【作者】 徐丽；

【导师】 牛侨；

【作者基本信息】 山西医科大学 ， 劳动卫生与环境卫生学， 2010， 硕士

【摘要】 [目的]观察和探讨Nee-1对染铝(A1)神经细胞的作用。[方法]1.体外原代培养小鼠神经细胞：选用新生1-3d的昆明小鼠,取脑,分离大脑皮质进行神经细胞培养。2.制造Al损伤神经细胞模型：在细胞培养第5天左右,选取生长良好的同批次神经细胞染铝使其终浓度为2mmol／L。3.加入Nee-1不同剂量0μmol/L、30μmol/L、60μmol/L进行干预,48小时后(1)观察神经细胞的形态学变化并用CCK-8法检测神经细胞生长状况；(2)流式细胞术(AnnexinV和PI双染法)检测神经细胞的坏死率和凋亡率；(3)荧光定量PCR法检测Nec-1不同剂量作用下神经细胞坏死、凋亡及自吞噬基因的表达量；(4)蛋白印迹法(Western-blot)检测神经细胞坏死、凋亡及自吞噬蛋白的表达量。[结果]1.Nec-1对染铝神经细胞形态学及细胞活力的影响：(1)光镜下观察神经细胞的形态学改变,结果显示正常神经细胞生长旺盛,有长且比较均一的轴突,有较多从胞体开始向末梢逐渐变细的树突,细胞与突触间,胞体与胞体间连接丰富；染铝(2mmol／L)使神经细胞的数量减少,胞核分裂,突触变少变短甚至消失；而随着Nec-1剂量的加大,神经细胞数量逐渐增多,体积逐渐增大,轴突变长,且树突的数量明显增多。(2)AO-EB染色结果显示,正常细胞的核染色质着绿色并呈正常结构,加入Al3+(2mmol/L)时神经细胞出现明显的凋亡和坏死现象,随着Nec-1剂量的增加,神经细胞的凋亡和坏死有明显的下降。(3)细胞活力结果显示,与染铝相比较,Nec-1(60μmol／L、90μmol／L)可使细胞活力升高(P<0.01)。2.Nec-1在铝致神经细胞坏死中的作用：(1)流式结果显示, A13+(2mmol／L)作用于神经细胞可使细胞的坏死率上升(P<0.01),而随着Nec-1剂量的增加,细胞的坏死率呈下降趋势,且有统计学意义(P<0.01)。(2)荧光定量PCR(qRT-PCR)结果显示,以A13+(2mmol／L)为对照,Nee-1(60μmol／L、90μmol／L)可使神经细胞的坏死相关基因RIP1、NFκB的mRNA表达下降(P<0.01)。(3)Western结果显示,同A13+(2mM)组比,Nec-1(60μmol／L、90μmol／L)组细胞NFκB蛋白的表达呈下降趋势,且有显著的统计学意义(P<0.01)；Nec-1(90μmol／L)亦使RIPl蛋白的表达下降(P<0.01)。3.Nec-1在铝致神经细胞凋亡中的作用：(1) JC-1染色结果显示,正常神经细胞线粒体膜电位较高,产生红色荧光；2mM染铝组神经细胞的线粒体膜电位明显变低(早期凋亡改变),产生绿色荧光的细胞增多；Nec-1(60μmol／L)又使其膜电位升高,产生红色荧光的细胞较染铝组明显增多。(2)流式结果显示,Al13+(2mmol／L)作用于神经细胞可使其凋亡率上升(P<0.01),而Nec-1(60μmo1／L、90μm01／L)可使细胞凋亡率下降(P<0.01)。(3)qRT-PCR结果显示,以A13+(2mmo1／L)为对照,Nec-1(60μmol／L、90μmol／L)可使神经细胞凋亡相关基因caspase-3、caspase-8、caspase-9的mRNA表达下降(P<0.01)。(4)Western结果显示,同A13+(2mmol／L)组比,Nec-1(60μmo1／L90μtmol/L)组细胞caspase-3、caspase-8、caspase-9蛋白的表达亦均呈下降趋势,且有统计学意义(P<0.01)。4.Nec-1对染铝神经细胞发生自吞噬的影响：同A13+(2mmol／L)比,Nee-1(60μmol/L)使LC3-Ⅱ基因mRNA的表达下降(P<0.05)；且Nee-1(90μmol／L)使LC3-Ⅱ蛋白的表达亦呈统计学下降(P<0.05)。[结论]Nec-1可减少铝导致的神经细胞死亡,尤其对坏死以及凋亡的作用比较明显,从而起到保护神经细胞的作用。更多还原

