铝致神经母细胞瘤细胞死亡方式及其干预研究

【作者】 张勤丽；

【导师】 牛侨；

【作者基本信息】 山西医科大学 ， 劳动卫生学和环境卫生学， 2008， 博士

【摘要】 目的铝是公认的神经毒素,长期慢性的铝接触与多种神经退行性变的发生有密切关系。进行性细胞死亡是神经退行性变的重要特征之一,大量的体内、体外实验表明,铝能导致神经细胞死亡。为了探讨铝致神经细胞死亡方式的类型及其干预效果,本课题对铝诱导的混合神经细胞研究模型细胞—神经母细胞瘤细胞（SH-SY5Y）的死亡方式及其干预措施的效果进行了研究。本课题的第一个目的是研究铝诱导的SH-SY5Y细胞的死亡方式和类型;在此基础上,第二个目的是用RNA干扰的方法阻抑凋亡相关基因bak,caspase-3的表达,研究RNA干扰的效果及其对铝诱导的神经细胞凋亡的阻抑作用。除凋亡和坏死之外,程序性坏死是2005年提出的一种存在于脑缺血损伤中的新型细胞死亡通路,研究程序性坏死是否也存在于铝诱导的SH-SY5Y细胞的死亡途径中,及其特异性的阻断剂Nec-1对铝诱导的SH-SY5Y细胞死亡的阻抑效果是本研究的第三个目的。第四个目的是研究不同死亡方式的阻抑剂对细胞死亡方式的影响及其联合作用的效果。另外,我们还单独探讨了神经胶质细胞在铝致神经细胞凋亡中的作用及其干预效果。方法①为了检测铝诱导的SH-SY5Y细胞的死亡方式和类型,我们在体外培养了SH-SY5Y细胞并用0-8mMAlCl₃·6H₂O染毒,作用24h后检测其细胞死亡方式。相差显微镜观察细胞的生长状态,AO-EB和Hoechst染色法观察铝染毒细胞的凋亡和坏死,流式细胞仪Annexin V-PI双染法定量测定细胞的凋亡率和坏死率,免疫组织化学染色定量测定凋亡相关蛋白NF-κB和Cty-c的表达量,透射电镜进一步观察染铝细胞的超微结构变化。②根据程序性坏死具有坏死的形态学特点和程序性的特征性变化,对培养的SH-SY5Y细胞用不同浓度铝染毒,和/或加入不同浓度的Nec-1,观察SH-SY5Y细胞的形态学特点,Hoechst-PI染色法观察细胞的凋亡和坏死变化,检测细胞活力、线粒体膜电位、活性氧含量、细胞的凋亡率和坏死率以及其对细胞自吞噬小体的影响等。③为了特异性降低细胞的凋亡率,采用RNA干扰的方法特异性阻断凋亡相关基因bak,caspase-3基因的表达。设计针对bak,caspase-3基因干扰的siRNA序列,用不同浓度铝染毒细胞制做铝致神经细胞死亡模型,分别测定不同的siRNA序列、不同的转染浓度及不同的转染时间对细胞活力的影响。用荧光染色法计数转染效率,用荧光定量PCR法检测干扰效率,用免疫组织化学染色法测定阻抑蛋白表达效果并检测其对细胞的凋亡率及坏死率的影响。④采用小干扰RNA和Nec-1联合作用同时对细胞的凋亡和程序性坏死途径进行阻抑。对不同浓度铝染毒SH-SY5Y细胞制作的铝致神经细胞死亡模型,用caspase-3RNAi抑制caspase-3基因的表达,同时用Nec-1抑制程序性坏死的产生,并检测了其对细胞的凋亡率、坏死率和自吞噬标志蛋白LC3表达的影响,以探索铝诱导的神经母细胞瘤细胞不同死亡方式间的相互影响和相互作用。⑤为了研究神经胶质细胞在铝致神经细胞凋亡中的作用,以神经胶质瘤细胞为研究对象,用不同浓度铝作用于该细胞系,用RNA干扰的方法对凋亡相关基因bax的表达进行了阻抑,分别测定不同的siRNA序列、不同的转染浓度及不同的转染时间对细胞活力的影响。用荧光染色法计数转染效率,用荧光定量PCR法检测干扰效率,用免疫组织化学染色法测定阻抑蛋白表达效果并检测其对细胞的凋亡率及坏死率的影响。结果①AO-EB和Hoechst染色法显示了铝染毒细胞的凋亡和坏死的形态学变化,免疫组织化学染色显示凋亡蛋白Cty-c的表达量随染铝剂量增加而显著上升（P＜0.05）,抗凋亡蛋白NF-κB的表达量随染铝剂量增加而显著下降（P＜0.05,P＜0.01）。流式细胞仪AnnexinV-PI双染法定量测定结果表明染铝细胞的凋亡率和坏死率显著上升（P＜0.05,P＜0.01）,细胞的超微结构变化进一步证实了凋亡、坏死和自噬性死亡的共存现象。②加入Nec-1可以显著改善染铝后细胞的坏死样形态学改变,不同浓度Nec-1可以显著提高细胞活力（P＜0.05,P＜0.01）、提高线粒体膜电位、保护线粒体膜完整性（P＜0.05,P＜0.01）,但对染铝后细胞活性氧含量的影响未见有显著的统计学意义（P＞0.05）。流式细胞仪测定结果显示,Nec-1可以显著降低SH-SY5Y细胞的坏死率（P＜0.05,P＜0.01）,但对凋亡率的影响未见有显著的统计学意义（P＞0.05）。③测定bak,caspase-3基因不同的siRNA序列在4mM铝染毒作用下,不同的转染浓度及不同的转染时间下细胞活力。对bak基因RNAi结果表明,siRNA1序列为最有效的序列,最适转染浓度为10nmol/L,最适作用时间为转染后48h。荧光染色并观察计数表明siRNA的转染效率＞90%,最大干扰效率为57.76%,并表现了对Bak蛋白表达的显著的阻抑效应（P＜0.05）。bak siRNA1作用于染铝后的神经瘤细胞,可以显著降低细胞的凋亡率（P＜0.05）,但对细胞坏死率的影响未见有显著的统计学意义（P＞0.05）。通过细胞活力在不同siRNA序列、不同转染剂量、不同作用时间点的变化,筛选出caspase-3基因RNAi的最有效序列为siRNA1序列,并找到其最适转染浓度为10nmol/L,最适作用时间为转染后48h。荧光染色并观察计数表明siRNA的转染效率为93%,其干扰效率为63.02%,并表现了对caspase-3蛋白表达的显著阻抑效应（P＜0.05）。④用bak RNAi和caspase-3 RNAi阻断细胞的凋亡和/或Nec-1阻断细胞的程序性坏死,并对处理后的细胞检测自噬小体的标志蛋白LC3的表达结果显示,染铝细胞的凋亡、坏死和自噬性死亡间具有相互作用。bak RNAi和caspase-3 RNAi虽然可以降低细胞的凋亡率但却同时增加了细胞的自噬性死亡（P＜0.05）;但Nec-1不仅可以显著降低细胞的坏死率（P＜0.05,P＜0.01）,也可以避免细胞自噬性死亡的增多（P＞0.05）,并通过神经细胞间的相互作用阻抑了细胞凋亡的发生。Nec-1和bak RNAi、caspase-3 RNAi共同作用的结果表明,共同作用后细胞的自噬性死亡低于bak RNAi、caspase-3 RNAi单独作用组,但仍高于Nec-1单独作用组（P＜0.05,P＜0.01）。Caspase-3 siRNA单独作用于染铝SH-SY5Y细胞可以显著提高细胞活力（P＜0.05）,降低其凋亡率（P＜0.05）;Nec-1单独作用于染铝SH-SY5Y细胞可以显著提高其细胞活力（P＜0.05,P＜0.01）,降低细胞坏死率（P＜0.05,P＜0.01）;共同作用在染铝SH-SY5Y细胞时两者有增强作用,可以显著提高细胞活力（P＜0.05,P＜0.01）,并同时降低SH-SY5Y细胞的凋亡率及坏死率（P＜0.05,P＜0.01）。