【摘要】：目的 采用SH-SY5Y細(xì)胞研究環(huán)境因素鋁和遺傳因素ApoE S4基因分別及聯(lián)合作用的情況下,對tau蛋白不同位點磷酸化水平和Aβ表達(dá)的影響,探討鋁和ApoEε4是否存在交互作用。 方法 培養(yǎng)并選取SH-SY5Y細(xì)胞,分別進(jìn)行AICl3染毒和ApoE ε4轉(zhuǎn)染處理,實驗分組包括空白對照組、200μM AlCl3的低劑量組、400μM AlCl3的中劑量組、800μM AlCl3的高劑量組、空質(zhì)粒組、ApoE ε4組、400μM AlCl3+ApoEε4組。AICl3染毒或ApoE ε4轉(zhuǎn)染48h后,采用CCK-8試劑盒測定細(xì)胞活力；采用ELISA試劑盒測定總tau蛋白、tau-181、tau-231、au-262、tau-396和Aβ-40含量；采用析因設(shè)計的方法分析AICl3和ApoE s4是否存在交互作用。 結(jié)果 1.隨著AlCl3染毒劑量的增高,SH-SY5Y細(xì)胞數(shù)量逐漸減少,突觸變短消失；CCK-8檢測結(jié)果顯示細(xì)胞活力逐漸下降,200μM AlCl3的低劑量組、400μM AlCl3的中劑量組、800μM AICl3的高劑量組活力顯著低于空白對照組(P0.05); ELISA檢測結(jié)果顯示,200μM AlCl3的低劑量組、400μM AlCl3的中劑量組、800μM AlCl3的高劑量組相較于空白對照組,總tau蛋白、tau-181、tau-231、tau-262、tau-396和Aβ-40含量均有顯著升高(P0.05)。 2.經(jīng)ApoE ε4轉(zhuǎn)染后,SH-SY5Y細(xì)胞形態(tài)趨于圓形,易崩解死亡；CCK-8檢測結(jié)果顯示細(xì)胞活力明顯下降,并顯著低于空白對照組(P0.05); ELISA檢測結(jié)果顯示,ApoE ε4組細(xì)胞的tau蛋白和Aβ-40含量均顯著高于空白對照組(P0.05)。 3.經(jīng)AICl3和ApoE ε4獨立和聯(lián)合染毒后可觀察到,兩種因素聯(lián)合作用下細(xì)胞喪失固有形態(tài),其數(shù)量較空白對照和兩因素獨立作用時更為稀少；CCK-8檢測結(jié)果顯示細(xì)胞活力低下,與空白對照組有顯著差異(P0.05); ELISA檢測結(jié)果顯示,ApoE s4組細(xì)胞的總tau蛋白、tau-181、tau-231、tau-262、tau-396和Aβ-40含量均顯著高于空白對照組(P0.05)；根據(jù)以上結(jié)果經(jīng)析因設(shè)計分析得出AlC13和ApoE ε4存在明顯的交互作用(P0.05),并表現(xiàn)為協(xié)同效應(yīng)。 結(jié)論 1.鋁和ApoE ε4基因均可導(dǎo)致SH-SY5Y細(xì)胞形態(tài)改變,降低細(xì)胞活力,具有明顯的細(xì)胞毒性作用； 2.鋁和ApoE ε4基因均促使總tau、tau-181、tau-231、tau-262和tau-396磷酸化水平升高,Ap沉積增多； 3.鋁和ApoE ε4基因同時存在的情況下兩種因素具有交互作用,且表現(xiàn)為協(xié)同效應(yīng)。
[Abstract]:Objective to study the effects of environmental factor aluminum and genetic factor ApoE S4 gene on the phosphorylation level of tau protein and the expression of A 尾 in SH-SY5Y cells, and to explore the interaction between aluminum and ApoE 蔚 4. Methods SH-SY5Y cells were cultured and treated with AICl3 and ApoE 蔚 4 transfection respectively. The experimental groups included blank control group, low dose group of 200 渭 M AlCl3, middle dose group of 400 渭 M AlCl3, high dose group of 800 渭 M AlCl3, empty plasmid group and ApoE 蔚 4 group. The cells were treated with AICl3 or ApoE 蔚 4 for 48 h, and the cell viability was measured by CCK-8 kit. The contents of total tau protein, tau-181,tau-231,au-262,tau-396 and A 尾 -40 were determined by ELISA kit, and the interaction between AICl3 and ApoE s4 was analyzed by factorial design. Result 1. With the increase of the dose of AlCl3, the number of SH-SY5Y cells gradually decreased, and the synapse became shorter and disappeared. The results of CCK-8 showed that the cell viability was decreased gradually. The activity of the low dose group of 200 渭 M AlCl3, the middle dose group of 400 渭 M AlCl3 group and the high dose group of 800 渭 M AICl3 group were significantly lower than that of the blank control group (P0.05). The results of ELISA showed that the low dose group of 200 渭 M AlCl3, the middle dose group of 400 渭 M AlCl3, and the high dose group of 800 渭 M AlCl3 compared with the control group, total tau protein, tau-181,tau-231,tau-262, The contents of tau-396 and A 尾 -40 were significantly increased (P0.05). 2. After transfection with ApoE 蔚 4, SH-SY5Y cells tended to be round in shape and prone to disintegration and death. The results of CCK-8 showed that the cell viability was significantly decreased and significantly lower than that of the blank control group (P0.05); ELISA results showed that the ApoE 蔚 4 cells tau protein and A 尾 -40 content were significantly higher than the blank control group (P0.05). 3. After AICl3 and ApoE 蔚 4 were treated independently and jointly, it was observed that the cells lost their inherent morphology under the combined treatment of two factors, and the number of the cells was less than that of the blank control and the two independent factors. The results of CCK-8 showed that the cell activity was low and there was significant difference between the control group and the control group (P0.05). ELISA results showed that the total tau protein, tau-181,tau-231,tau-262,tau-396 and A 尾 -40 contents in ApoE s4 cells were significantly higher than those in the blank control group (P0.05). According to the analysis of factorial design, the interaction between AlC13 and ApoE 蔚 4 was obvious (P0.05), and showed synergistic effect. Conclusion 1. Both aluminum and ApoE 蔚 4 gene can cause morphological changes and decrease cell viability of SH-SY5Y cells. 2. Both Al and ApoE 蔚 4 genes increased the phosphorylation level of total tau,tau-181,tau-231,tau-262 and tau-396, and increased Ap deposition. In the presence of both aluminum and ApoE 蔚 4 genes, the two factors have interaction and show synergistic effect.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R749.16

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