铝致痴呆模型中Caspase-3基因的干预研究
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摘要
目的通过运用RNA干扰技术下调铝致痴呆模型小鼠体内Caspase-3基因的表达水平,研究RNA干扰对铝神经毒性作用的影响及对神经元其他死亡方式的影响。
方法取健康3月龄雄性昆明小鼠48只,体重g,随机分为6组,每组8只,分别为:空白对照组、假手术组、生理盐水组(生理盐水4μl)、染铝组(0.5%AlCl3 3μl+生理盐水1μl)、Al+阴性对照组(0.5% AlCl3 3μl和对照siRNA表达载体1μl)和Al+RNAi组(0.5%三氯化铝3μl和caspase-3 siRNA表达载体1μl),采用侧脑室注射方法染毒,连续染毒5天,染毒期结束后第15天进行跳台实验、旷场实验和Morris水迷宫实验,测定动物的学习记忆能力,第20天处死小鼠,取脑,切取一半大脑浸泡在10%福尔马林中固定24h,做病理切片,并观察转染效率,进一步做HE染色、硫堇染色;另一半大脑分离出海马做电镜,用流式细胞仪测定凋亡率和线粒体膜电位,大脑皮质置于EP管中,-80℃保存。用Western-blot法检测AD相关蛋白:Tau、APP、Aβ,凋亡相关蛋白:Bax、Bcl-2、NF-κB、Caspase-3及活化的Caspase-3 ,自吞噬相关蛋白LC3-Ⅱ及坏死状凋亡相关蛋白RIP1的表达水平,用QRT-PCR检测APP、Bax、Bcl-2、NF-κB、LC3-Ⅱ、Caspase-3及RIP1基因在小鼠脑内的表达。
结果1、小鼠神经行为学测试:与空白对照组相比,假手术组和生理盐水组小鼠水迷宫的逃避潜伏期增加(P<0.05),其余指标均无显著差异(P>0.05);染铝组和Al+阴性对照组小鼠的各项指标较生理盐水组均有显著差异(P<0.05),Al+RNAi组小鼠无明显改变(P>0.05),但比染铝组小鼠的学习记忆能力显著增强(P<0.05)。2、转染效率及基因沉默效率结果:荧光镜下观察并计算caspase-3 siRNA转染神经细胞的转染效率大于90%,并且caspase-3基因的抑制效率为64.92%。3、各组小鼠海马凋亡率和线粒体膜电位检测结果均显示:假手术组的凋亡情况与空白对照组无显著差异(P>0.05),生理盐水组的凋亡现象较空白对照组多(P<0.05);染铝组和Al+阴性对照组凋亡现象较生理盐水组显著增加(P<0.05),而Al+RNAi组凋亡情况无显著差别(P>0.05),较染铝组显著减少(P<0.05)。4、HE染色、硫瑾染色、电镜结果均表明:空白对照组和假手术组小鼠海马CA3区情况相近,细胞无明显凋亡;生理盐水组在该区域出现轻微的病理改变,电镜结果无明显差异;染铝组和Al+阴性对照组病理改变明显,出现典型的凋亡形态;Al+RNAi组海马区细胞的上述病理改变得到修复,电镜结果与生理盐水组细胞形态较接近。5、相关蛋白的检测:凋亡相关基因、蛋白结果显示,染铝组和Al+阴性对照组较生理盐水组有显著差异(P<0.05),而Al+RNAi组较生理盐水组无明显变化(P>0.05),与染铝组的差异有统计学意义(P<0.05);AD相关蛋白、基因结果显示:染铝后导致APP、Aβ、Tau蛋白及相应基因表达显著增高,caspase-3 siRNA干扰后均显著减少(P<0.05);LC3-Ⅱ和RIP1基因、蛋白表达水平在染铝组、Al+阴性对照组表达较生理盐水组显著增高(P<0.05),Al+RNAi组则无显著差别(P>0.05),但比染铝组显著减少(P<0.05)。
结论1、铝能够诱导体内神经细胞凋亡,产生神经毒性作用,caspase-3 siRNA在动物模型体内能够有效的阻断凋亡过程,从而在一定程度上减轻了铝的毒性作用,对AD等神经退行性疾病的治疗和干预有重要意义;2、以慢病毒为载体,并标记有GFP的caspase-3 siRNA通过侧脑室注射方法能有成功转染神经细胞;3、凋亡和坏死、自吞噬等死亡方式之间存在交互作用,实验结果提示凋亡的减少可能会抑制其他细胞死亡方式。
Objective Through reducing the expression of Caspase-3 gene in aluminum-induced apoptosis by RNA interference,study the effect of RNAi on the neurotoxicity of aluminum,and effects on other modes of cell death.
