摘要
目的:观察α-Synuclein表达对6-羟基多巴诱导的多巴胺能细胞凋亡的影响及分析其在帕金森病发病机制中所起的作用。方法:实验于2001-12/2004-10于中山大学第一附属医院神经科实验室完成。用100μmol/L的6-羟基多巴诱导PC12细胞凋亡。采用多组样本的完全随机设计方法,将1×105个细胞接种于4块四孔培养板中,共16个孔,随机分为4组:反义、正义、无义寡核苷酸治疗组和空白对照组。前3组分别加入相应的寡核苷酸5μmol/L治疗24h,空白对照组不加。反义寡核苷酸阻断PC12细胞α-Synuclein表达,用原位杂交和定量Westernplot检测其基因和蛋白表达;凋亡原位末端标记检测试剂盒检测6-羟基多巴诱导的PC12细胞凋亡。全自动图象分析系统测量有关平均积分吸光度。用SPSS10.0软件进行统计学分析。结果:①反义阻断后α-SynucleinmRNA表达:反义寡核苷酸治疗组较空白对照组明显减少(P<0.01),正义寡核苷酸治疗组和无义寡核苷酸治疗组与空白对照组比较无差异(P>0.05)。②α-Synuclein蛋白表达:反义寡核苷酸治疗组明显减少,较空白对照组减少约53.93%(P<0.05)。正义寡核苷酸治疗组、无义寡核苷酸治疗组和空白对照组之间差异不显著(P>0.05)。③6-羟基多巴治疗后的反义寡核苷酸治疗组、正义寡核苷酸治疗组、无义寡核苷酸治疗组和空白对照组随机计数的200个细胞中,凋亡细胞数量间相互比较无显著统计学意义(P>0.05)。结论:α-Synuclein表达减少对6-羟基多巴诱导的PC12细胞凋亡无影响。推测在帕金森病的多巴胺神经元凋亡的发生机制中,α-Synuclein在神经元内的异常聚集较其表达量的多少更重要。
AIM: To observe the effect of obstructed α-Synuclein expression on the apoptosis of PC12 cells induced by 6-hydroxydopamine (6-OHDA), and explore its role in the mechanism of Parkinson disease. METHODS: The experiment was conducted at the Neurological Laboratory, the First Affiliated Hospital of Sun Yat-sen University between December 2001 and October 2004. Apoptosis of PC12 cell was induced by 100μmol/L 6-OHDA. With the complete randomized method of multi-group samples, 1×10^5 cells were seeded in 4 cultivated plates with 4 wells (totally 16 wells) and divided into 4 groups: antisense, sense and no-sense oligonucleotides groups and blank control group. The former three groups were treated with corresponding oligonucleotides 5 μmol/L for 24 hours, but the control group was treated without oligonucleotides, α-Synuclein expression of PC12 cell was obstructed with antisense oligonueleotides, which was detected by in situ hybridization and Western blot quantitative analysis; TUNEL in situ apoptosis reagent was used to detect the apoptosis of PC12 cell induced with 6-OHDA. The computerized automatic image analysis was adopted to measure the relative mean integral absorbance (A) and processed with SPSS 10.0 software. RESULTS: ①mRNA expression of obstructed α-Synuclein: The expression of antisense oligonucleotides group was decreased significantly compared with the control group (P 〈 0.01); and no obvious difference was found in the sense and no-sense oligonucleotides groups and blank control group (P 〉 0.05). ②Expression of α-Synuclein protein: The expression of the antisense oligonucleotides group was decreased by 53.93% of that of the control group (P 〈 0.05). No obvious difference was found in the sense and no-sense oligonucleotides groups and blank control group (P 〉 0.05). ③In the 200 cells randomly counted from the antisense, sense and nosense oligonucleotides groups after induced with 6-OHDA and blank control group, no significant difference was found in the number of apoptotic cells (P 〉 0.05). CONCLUSION: Decreased α-Synuelein expression has no effect on the apoptosis of PC12 cells induced by 6-OHDA. It suggests that the abnormal congregation of α-Synuclein in neurons may be more important than its expressive quantity in the mechanism of apoptosis of the dopaminergic neurons of Parkinson disease.
出处
《中国临床康复》
CSCD
北大核心
2006年第34期79-81,i0002,共4页
Chinese Journal of Clinical Rehabilitation
基金
中山大学211工程基金资助项目[(1998)98119]~~