铝致痴呆模型中Caspase-3基因的干预研究
发布时间：2018-08-11 17:50
【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(P0.05),其余指标均无显著差异(P0.05);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(P0.05),Al+RNAi组小鼠无明显改变(P0.05),但比染铝组小鼠的学习记忆能力显著增强(P0.05)。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(P0.05),生理盐水组的凋亡现象较空白对照组多(P0.05);染铝组和Al+阴性对照组凋亡现象较生理盐水组显著增加(P0.05),而Al+RNAi组凋亡情况无显著差别(P0.05),较染铝组显著减少(P0.05)。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(P0.05),而Al+RNAi组较生理盐水组无明显变化(P0.05),与染铝组的差异有统计学意义(P0.05);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(P0.05);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(P0.05),Al+RNAi组则无显著差别(P0.05),但比染铝组显著减少(P0.05)。 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。
[Abstract]:Objective To study the effect of RNA interference on the neurotoxicity of aluminum and other neuronal death modes by down-regulating the expression of Caspase-3 gene in dementia mice induced by aluminum.
Methods Forty-eight healthy three-month-old male Kunming mice weighing g were randomly divided into 6 groups: blank control group, sham-operation group, saline group (saline 4 mul), aluminum group (0.5% AlCl 3 mul + saline 1 mul), Al + negative control group (0.5% AlCl 3 mul and control siRNA expression vector 1 mul) and Al + RNA group (0.5% AlCl 3 mul) respectively. And caspase-3 siRNA expression vector 1 mul were injected into the lateral ventricle for 5 consecutive days. On the 15th day after the end of the exposure period, step-down test, open-field test and Morris water maze test were performed to determine the learning and memory ability of the animals. On the 20th day, the mice were sacrificed. Half of the brains were immersed in 10% formalin and fixed for 24 hours. The other half of the brain was separated from the hippocampus for electron microscopy, and the apoptosis rate and mitochondrial membrane potential were measured by flow cytometry. The cerebral cortex was stored in EP tube at - 80 C. The AD-related proteins: Tau, APP, Abeta, apoptosis-related proteins: Bax, Bcl-2, NF-kappa B, Caspase-3 and activity were detected by Western-blot. The expression levels of transformed caspase-3, autophagy-related protein LC3-II and necrotic apoptosis-related protein RIP1 were detected by QRT-PCR. The expressions of APP, Bax, Bcl-2, NF-kappa B, LC3-II, Caspase-3 and RIP1 genes in mouse brain were detected by QRT-PCR.
Results 1. Neurobehavioral test: Compared with the control group, the escape latency of water maze in sham-operated group and normal saline group increased (P 0.05), while the other indexes were not significantly different (P 0.05); the indexes of Al-exposed group and Al + negative control group were significantly different (P 0.05), and the mice in Al + RNAi group did not change significantly (P 0.05). Transfection efficiency and gene silencing efficiency: The transfection efficiency of Caspase-3 siRNA transfected nerve cells was more than 90%, and the inhibition efficiency of Caspase-3 gene was 64.92%. The results showed that there was no significant difference in apoptosis between sham operation group and blank control group (P 0.05). The apoptosis of normal saline group was more than that of blank control group (P 0.05). The apoptosis of aluminum group and Al + negative control group was significantly higher than that of normal saline group (P 0.05), but there was no significant difference in apoptosis of Al + RNAi group (P 0.05). P 0.05).4, HE staining, thiophene staining, electron microscopy results showed that: the blank control group and sham operation group mice hippocampal CA3 region was similar, no obvious cell apoptosis; normal saline group in the region of slight pathological changes, electron microscopy results showed no significant difference; aluminum staining group and Al + negative control group pathological changes were obvious, typical morphology of apoptosis; The pathological changes of hippocampal cells in RNAi group were repaired. The results of electron microscopy were close to those in normal saline group. The expression of apoptosis-related genes and proteins were detected. The results showed that there was a significant difference between Al + negative control group and Al + negative control group (P 0.05), but there was no significant difference between Al + RNAi group and normal saline group (P 0.05). The expression of APP, Abeta, Tau protein and their related genes increased significantly after aluminum exposure, and caspase-3 siRNA interference decreased significantly (P 0.05). The expression of LC3-II and RIP1 gene was significantly higher in aluminum exposure group and Al + negative control group than in normal saline group (P 0.05), but there was no significant difference in group Al+RNAi (P0.05), but it was significantly lower than that in Aluminum Group (P0.05).