Methods 48 maleness KunMing mice, 3 months old,were divided into 6 groups randomly by weight,8 in each batch:blank control group、Sham group、Normal saline group(NS 4μl)、aluminum exposed group(0.5%AlCl3 3μl+NS 1μl)、Al plus empty vector group(0.5% AlCl3 3μl plus control siRNA expression vector)and Al plus RNAi group(0.5% AlCl3 3μl plus targeted siRNA expression vector). Every group was treated by lateral cerebral ventricle micro-injection for 5 days. On the 15th day after exposure,the step-down test、open-field test、Morris water maze test were performed to test the learning and memory abilities of mice. Then on the 20th day after exposure,mice were sacrificed. Half of the brain is soaked in 10% formalin 24h which morphological changes were observed by optical microscope,and observed the transfection efficiency,further to do HE staining and thionin staining. The cerebral cortex and hippocampus was removed from the other half. The hippocampus was for observation by electron microscope,and apoptosis rate by flow cytometry and mitochondrial membrane potential detection. The cerebral cortex was placed in EP tube at -80℃to save for the Western-blot to detect the protein expression :AD-related protein:Tau、APP、Aβ,apoptosis-related protein:Bax、Bcl-2、NF-κB、Caspase-3 and activated Caspase-3,autophagy-related protein:LC3-Ⅱ,necroptosis -related protein:RIP1,and the gene expression of APP、Bax、bcl-Ⅱ、NF-κB、LC3-Ⅱ、Caspase-3 and RIP1 by QRT-PCR.
Results: 1、learning and memory capability test in mice:Compared with the control group, water maze escape latency of mice in sham group and the normal saline group increased(P<0.05), the other indexes were not significantly different(P>0.05); Compared with saline group,the indicators of mice in aluminum exposed group and the Al+negative control group were significantly different(P<0.05), Al+RNAi group were no significant changes(P>0.05), but enhanced the learning and memory ability was significantly higher than mice in aluminum exposed group(P<0.05). 2、Transfection efficiency and the efficiency of gene silencing:observation by fluorescence microscope and calculate the number of neural cells transfected by caspase-3 siRNA,and the transfection efficiency was more than 90%.The inhibition efficiency of caspase-3 gene was 64.92%.3、Results of apoptosis rate of hippocampus in each group and the mitochondrial membrane potential show: apoptosis of the sham group and blank control group were no significant difference(P>0.05),apoptosis of normal saline group was more than blank control group(P<0.05); compared with normal saline group,apoptosis of aluminum exposed group and the Al+negative control group increased significantly (P<0.05), while apoptosis of the Al+RNAi group was no significant difference(P>0.05), and significantly less than aluminum exposed group(P<0.05).4、HE staining, histological thionin staining, electron microscopy results show that: hippocampal CA3 area of sham group was close to the blank control group, and cells had no obvious apoptosis; the region of normal saline group had slight pathological changes, and results of electron microscopy showed no significant differences; the pathological changes in aluminum exposed group and the Al+negative control group was the typical apoptotic morphology; while the pathological changes of the hippocampus cells in Al+RNAi group had been restored, results of electron microscopy was close to the normal saline group.5、Associated proteins: apoptosis-related genes、proteins showed:aluminum exposed group and the Al+negative control group were significantly different from normal saline group(P<0.05), while the Al+RNAi group had no significant change(P>0.05), and significantly different from aluminum exposed group(P<0.05); AD-related proteins、genes results:aluminum led to significant increase with protein of APP, Aβ, Tau and the corresponding gene expression, but caspase-3 siRNA can significantly reduce their expression(P<0.05);expression levels of LC3-Ⅱand RIP1 in aluminum exposed group and Al+negative control group were significantly higher than normal saline group(P<0.05),while Al+RNAi group were no significantly different(P>0.05),and significantly less than aluminum exposed group(P<0.05).
Conclusion: 1 ) Aluminum can induce neuronal apoptosis in vivo, and have neurotoxicity.Caspase-3 siRNA can effectively block the apoptotic process in animal models, to some extent which can reduce the toxic effects of aluminum,and this is important to the treatment and intervention of AD and other neurodegenerative diseases;2)Lentivirus as vector, and labeled with GFP, neural cells can be successful transfected of caspase-3 siRNA by intracerebroventricular injection method;3)Apoptosis、necrosis、phagocytosis and other forms of death have the interaction.Results show that the reduction of apoptosis can inhibit the other cell death forms.
