【作者】 周伟；

【导师】 袁崇刚；

【作者基本信息】 华东师范大学 ， 分子神经生物学， 2015， 硕士

【摘要】 目的：研究L-DOPA (Levodopa, L-DOPA)对神经细胞增殖损伤影响及醌氧化还原酶(Quinone oxidoreductase, NQO1)对神经细胞损伤的保护作用,进一步探究帕金森病发病机理及相关左旋多巴衰竭综合症防治方法。方法：运用MTT比色法(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT)检测左旋多巴(Levodopa, L-DOPA)对神经母细胞瘤细胞(SH-SY5Y)的增殖损伤影响,检测利血平(Reserpine, RE)与不同浓度左旋多巴(Levodopa, L-DOPA)共同作用对细胞毒性的影响以及萝卜硫素(Sulforaphane, SF)对不同浓度左旋多巴(Levodopa, L-DOPA)的细胞毒性影响的保护作用；运用醌蛋白检测法(氯化硝基四氮唑蓝NBT/甘氨酸法)检测左旋多巴(Levodopa, L-DOPA)、左旋多巴(L-DOPA)与利血平(RE)、左旋多巴(L-DOPA)与萝卜硫素(SF)作用于SH-SY5Y细胞后醌化蛋白含量变化；运用蛋白质印迹(Western Blot)方法检测萝卜硫素(SF)作用SH-SY5Y细胞后,细胞内NQO1的表达量变化；运用乳酸脱氢酶(LDH)还原NADH至NAD+, NADH在340nm有吸光值原理,用酶标仪检测左旋多巴(Levodopa, L-DOPA)作用SH-SY5Y细胞后乳酸脱氢酶酶活性变化。结果：左旋多巴(Levodopa, L-DOPA)对SH-SY5Y细胞的毒性作用随浓度的增大而对细胞毒性作用增强,在400μmol/L时SH-SY5Y细胞呈现明显损伤,细胞内醌化蛋白含量随L-DOPA作用浓度增加而增加；左旋L-DOPA与利血平(Reserpine, RE)共同作用SH-SY5Y细胞,与相同剂量的单独L-DOPA作用相比,对细胞的毒性作用增大,且细胞内醌化蛋白含量随之增加；L-DOPA作用SH-SY5Y细胞后,细胞内乳酸脱氢酶酶活性下降；萝卜硫素(Sulforaphane, SF)作用SH-SY5Y细胞后,会使胞内NQO1表达量增加,缓解L-DOPA对细胞的毒性作用；SF预处理后与L-DOPA共同作用产生的醌蛋白含量,同L-DOPA单独作用细胞所产生醌蛋白含量相比无明显差异。结论：L-DOPA对SH-SY5Y细胞具有剂量依赖性的毒性效应,随着L-DOPA浓度增加,SH-SY5Y细胞醌化蛋白含量增加,其引起的细胞毒性与胞内醌化蛋白的形成呈一定程度的正相关性；利血平(Reserpine, RE)能够加大L-DOPA的细胞毒性作用,并引起细胞醌蛋白含量进一步增加；L-DOPA能够抑制乳酸脱氢酶活性,推澳L-DOPA作用细胞后使乳酸脱氢酶醌化,进而影响细胞氧化代谢,导致细胞损伤死亡；90μmol/L羟化酪醇(HT)对L-DOPA的细胞毒性影响未见明显的保护作用；SF能诱导产生NQO1,在一定程度上起到对L-DOPA细胞毒性的保护作用。SF对于降低L-DOPA作用SH-SY5Y所产生的醌化蛋白含量并不显著。更多还原

【Abstract】 AIM:To study the influence of L-DOPA (Levodopa, L-DOPA) on dopaminergic cells proliferation and survival, and the protective effect of Quinone oxidoreductase (Quinone oxidoreductase, NQO1) on L-DOPA cytotoxicity. To further investigate the pathogenesis of Parkinson’s disease and explore the potential therapeutic prevention methods of Levodopa failure syndrome.METHODS:The MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) was used to detect the proliferation injury affection of levodopa (L-DOPA) on neuroblastoma cells (SH-SY5Y), to detect the combined cytotoxic effects of reserpine (Reserpine, RE) and different concentrations of L-DOPA on SH-SY5Y and the protective effect of sulforaphane (Sulforaphane, SF) on cytotoxicity induced by different concentrations of L-DOPA. The quinone protein was detected by the NBT/glycinate assay. Intracellular expression of NQO1 induced by sulforaphane (SF) was detected by Western Blots. Microplate Reader was used to detect the changes of lactate dehydrogenase activity.RESULTS:With the increasing concentration of L-DOPA, the cytotoxic effects on SH-SY5Y cells were enhanced. SH-SY5Y cells showed significant damage when the concentration of L-DOPA is 400μmol/L, the content of intracellular quinone protein increased with L-DOPA concentration increasing. The cytotoxicity effects on SHSY5Y much more increased when L-DOPA induced with reserpine, the same as quinone protein content increased; intracellular lactate dehydrogenase activity was decreased after L-DOPA effects on SH-SY5Y; sulforaphane increases the expression of NQO1 and alleviates L-DOPA-induced cytotoxicity in SH-SY5Y cells; there are no significant differences in content of quinone protein between L-DOPA plus SF and L-DOPA alone group.CONCLUSIONS:L-DOPA cytotoxic effects on SH-SY5Y cells in a dose-dependent manner, with increasing concentration of L-DOPA, the content of quinone protein increased, which can be speculated that there are a degree of correlation between L-DOPA cytotoxic effects and the formation of quinone protein. Reserpine can increases the cells cytotoxicity of L-DOPA, which also increases the content of quinone protein. L-DOPA can effectively reduce the Intracellular lactate dehydrogenase activity, suggesting that L-DOPA could convert lactate dehydrogenase to quinone protein, then affecting cellular oxidative metabolism and leading to cell injury.90μmol/L HT can’t protect the cytotoxicity effects of L-DOPA; SF can induce NQO1, which may play a protective effect on the cytotoxicity of L-DOPA; there are no significant effects on SF’s preventing the increasing of quinone protein induced by L-DOPA.更多还原