⑤对染铝后神经胶质细胞瘤细胞的bax siRNA转染结果表明,最有效的针对bax基因的siRNA序列为siRNA1,该序列的转染效率＞90%,对bax基因表达的干扰效率为62.3%,最佳转染后检测点为转染后72小时,最适转染剂量为20nmol/L。bax siRNA1作用于染铝后的神经瘤细胞,可以显著降低细胞的凋亡率（P＜0.05）,但对细胞坏死率的影响并不显著（P＞0.05）。结论铝诱导的SH-SY5Y细胞死亡方式除了凋亡、坏死和自噬性死亡之外,还有程序性坏死的存在。凋亡是铝致SH-SY5Y细胞死亡的重要途径,针对bak,caspase-3基因,用化学法合成siRNA并将其导入神经细胞,可以有效降低bak,caspase-3基因和其蛋白的表达,提高细胞活力,降低细胞的凋亡率,但同时却增加了细胞的自噬性死亡的发生。Nec-1作为程序性坏死的特异阻断剂可以有效降低铝致神经细胞死亡的坏死率,而且不会增加铝诱导的自噬性死亡的发生。caspase-3 siRNA与Nec-1的协同作用可以显著改善细胞的生存状态,同时降低染铝细胞的凋亡率和坏死率。对染铝神经胶质细胞的凋亡阻断结果提示,铝致神经细胞的损伤主要是铝对神经元的损伤作用,神经胶质细胞因其较大的数量和对铝神经毒作用的较强的耐受力,可能是铝中毒机制研究和神经元保护的另一个重要靶点。更多还原

【Abstract】 Objective Aluminum is known as a kind of neurological toxin,it is related to kinds of degenerative diseases if being chronically exposed in a long-term lifespan.Processing cell death is one of the most important features of neural cells degeneration,a lot of studies have shown that aluminum might induce neural cells death.To investigate the mode of cell death induced by aluminum and effect of inhibition,the present study focus on the types of cell death in SH-SY5Y cells,a kind of common used model of mixed neural cells,and the effects of inhibitions. Therefore,the first aim of the present study is to investigate the cell death types in SH-SY5Y cells induced by aluminum.Based on this,the next aim is to inhibit apoptosis by RNA interference to apoptotic related genes:bak and caspase-3.Besides,Necroptosis is a type of novel cell death pathway apart from apoptosis and necrosis,which was found in ischemic brain injury by Prof.Jun Yingyuan’s group in 2005.To probe whether necroptosis exists in neural cell death induced by aluminum and effect of its specific inhibitor,Nec-1,are the third aim of the study.Concerning interaction of the cell death types and combined effect of the inhibitors,there comes the forth aim of the present work.Finally,to investigate the role of neuroglias on aluminum induced neural cells apoptosis and RNA interference effect on specific bax gene are the fifth aim of the research.Methods①To find out the mode of cell death induced by aluminum,SH-SY5Y cells were cultured in vitro and treated with 0-8mM AlCl₃·6H₂O,types of cell death were detected 24h after treatment.The cell status was observed under a phase contrast microscope,apoptosis and necrosis were shown by AO-EB fluorescent double staining and Hoechst sigle staining respectively.Furthermore,flow cytometry by Annexin V-PI double staining was used to quantify the apoptotic and necrotic rates,expressions of apoptotic related proteins NF-κB and Cty-c were detected by immunohistochemistry.Finally,ultrastructure images of transmission electronic microscope were used to confirme the cell death types of aluminum treated SH-SY5Y cells.②According to the necrotic like morphous and programmed cell death characteristics,neuroptosis was recognized in SH-SY5Y cells,which were incubated with various concentrations of aluminum and/or Nec-1.Morphous of aluminum treated SH-SY5Y cells was observed under a phase contrast microscope,apoptosis and necrosis were shown by Hoechst-PI double staining,cytometry was used to quantify cell viability, mitochondria membrane potential（MMP）,content of reactive oxygen species（ROS）,and apoptotic rate/necrotic rate.Autophogosomes were imagined in electronic microscope photographs.