方法取健康3月龄雄性昆明小鼠48只,体重g,随机分为6组,每组8只,分别为:空白对照组、假手术组、生理盐水组(生理盐水4μl)、染铝组(0.5%AlCl3 3μl+生理盐水1μl)、Al+阴性对照组(0.5% AlCl3 3μl和对照siRNA表达载体1μl)和Al+RNAi组(0.5%三氯化铝3μl和caspase-3 siRNA表达载体1μl),采用侧脑室注射方法染毒,连续染毒5天,染毒期结束后第15天进行跳台实验、旷场实验和Morris水迷宫实验,测定动物的学习记忆能力,第20天处死小鼠,取脑,切取一半大脑浸泡在10%福尔马林中固定24h,做病理切片,并观察转染效率,进一步做HE染色、硫堇染色;另一半大脑分离出海马做电镜,用流式细胞仪测定凋亡率和线粒体膜电位,大脑皮质置于EP管中,-80℃保存。用Western-blot法检测AD相关蛋白:Tau、APP、Aβ,凋亡相关蛋白:Bax、Bcl-2、NF-κB、Caspase-3及活化的Caspase-3 ,自吞噬相关蛋白LC3-Ⅱ及坏死状凋亡相关蛋白RIP1的表达水平,用QRT-PCR检测APP、Bax、Bcl-2、NF-κB、LC3-Ⅱ、Caspase-3及RIP1基因在小鼠脑内的表达。
结果1、小鼠神经行为学测试:与空白对照组相比,假手术组和生理盐水组小鼠水迷宫的逃避潜伏期增加(P<0.05),其余指标均无显著差异(P>0.05);染铝组和Al+阴性对照组小鼠的各项指标较生理盐水组均有显著差异(P<0.05),Al+RNAi组小鼠无明显改变(P>0.05),但比染铝组小鼠的学习记忆能力显著增强(P<0.05)。2、转染效率及基因沉默效率结果:荧光镜下观察并计算caspase-3 siRNA转染神经细胞的转染效率大于90%,并且caspase-3基因的抑制效率为64.92%。3、各组小鼠海马凋亡率和线粒体膜电位检测结果均显示:假手术组的凋亡情况与空白对照组无显著差异(P>0.05),生理盐水组的凋亡现象较空白对照组多(P<0.05);染铝组和Al+阴性对照组凋亡现象较生理盐水组显著增加(P<0.05),而Al+RNAi组凋亡情况无显著差别(P>0.05),较染铝组显著减少(P<0.05)。4、HE染色、硫瑾染色、电镜结果均表明:空白对照组和假手术组小鼠海马CA3区情况相近,细胞无明显凋亡;生理盐水组在该区域出现轻微的病理改变,电镜结果无明显差异;染铝组和Al+阴性对照组病理改变明显,出现典型的凋亡形态;Al+RNAi组海马区细胞的上述病理改变得到修复,电镜结果与生理盐水组细胞形态较接近。5、相关蛋白的检测:凋亡相关基因、蛋白结果显示,染铝组和Al+阴性对照组较生理盐水组有显著差异(P<0.05),而Al+RNAi组较生理盐水组无明显变化(P>0.05),与染铝组的差异有统计学意义(P<0.05);AD相关蛋白、基因结果显示:染铝后导致APP、Aβ、Tau蛋白及相应基因表达显著增高,caspase-3 siRNA干扰后均显著减少(P<0.05);LC3-Ⅱ和RIP1基因、蛋白表达水平在染铝组、Al+阴性对照组表达较生理盐水组显著增高(P<0.05),Al+RNAi组则无显著差别(P>0.05),但比染铝组显著减少(P<0.05)。
结论1、铝能够诱导体内神经细胞凋亡,产生神经毒性作用,caspase-3 siRNA在动物模型体内能够有效的阻断凋亡过程,从而在一定程度上减轻了铝的毒性作用,对AD等神经退行性疾病的治疗和干预有重要意义;2、以慢病毒为载体,并标记有GFP的caspase-3 siRNA通过侧脑室注射方法能有成功转染神经细胞;3、凋亡和坏死、自吞噬等死亡方式之间存在交互作用,实验结果提示凋亡的减少可能会抑制其他细胞死亡方式。
Objective Through reducing the expression of Caspase-3 gene in aluminum-induced apoptosis by RNA interference,study the effect of RNAi on the neurotoxicity of aluminum,and effects on other modes of cell death.