【Abstract】 [Objective] To observe and investigate the effect of Nec-1 on nerve cells poisoned by alumnium.[Methods] 1. The study of aluminum in vitro:cultured primary neurocytes of 1-3 days Kunming mouse and dissociated cerebral cortex, cell density was adjusted at 1×105 cells/ml.2. Manufacturing Al injury model of nerve cells:After 5 days, neurocytes were poisoned with Al3+ of dose 2mmol/L.3. Adding different doses of Nec-1(Oμmol/L、30μmol/L、60μmol/L、90μmol/L). After 48 hours, (1) Observating the morphological changes of nerve cells and the cell viability of neurocytes was quantified by method of Cell Counting Kit-8; (2) the necrotic and apoptosis rate of neurocytes was measured by method of fluorescent analysis; (3) the genes expression of necrosis, apoptosis and autophagy were detected by qRT-PCR; (4) the expressions of necrosis, apoptosis and autophagy in protein levels were detected by Western blot.[Results] The results presented here, indicate that 1. (1) In light microscopy, normal nerve cells growed vigorously, they had long and relatively homogeneous axons and there were more dendrites which became tapered from cell body to peripheral,and there were rich connection between cell bodies. While in aluminum (2mmol/L) group, nucleus splitted and synaptic became fewer and shorter or even disappeared. When the doses of Nec-1 increased, cell volume gradually increased, axons became longer, and the dendritic markedly increased, and the connection between cell body to cell body and cell body to synaptic became significantly increased. (2) Chromatin of normal cells showed green and a normal structure, while adding Al3+(2mmol/L), nerve cells appeared apoptosis and necrosis. With the dose of Nec-1 increasing, apoptosis and necrosis of cells decrease. (3) Compared with aluminum group, Nec-1 (60μmol/L.90μmol/L) increased the viability of cultured neurocytes significantly (P<0.01).2. (1) FCM indicated that after Al3+poisoning nerve cells and with Nec-1 dose increasing, the necrotic rate of cells declined(P<0.01). (2) The result of qRT-PCR showed that compared with Al3+(2mmol/L), the expressions of mRNA of RIP1、NFκB genes decreased significantly in the doses of Nec-1 (60μmol/L,90μmol/L)(P<0.01). (3) The Western-blot demonstrated that compared with Al3+(2mmol/L) group, Nec-1 (60μmol/L、90μmol/L) declined the expression of NFκB(P<0.01); the expression of RIP 1 protein decrease significantly in Nec-1 (90μmol/L) group(P＜0.01).3. (1) JC-1 staining showed that normal nerve cells had a relative high mitochondrial membrane potential, resulting in red fluorescence, and in 2mM aluminum group of nerve cells, which had low mitochondrial membrane potential, resulting in an increase in green fluorescence, while membrane potential of neve cells of treated with Nec-1 (60μmol/L) had increased, resulting in red fluorescent cells were significantly increased compared with anodized aluminum. (2) FCM indicated that after Al3+ poisoning nerve cells and with Nec-1 dose increasing, the apoptosis rate of cells declined (P＜0.01). (3) The result of qRT-PCR showed that compared with Al3+(2mmol/L), the expressions of mRNA of caspase-3、caspase-8、caspase-9 genes decreased significantly in the doses of Nec-1 (60μmol/L、90μmol/L) (P<0.01). (4) The Western-blot demonstrated that compared with Al3+(2mmol/L) group, Nec-1 (60μmol/L、90μmol/L) declined the expression of caspase-3、caspase-8、caspase-9 (P<0.01).4. Compared with Al3+(2mmol/L), Nec-1 (60μmol/L) declined the expression of LC3-Ⅱgene (P＜0.05), and Nec-1 (90μmol/L) decreased the protein expression of LC3-Ⅱ(P＜0.05).[Conclusion] Nec-1 can reduce aluminum-induced death of nerve cells, especially in the pathway of apoptosis and necrosis, accordingly protect nerve cells.更多还原