【作者】 刘楠楠；

【导师】 郭松超；

【作者基本信息】 广西医科大学 ， 营养与食品卫生学， 2011， 硕士

【摘要】 【目的】应用新生大鼠海马神经元细胞原代培养的方法,探讨ERK 1/2在介导牛磺酸保护锰诱导海马神经元细胞氧化损伤的机制。【方法】取SPF级新生24h以内的Wistar大鼠脑部海马组织进行海马神经元细胞原代培养,培养至最佳状态后,用于实验。用于培养的海马神经元细胞分组为:①空白对照组;②牛磺酸对照组(Tau:4mmol/L);③低浓度染锰组(Mn:0.2mmol/L);④中浓度染锰组(Mn:0.8mmol/L);⑤高浓度染锰组(Mn:1.2mmol/L);⑥低浓度干预组(Tau:4mmol/L,Mn:0.2mmol/L);⑦中浓度干预组(Tau:4mmol/L,Mn:0.8mmol/L);⑧高浓度干预组(Tau: 4mmol/L,Mn:1.2mmol/L)。各处理组在加以处理因素24小时后终止培养,在普通光学显微镜下观察后进行以下检测:MTT实验测试细胞活力、荧光测定法检测细胞内活性氧族的生成和增加、Western Blot法检测ERK 1/2蛋白表达、实时荧光定量PCR检测ERK1/2蛋白mRNA的相对表达。【结果】①锰可致海马神经元细胞形态学改变,细胞形态改变随着染锰浓度的增加而增大,对应染锰浓度的牛磺酸干预组细胞形态较为完整,存活细胞量较多;②MTT结果显示:海马神经元细胞经锰损伤后,细胞活力明显下降,与空白对照组和牛磺酸对照组比较,有显著性差异(P<0.01)。使用牛磺酸干预后,低、中、高浓度的牛磺酸干预组的活力与对应浓度的低、中、高染锰组对比,具有显著性差异(P<0.01),活力均比后者提高;③ROS检测显示:与空白组和牛磺酸对照组比较,锰损伤后各组ROS均明显增高。空白对照组与牛磺酸对照组比较,无统计学意义。牛磺酸干预各组细胞ROS降低,与同等浓度的染锰组对比,均有显著性差异(P值均<0.01)。4个时间点测量ROS含量存在显著性差异,F= 18.107,P<0.01;④低浓度干预组ERK1/2蛋白表达量与空白对照组、牛磺酸对照组无差别;在低浓度染锰组、中浓度染锰组的ERK1/2蛋白表达量均高于对应同等染锰浓度的干预组;在高浓度染锰组与高浓度干预组的ERK1/2蛋白表达量相比无差别;⑤低浓度干预组ERK1/2的mRNA相对表达量与空白对照组、牛磺酸对照组无差别,在低浓度染锰组、中浓度染锰组和高浓度的染锰组的ERK1/2mRNA相对表达量均低于对应同等染锰浓度的干预组。【结论】锰可致海马神经元细胞氧化应激损伤,牛磺酸可拮抗锰致海马神经元氧化应激损伤,ERK1/2在牛磺酸拮抗锰致海马神经元细胞氧化应急损伤保护作用中起到重要作用。更多还原

【Abstract】 Objective: By applying the primary culture method on hippocampal neurons, this paper discusses the mechanism of ERK1 / 2 in mediating the protection of taurine on hippocampal neuronal cells in manganese oxidation injury.Methods:Take the hippocampal neurons from the brain hippocampus of SPF Wistar rats which time of birth are less than 24h’s. Then put the hippocampal neurons in primary culture till the optimum state.The hippocampal neurons used culture can be divided to several groups as follows:①the control group;②control group taurine (Tau: 4mmol / L);③low concentration of Mn group (Mn: 0.2mmol / L);④medium concentration of Mn group (Mn: 0.8mmol / L);⑤high concentration of Mn group (Mn: 1.2mmol / L);⑥low concentrations of the intervention group (Tau: 4mmol / L, Mn: 0.2mmol / L);⑦medium concentration intervention group(Tau: 4mmol / L, Mn: 0.8mmol / L);⑧high concentration of the intervention group (Tau: 4mmol / L, Mn: 1.2mmol / L).Terminated the culture of each group after 24 hours addressed by treatment factors. Use ordinary optical microscopet to observe and carry out the follow testing: MTT tested for the living rate of hippocampal neurons, Fluorescence detection for intracellular reactive oxygen species generation and increment, Western Blot Detection for ERK1 /2 protein expression, real-time fluorescence quantitative PCR detection for ERK1 / 2 protein mRNA relative expression.Results:①Manganese can cause the morphological changes of hippocampal neurons. And with the concentration increasing,the change can be amplified. The cells form of the control group with intervention was more complete and have more viable cells;②MTT result shows that after damaged by manganese , the viability of hippocampal neurons decreased significantly. There was significant difference (P <0.01) comparing with the control group and taurine control group. When using taurine to intervene, low, medium and high concentrations of Mn groups’ activities have significant differences(P <0.01) compared with the corresponding concentration contrast groups. Their activities were better than those exposed to manganese groups;③ROS test shows that comparing with the control group and taurine control groups, the ROS of each adding manganese group increased significantly. The comparison of control group and taurine control group was not statistically significant. The ROS of taurine control groups decreased. There were significant differences(P<0.01)compared with the same concentration of manganese groups. 4 time points measured ROS levels were significantly different,F=18.107,P<0.01;④there was no difference of ERK1/2 protein expression between among low concentration of manganese intervention group,control group and taurine control group. The ERK1/2 protein expression of low,medium concentration manganese groups were lower than those corresponding to the same concentration of manganese in the intervention groups; When come to the high concentration of manganese group and the same concentration of manganese in the intervention group’s, ERK1/2 protein expressions have no difference.⑤there were no difference between ERK1/2’s mRNA relative expression among low concentration group ,control group and taurine control group,the ERK1/2’s mRNA relative expression of low,medium,high concentration manganese groups were lower than those corresponding to the same concentration of manganese in the intervention groups.Conclusions: Manganese can cause oxidative stress injury of hippocampal neurons. Taurine can inhibit the hippocampal neurons oxidative stress caused by manganese, ERK1 / 2 plays an important role in the taurine antagonist hippocampal neurons oxidative stress injury induced by manganese.更多还原