Methods 48 maleness KunMing mice, 3 months old,were divided into 6 groups randomly by weight,8 in each batch:blank control group、Sham group、Normal saline group(NS 4μl)、aluminum exposed group(0.5%AlCl3 3μl+NS 1μl)、Al plus empty vector group(0.5% AlCl3 3μl plus control siRNA expression vector)and Al plus RNAi group(0.5% AlCl3 3μl plus targeted siRNA expression vector). Every group was treated by lateral cerebral ventricle micro-injection for 5 days. On the 15th day after exposure,the step-down test、open-field test、Morris water maze test were performed to test the learning and memory abilities of mice. Then on the 20th day after exposure,mice were sacrificed. Half of the brain is soaked in 10% formalin 24h which morphological changes were observed by optical microscope,and observed the transfection efficiency,further to do HE staining and thionin staining. The cerebral cortex and hippocampus was removed from the other half. The hippocampus was for observation by electron microscope,and apoptosis rate by flow cytometry and mitochondrial membrane potential detection. The cerebral cortex was placed in EP tube at -80℃to save for the Western-blot to detect the protein expression :AD-related protein:Tau、APP、Aβ,apoptosis-related protein:Bax、Bcl-2、NF-κB、Caspase-3 and activated Caspase-3,autophagy-related protein:LC3-Ⅱ,necroptosis -related protein:RIP1,and the gene expression of APP、Bax、bcl-Ⅱ、NF-κB、LC3-Ⅱ、Caspase-3 and RIP1 by QRT-PCR.
Results: 1、learning and memory capability test in mice:Compared with the control group, water maze escape latency of mice in sham group and the normal saline group increased(P<0.05), the other indexes were not significantly different(P>0.05); Compared with saline group,the indicators of mice in aluminum exposed group and the Al+negative control group were significantly different(P<0.05), Al+RNAi group were no significant changes(P>0.05), but enhanced the learning and memory ability was significantly higher than mice in aluminum exposed group(P<0.05). 2、Transfection efficiency and the efficiency of gene silencing:observation by fluorescence microscope and calculate the number of neural cells transfected by caspase-3 siRNA,and the transfection efficiency was more than 90%.The inhibition efficiency of caspase-3 gene was 64.92%.3、Results of apoptosis rate of hippocampus in each group and the mitochondrial membrane potential show: apoptosis of the sham group and blank control group were no significant difference(P>0.05),apoptosis of normal saline group was more than blank control group(P<0.05); compared with normal saline group,apoptosis of aluminum exposed group and the Al+negative control group increased significantly (P<0.05), while apoptosis of the Al+RNAi group was no significant difference(P>0.05), and significantly less than aluminum exposed group(P<0.05).4、HE staining, histological thionin staining, electron microscopy results show that: hippocampal CA3 area of sham group was close to the blank control group, and cells had no obvious apoptosis; the region of normal saline group had slight pathological changes, and results of electron microscopy showed no significant differences; the pathological changes in aluminum exposed group and the Al+negative control group was the typical apoptotic morphology; while the pathological changes of the hippocampus cells in Al+RNAi group had been restored, results of electron microscopy was close to the normal saline group.5、Associated proteins: apoptosis-related genes、proteins showed:aluminum exposed group and the Al+negative control group were significantly different from normal saline group(P<0.05), while the Al+RNAi group had no significant change(P>0.05), and significantly different from aluminum exposed group(P<0.05); AD-related proteins、genes results:aluminum led to significant increase with protein of APP, Aβ, Tau and the corresponding gene expression, but caspase-3 siRNA can significantly reduce their expression(P<0.05);expression levels of LC3-Ⅱand RIP1 in aluminum exposed group and Al+negative control group were significantly higher than normal saline group(P<0.05),while Al+RNAi group were no significantly different(P>0.05),and significantly less than aluminum exposed group(P<0.05).
Conclusion: 1 ) Aluminum can induce neuronal apoptosis in vivo, and have neurotoxicity.Caspase-3 siRNA can effectively block the apoptotic process in animal models, to some extent which can reduce the toxic effects of aluminum,and this is important to the treatment and intervention of AD and other neurodegenerative diseases;2)Lentivirus as vector, and labeled with GFP, neural cells can be successful transfected of caspase-3 siRNA by intracerebroventricular injection method;3)Apoptosis、necrosis、phagocytosis and other forms of death have the interaction.Results show that the reduction of apoptosis can inhibit the other cell death forms.
引文
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