③To specific decrease apoptotic rate,3 siRNA sequences were designed for inhibition of bak and caspase-3 genes respectively,which were transfected into SH-SY5Y cells. Cell viabilities on siRNAs,concentrations of transfected reagent,time courses of transfection were assayed by CCK-8 method.Interference rates of genes were detected by QRT-PCR, inhibition on proteins was measured by immunohistochemistry,apoptotic rate and necrostic rate were quantified by flow cytometer.④To inhibite apoptosis and necrosis simultaneously,bak RNAi and caspase-3 RNAi were performed to reduce the apoptotic rate and Nec-1 was used to decline the necrosis rate,LC3 expression was detected by western blot after performation of bak RNAi,caspase-3 RNAi and/or Nec-1 treatment.⑤To investigate the role of neuroglias on aluminum induced apoptosis,bax RNA interference was performed on gliocytoma cells,cell viability was detected by CCK-8 kit on different siRNA sequences,various transfection concentrations,and diverse transfection courses.Transfection efficiency was determined by percentage of CY3 fluorescent staining cells in DAPI numbered cells,and interference efficiency was measured by QRT-PCR,immunohistochemistry was used to quantify protein content. Besides,flow cytometry was used to measure apoptotic rate and necrotic rate in aluminum treated SH-SY5Y cells transfected by the selected bax siRNA.Results①The co-exist status of apoptosis and necrosis was present by AO-EB fluorescent staining and Hoechst staining, expression of apoptotic related protein Cty-c was significantly upregulated with aluminum concentration increasing（P＜0.05）,while which of apoptotic preventive protein NF-κB downregulated（P＜0.05,P＜0.01）.Apoptotic rate and necrotic rate detected by flow cytometer quantified their significant upregulation（P＜0.05,P＜0.01）.Finally,ultrastracture in aluminum treated SH-SY5Y cells confirmed the apoptosis and autophagy features.②Nec-1 ameliorated the necrotic-like cell morphology,enhanced cell viability（P＜0.05,P＜0.01）,increased cell MMP （P＜0.05,P＜0.01）,and retained ROS content in aluminum treated SH-SY5Y cells（P＞0.05）. Besides,Nec-1 could significantly decrease the necrotic rate（P＜0.05,P＜0.01）,while it has no significant effect on apoptotic rate（P＞0.05）.③Based on the viabilities and gene inhibitive rates of bak siRNA sequences,siRNA1 was selected as the optimal siRNA sequence,the optimal transfection concentration was 10nmol/L,and the optimal time course was 24 h after transfection. The transfection efficiency was above 90%,the interference efficiency was 57.76%,significant inhibitive effect on Bak protein was found as well（P＜0.05）.Furthermore,the apoptotic rate in aluminum treated SH-SY5Y cells were significantly