摘要
目的观察突变型α-核突触蛋白对PCI2细胞增殖的影响和可能的降解途径,探讨其在帕金森病发病机制中的作用。方法对转染了α-核突触蛋白(A30P)的PCI2细胞进行药物干预,检测细胞的增殖活性,并采用透射电镜观察细胞超微结构改变以及自噬的特征性改变,同时检测α-核突触蛋白的表达和超氧化物歧化酶(SOD)的水平。结果(1)Western Blot法检测α-核突触蛋白的表达:A30P+渥曼青霉素组(A30P+W组)、A30P+1-甲基-±苯基吡啶组(A30P+MPP^+组)较A30P组明显增高,以A30P+W组最为明显;而A30P+雷帕霉素组(A30P+R组)条带较A30P组减低(P〈0.01);(2)不同时间点细胞培养液中SOD水平(U/ml)的测定:用MPP^+处理转染了突变型α-核突触蛋白的PC12细胞后,培养液中SOD水平(A30P+MPP^+组:3h:97.49±13.8;12 h:102.7±12.7;24 h:101.5±11.8;48 h:104.3±12.4)较A30P组在各时间点显著下调(t=3.7721,P=0.0017);A30P+R组在给药12h以后,培养液中SOD水平逐渐升高,其中在24h(121.2±13.0)、48h(124.3±14.1)和72h(127.7±13.7)时与A30P+W组比较差异有统计学意义(t=2.9746,P=0.0083);突变型α-核突触蛋白激活了自噬途径,并介导了MPP^+的毒性作用,自噬抑制剂渥曼青霉素可通过抑制自噬而加剧α-核突触蛋白积聚,导致细胞死亡;而自噬诱导剂雷帕霉素则可以通过诱导自噬的发生而促进α-核突触蛋白的降解和细胞生长。结论α-核突触蛋白的异常积聚导致PC12细胞的自噬性细胞死亡,促进自噬有助于突变型α-核突触蛋白降解,对细胞具有保护作用。
Objective To observe the effect of mutant α-synuclein (A30P) in autophagic programmed cell death by transfected PC12 cells and explore its probable role and pathway in PD. Methods The definite PC12 cells which were transfected mutant α-synuclein (A30P) were constructed at first and MPP^+, Rapamycin and Wortmanin were administrated to transfected PC12 cells with mutant α-synuclein. Not only the proliferative activity of cells was detected with MTF method but also the uhrastructure changes of cells and expression of α-synuclein in different circumstance were observed by transmission electron microscopy (TEM), Western Blot and the level of SOD. Results ( 1 ) The expression of α-synuclein in groups A30P +Wortmannin and A30P +MPP^+was higher than that in group A30P ( P 〈 0. 01 ), particularly, there was more significant expression of α-synuclein in group A30P +Wortmannin. The expression of α-synuclein in group A30P +Rapamycin was weaker than that in group A30P ( P 〈 0. 01 ) ; (2) The results showed that the SOD level ( group A30P +MPP^+:3 h: 97.49 ± 13.8; 12 h: 102. 7 ± 12.7 ; 24 h: 101.5 ± 11.8; 48 h: 104. 3 ± 12. 4) was significantly decreased at various time points after MPP^+treatment compared that of group A30P (t =3. 7721, P =0. 0017). SOD level gradually increased in A30P + Rapamycin 12 h and showed significant difference at 24 h ( 121.2 ± 13.0), 48 h ( 124. 3 ± 14. 1 ) and 72 h (127. 7 ± 13. 7) after drug treatment compared with that in group A30P + Wortmannin (t = 2. 9746, P =0. 0083) ; (3) Mutant α-synuclein (A30P) leading to PC12 cells death by means of autophagy involved α-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutant α- synuclein (A30P) mediated the toxicity of MPP^+. Rapamycin, an inducer of autophagy, reduced the aggregation of α-synuclein in transfected cells. Meanwhile, Wortmanin, an inhibitor of autophagy, promoted the aggregation of α-synuclein in transfected cells and induced cells to die. Conclusions The abnormal aggregation of α-synuclein induces autophagic programmed cell death in PC12 ceils and mutant α-synuclein (A30P) mediates the toxicity of MPP^+ Meanwhile, Rapamycin may reduce the aggregation of α-synuclein in transfected cells by activation of autophagic pathway.
出处
《中华神经科杂志》
CAS
CSCD
北大核心
2008年第1期51-55,共5页
Chinese Journal of Neurology
基金
卫生部科学研究基金资助项目(WKJ2005-2-030)