Conclusion 1. Aluminum can induce apoptosis of nerve cells in vivo and produce neurotoxicity. Caspase-3 siRNA can effectively block the apoptosis process in animal models, thus reducing the toxicity of aluminum to a certain extent. 2. Lentiviruses are used as carriers and labeled with GFP. Caspase-3 siRNA can be successfully transfected into nerve cells by intraventricular injection. 3. There is interaction between apoptosis and necrosis, autophagy and other death modes. The results suggest that the decrease of apoptosis may inhibit other cell death modes.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R749.16;R-332
[Abstract]:Objective To study the effect of RNA interference on the neurotoxicity of aluminum and other neuronal death modes by down-regulating the expression of Caspase-3 gene in dementia mice induced by aluminum.
Methods Forty-eight healthy three-month-old male Kunming mice weighing g were randomly divided into 6 groups: blank control group, sham-operation group, saline group (saline 4 mul), aluminum group (0.5% AlCl 3 mul + saline 1 mul), Al + negative control group (0.5% AlCl 3 mul and control siRNA expression vector 1 mul) and Al + RNA group (0.5% AlCl 3 mul) respectively. And caspase-3 siRNA expression vector 1 mul were injected into the lateral ventricle for 5 consecutive days. On the 15th day after the end of the exposure period, step-down test, open-field test and Morris water maze test were performed to determine the learning and memory ability of the animals. On the 20th day, the mice were sacrificed. Half of the brains were immersed in 10% formalin and fixed for 24 hours. The other half of the brain was separated from the hippocampus for electron microscopy, and the apoptosis rate and mitochondrial membrane potential were measured by flow cytometry. The cerebral cortex was stored in EP tube at - 80 C. The AD-related proteins: Tau, APP, Abeta, apoptosis-related proteins: Bax, Bcl-2, NF-kappa B, Caspase-3 and activity were detected by Western-blot. The expression levels of transformed caspase-3, autophagy-related protein LC3-II and necrotic apoptosis-related protein RIP1 were detected by QRT-PCR. The expressions of APP, Bax, Bcl-2, NF-kappa B, LC3-II, Caspase-3 and RIP1 genes in mouse brain were detected by QRT-PCR.
Results 1. Neurobehavioral test: Compared with the control group, the escape latency of water maze in sham-operated group and normal saline group increased (P 0.05), while the other indexes were not significantly different (P 0.05); the indexes of Al-exposed group and Al + negative control group were significantly different (P 0.05), and the mice in Al + RNAi group did not change significantly (P 0.05). Transfection efficiency and gene silencing efficiency: The transfection efficiency of Caspase-3 siRNA transfected nerve cells was more than 90%, and the inhibition efficiency of Caspase-3 gene was 64.92%. The results showed that there was no significant difference in apoptosis between sham operation group and blank control group (P 0.05). The apoptosis of normal saline group was more than that of blank control group (P 0.05). The apoptosis of aluminum group and Al + negative control group was significantly higher than that of normal saline group (P 0.05), but there was no significant difference in apoptosis of Al + RNAi group (P 0.05). P 0.05).4, HE staining, thiophene staining, electron microscopy results showed that: the blank control group and sham operation group mice hippocampal CA3 region was similar, no obvious cell apoptosis; normal saline group in the region of slight pathological changes, electron microscopy results showed no significant difference; aluminum staining group and Al + negative control group pathological changes were obvious, typical morphology of apoptosis; The pathological changes of hippocampal cells in RNAi group were repaired. The results of electron microscopy were close to those in normal saline group. The expression of apoptosis-related genes and proteins were detected. The results showed that there was a significant difference between Al + negative control group and Al + negative control group (P 0.05), but there was no significant difference between Al + RNAi group and normal saline group (P 0.05). The expression of APP, Abeta, Tau protein and their related genes increased significantly after aluminum exposure, and caspase-3 siRNA interference decreased significantly (P 0.05). The expression of LC3-II and RIP1 gene was significantly higher in aluminum exposure group and Al + negative control group than in normal saline group (P 0.05), but there was no significant difference in group Al+RNAi (P0.05), but it was significantly lower than that in Aluminum Group (P0.05).
Conclusion 1. Aluminum can induce apoptosis of nerve cells in vivo and produce neurotoxicity. Caspase-3 siRNA can effectively block the apoptosis process in animal models, thus reducing the toxicity of aluminum to a certain extent. 2. Lentiviruses are used as carriers and labeled with GFP. Caspase-3 siRNA can be successfully transfected into nerve cells by intraventricular injection. 3. There is interaction between apoptosis and necrosis, autophagy and other death modes. The results suggest that the decrease of apoptosis may inhibit other cell death modes.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R749.16;R-332
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