decreased by bak siRNA1 transfection （P＜0.05）,while there was no significant effect on necrotic rate（P＞0.05）.Similarly,based on the viabilities and gene inhibitive rates of caspase-3 siRNA sequences,siRNA1 was selected as the effective sequence,the optimal transfect concentration was 10nmol/L,the optimal transfection time was 48h after transfection.Furthermore,the transfection efficiency was 93%,the interference efficiency was 63.02%,and there was also a significant difference in the expression of Caspase-3 protein among the various siRNA transfection concentrations（P＜0.05）.④Results of LC3 expression in bak RNAi,caspase-3 RNAi and/or Nec-1 treatment shown that there was interaction among apoptosis,necroptosis and autophagy,bak RNAi and caspase-3 RNAi could inhibite cell apoptosis while LC3 marked autophagy upregulated（P＜0.05）.However,Nec-1 could reduce not only necrosis（P＜0.05,P＜0.01） but also LC3 expression of autophagy（P＞0.05）, thereafter,it could decrease sequenced apoptotic rate through cell signal transfer.The coeffect of Nec-1 and bak siRNA or caspase-3 siRNA shown that LC3 expression in coeffect cells was lower than that in Nec-1 treated alone cells,but higher than those in bak siRNA or caspase-3 siRNA treatment alone cells（P＜0.05,P＜0.01）.There was significant increase in cell viability of aluminum treated cells（P＜0.05,P＜0.01）,and significant decrease in apoptotic rate and necrotic rate（P＜0.05,P＜0.01）.⑤The optimal bax siRNA sequence was selected based on the cell viability and the gene inhibitive rate.The transfection efficiency was above 90%,the interference efficiency of bax gene was 62.3%,and the optimal transfection time was 72h after transfection, the optimal transfection concentration was 20nmol/L,there was a significant decrease in Bax protein content after transfection（P＜0.05）.Besides,apoptotic rate was significantly decreased （P＜0.05）,while necrotic rate showed no significant difference after transfection（P＞0.05）. Conclusion Apart from apoptosis,necrosis and autophagy,there is necroptosis in aluminum induced cell death.Apoptosis is one of the major cell death pathways in SH-SY5Y cells induced by aluminum,it can be specitifically reduced by transfection of bak and caspase-3 siRNAs. When chemically synthesized siRNAs are inducted to neural cells,it can significantly reduce bak and caspase-3 gene level,decrease Bak and Caspase-3 protein expression.Nec-1,as a specific inhibitor of necroptosis,can effectively block the necroptosis induced by aluminum,it indicates that necroptosis plays an important role in aluminum induced cell death.The cell viability could be enhanced significantly by caspase-3 siRNA combined Nec-1,besides apoptotic rate and necrotic rate decrease simultaneously.The present study also hints that neuroglia cells play a major role in aluminum induced neuron injury.Being mass number in nervous system and extraordinary resistence to aluminum toxicity,the neuroglia cells may be another target in performing neuron protection.更多还原