탈/재관주신경원적세포조망솔.차외,c-Met억제제SU11274(5 μmol/L)완전조단HGF적신경보호작용.결과표명,HGF대피층신경원양당박탈/재관주손상구유직접적보호작용,정일정적제량의뢰관계,병능유효대항신경원조망.
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% N2 and 5% CO2 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 (OGD2/R24) condition, the cultures were pretreated with HGF at differentconcentrations (5-120 ng/mL) 2 h prior to OGD2/R24 The results showed that OGD2/R24 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 OGD2/R24 In the presence of 20 nedmL HGF,the increase in cell viability in corticalneurons exposed to OGD2/R24 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 OGD2/R24 In the presence of 40 nedmL HGF,the decrease in LDHleakage rate in cortical neurons subjected to OGD2/R24 began to appear,and 80 ne/mL of HGF displayed the maximal effect.In addition,HGF at 80 ne/mL significantly attenuated cell apoptosis resulting from OGD2/R24.As detected by semi-quantitative RT-PCR andWestern blot analysis.c-Met mRNA and protein were expressed in cerebral cortical neurons cultured for 12 d in vitro.c-Met mRNAand protein expressions in cortical neurons exposed to OGD2/R24 were significantly upregulated and were not affected by pretreatmentof HGF at 80 ne/mL.Treatment with c-Met inhibitor SU11274 (5 μmol/L)completely eliminated HGF-mediated protection of corticalneurons subjected to OGD2/R24 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.