【摘要】：目的通過(guò)運(yùn)用RNA干擾技術(shù)下調(diào)鋁致癡呆模型小鼠體內(nèi)Caspase-3基因的表達(dá)水平,研究RNA干擾對(duì)鋁神經(jīng)毒性作用的影響及對(duì)神經(jīng)元其他死亡方式的影響。 方法取健康3月齡雄性昆明小鼠48只,體重g,隨機(jī)分為6組,每組8只,分別為:空白對(duì)照組、假手術(shù)組、生理鹽水組(生理鹽水4μl)、染鋁組(0.5%AlCl3 3μl+生理鹽水1μl)、Al+陰性對(duì)照組(0.5% AlCl3 3μl和對(duì)照siRNA表達(dá)載體1μl)和Al+RNAi組(0.5%三氯化鋁3μl和caspase-3 siRNA表達(dá)載體1μl),采用側(cè)腦室注射方法染毒,連續(xù)染毒5天,染毒期結(jié)束后第15天進(jìn)行跳臺(tái)實(shí)驗(yàn)、曠場(chǎng)實(shí)驗(yàn)和Morris水迷宮實(shí)驗(yàn),測(cè)定動(dòng)物的學(xué)習(xí)記憶能力,第20天處死小鼠,取腦,切取一半大腦浸泡在10%福爾馬林中固定24h,做病理切片,并觀察轉(zhuǎn)染效率,進(jìn)一步做HE染色、硫堇染色;另一半大腦分離出海馬做電鏡,用流式細(xì)胞儀測(cè)定凋亡率和線粒體膜電位,大腦皮質(zhì)置于EP管中,-80℃保存。用Western-blot法檢測(cè)AD相關(guān)蛋白:Tau、APP、Aβ,凋亡相關(guān)蛋白:Bax、Bcl-2、NF-κB、Caspase-3及活化的Caspase-3 ,自吞噬相關(guān)蛋白LC3-Ⅱ及壞死狀凋亡相關(guān)蛋白R(shí)IP1的表達(dá)水平,用QRT-PCR檢測(cè)APP、Bax、Bcl-2、NF-κB、LC3-Ⅱ、Caspase-3及RIP1基因在小鼠腦內(nèi)的表達(dá)。 結(jié)果1、小鼠神經(jīng)行為學(xué)測(cè)試:與空白對(duì)照組相比,假手術(shù)組和生理鹽水組小鼠水迷宮的逃避潛伏期增加(P0.05),其余指標(biāo)均無(wú)顯著差異(P0.05);染鋁組和Al+陰性對(duì)照組小鼠的各項(xiàng)指標(biāo)較生理鹽水組均有顯著差異(P0.05),Al+RNAi組小鼠無(wú)明顯改變(P0.05),但比染鋁組小鼠的學(xué)習(xí)記憶能力顯著增強(qiáng)(P0.05)。2、轉(zhuǎn)染效率及基因沉默效率結(jié)果:熒光鏡下觀察并計(jì)算caspase-3 siRNA轉(zhuǎn)染神經(jīng)細(xì)胞的轉(zhuǎn)染效率大于90%,并且caspase-3基因的抑制效率為64.92%。3、各組小鼠海馬凋亡率和線粒體膜電位檢測(cè)結(jié)果均顯示:假手術(shù)組的凋亡情況與空白對(duì)照組無(wú)顯著差異(P0.05),生理鹽水組的凋亡現(xiàn)象較空白對(duì)照組多(P0.05);染鋁組和Al+陰性對(duì)照組凋亡現(xiàn)象較生理鹽水組顯著增加(P0.05),而Al+RNAi組凋亡情況無(wú)顯著差別(P0.05),較染鋁組顯著減少(P0.05)。4、HE染色、硫瑾染色、電鏡結(jié)果均表明:空白對(duì)照組和假手術(shù)組小鼠海馬CA3區(qū)情況相近,細(xì)胞無(wú)明顯凋亡;生理鹽水組在該區(qū)域出現(xiàn)輕微的病理改變,電鏡結(jié)果無(wú)明顯差異;染鋁組和Al+陰性對(duì)照組病理改變明顯,出現(xiàn)典型的凋亡形態(tài);Al+RNAi組海馬區(qū)細(xì)胞的上述病理改變得到修復(fù),電鏡結(jié)果與生理鹽水組細(xì)胞形態(tài)較接近。5、相關(guān)蛋白的檢測(cè):凋亡相關(guān)基因、蛋白結(jié)果顯示,染鋁組和Al+陰性對(duì)照組較生理鹽水組有顯著差異(P0.05),而Al+RNAi組較生理鹽水組無(wú)明顯變化(P0.05),與染鋁組的差異有統(tǒng)計(jì)學(xué)意義(P0.05);AD相關(guān)蛋白、基因結(jié)果顯示:染鋁后導(dǎo)致APP、Aβ、Tau蛋白及相應(yīng)基因表達(dá)顯著增高,caspase-3 siRNA干擾后均顯著減少(P0.05);LC3-Ⅱ和RIP1基因、蛋白表達(dá)水平在染鋁組、Al+陰性對(duì)照組表達(dá)較生理鹽水組顯著增高(P0.05),Al+RNAi組則無(wú)顯著差別(P0.05),但比染鋁組顯著減少(P0.05)。 結(jié)論1、鋁能夠誘導(dǎo)體內(nèi)神經(jīng)細(xì)胞凋亡,產(chǎn)生神經(jīng)毒性作用,caspase-3 siRNA在動(dòng)物模型體內(nèi)能夠有效的阻斷凋亡過(guò)程,從而在一定程度上減輕了鋁的毒性作用,對(duì)AD等神經(jīng)退行性疾病的治療和干預(yù)有重要意義;2、以慢病毒為載體,并標(biāo)記有GFP的caspase-3 siRNA通過(guò)側(cè)腦室注射方法能有成功轉(zhuǎn)染神經(jīng)細(xì)胞;3、凋亡和壞死、自吞噬等死亡方式之間存在交互作用,實(shí)驗(yàn)結(jié)果提示凋亡的減少可能會(huì)抑制其他細(xì)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R749.16;R-332

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