【摘要】：目的：在SCA3/MJD細(xì)胞模型中明確ASIC1a與ataxin-3蛋白表達(dá)量之間的相互影響,在細(xì)胞水平初步探討ASIC1a在SCA3/MJD發(fā)病機(jī)制中的作用,以期為SCA3的發(fā)病機(jī)制及臨床治療研究提供新的線索。 方法： 1、將質(zhì)粒pEGFP-N1-ataxin-3-20Q/70Q轉(zhuǎn)染HEK293細(xì)胞,構(gòu)建SCA3/MJD細(xì)胞模型。 2、調(diào)節(jié)細(xì)胞培養(yǎng)液分別為DMEM培養(yǎng)基(pH=7.4,以下簡稱DMEM培養(yǎng)基)、pH=7.4的緩沖液、pH=6.0的緩沖液,Western印跡檢測ataxin-3蛋白的表達(dá)量,明確細(xì)胞培養(yǎng)液pH值改變是否影響ataxin-3蛋白的表達(dá)量。 3、在細(xì)胞培養(yǎng)液中依次加入不加和加入50μM、100μM、150μM的ASIC1a抑制劑鹽酸阿米洛利(5-N,N-dimethyl amiloride, DMA), Western印跡檢測ataxin-3蛋白的表達(dá)量,明確DMA對ataxin-3蛋白表達(dá)量的影響。 4、將質(zhì)粒pCMV-HA-ASIC1a和pEGFP-N1-ataxin-3-20Q/70Q共轉(zhuǎn)HEK293細(xì)胞,構(gòu)建表達(dá)ASIC1a的SCA3/MJD細(xì)胞模型。通過調(diào)節(jié)細(xì)胞培養(yǎng)液pH值激活A(yù)SIC1a, Western印跡檢測ataxin-3蛋白的表達(dá)量；在細(xì)胞培養(yǎng)液中加入不同濃度DMA, Western印跡檢測ataxin-3蛋白的表達(dá)量,明確激活或抑制ASIC1a對ataxin-3蛋白表達(dá)量的影響。 5、將質(zhì)粒pCMV-HA-ASIC1a和pEGFP-N1-ataxin-3-20Q/70Q共轉(zhuǎn)HEK293細(xì)胞,Western印跡檢測ASIC1a蛋白表達(dá)量,明確ataxin-3的polyQ擴(kuò)展突變是否影響ASICla蛋白的表達(dá)量。 結(jié)果： 1、在單轉(zhuǎn)僅表達(dá)ataxin-3蛋白的細(xì)胞(無ASICla蛋白表達(dá))中：不同pH處理后的各組細(xì)胞之間,ataxin-3-20Q和ataxin-3-70Q的表達(dá)量在不同pH值組間均無明顯差異；不同濃度DMA處理后的各組細(xì)胞之間,ataxin-3-20Q和ataxin-3-70Q的表達(dá)量亦無明顯的組間差異。 2、在ASIC1a與ataxin-3蛋白共表達(dá)的細(xì)胞中：經(jīng)過不同pH處理后的各組細(xì)胞之間,ataxin-3-20Q的表達(dá)量無明顯的組間差異,pH=6.0組其ataxin-3-70Q的表達(dá)量明顯高于另外兩組細(xì)胞(P0.01)；pH=6.0時(shí),ataxin-3-70Q的表達(dá)量在加50μM的DMA組與不加DMA組間無明顯差異,但加100μM和150μMDMA的兩組其ataxin-3-70Q表達(dá)量均比不加DMA組明顯減少(P0.05和P0.01)。 3. ASIC1a與ataxin-3共轉(zhuǎn)細(xì)胞經(jīng)不同pH處理后,ataxin-3-70Q組的ASIC1a表達(dá)量均較ataxin-3-20Q組高(P0.05)。 結(jié)論： 1、野生型ataxin-3蛋白和ASIC1a蛋白的表達(dá)量之間無相互影響。 2、ASIC1a在酸性環(huán)境中被激活后,可明顯上調(diào)擴(kuò)展突變型ataxin-3蛋白的表達(dá)量。 3、ASIC1a抑制劑DMA可逆轉(zhuǎn)激活狀態(tài)ASIC1a對擴(kuò)展突變型ataxin-3蛋白表達(dá)量的影響。 4、擴(kuò)展突變型的ataxin-3蛋白可上調(diào)ASIC1a的表達(dá)量。
[Abstract]:Objective: To clarify the interaction between the expression of ASIC1a and ataxin-3 protein in SCA 3/MJD cell model and to explore the role of ASIC1a in the pathogenesis of SCA 3/MJD at the cellular level, so as to provide new clues for the pathogenesis and clinical treatment of SCA 3.
Method:
1, plasmid pEGFP-N1-ataxin-3-20Q/70Q was transfected into HEK293 cells, and SCA3/MJD cell model was constructed.
2. Regulating the expression of ataxin-3 protein in DMEM medium (pH=7.4, hereinafter referred to as DMEM medium), buffer of pH=7.4, buffer of pH=6.0, Western blotting was used to detect the expression of ataxin-3 protein and determine whether the change of pH value in cell culture medium affected the expression of ataxin-3 protein.
3. The expression of ataxin-3 protein was detected by Western blot, and the effect of DMA on the expression of ataxin-3 protein was determined.
4. Plasmid pCMV-HA-ASIC1a and pEGFP-N1-ataxin-3-20Q/70Q were co-transfected into HEK293 cells to construct a SCA 3/MJD cell model expressing ASIC1a. ASIC1a was activated by adjusting the pH value of cell culture medium, and the expression of ataxin-3 protein was detected by Western blotting. Clearly activate or inhibit the effect of ASIC1a on the expression of ataxin-3 protein.
5. Plasmid pCMV-HA-ASIC1a and pEGFP-N1-ataxin-3-20Q/70Q were co-transfected into HEK293 cells. Western blotting was used to detect the expression of ASIC1a protein and to determine whether the polyQ expansion mutation of ataxin-3 affected the expression of ASICla protein.
Result:
1. The expression of ataxin-3-20Q and ataxin-3-70Q were not significantly different among the cells treated with different pH, and the expression of ataxin-3-20Q and ataxin-3-70Q was not significantly different among the cells treated with different concentrations of DMA. Difference.
2. In ASIC1a and ataxin-3 co-expressed cells, there was no significant difference in the expression of ataxin-3-20Q between the groups treated with different pH, the expression of ataxin-3-70Q in the group of pH=6.0 was significantly higher than that in the other two groups (P 0.01); at pH=6.0, the expression of ataxin-3-70Q in the DMA group with or without DMA was not found. The expression of ataxin-3-70Q in the two groups with 100 and 150 mu MDMA was significantly lower than that without DMA (P 0.05 and P 0.01).
3. ASIC1a and ataxin-3 co-transfected cells were treated with different pH. The expression of ASIC1a in ataxin-3-70Q group was higher than that in ataxin-3-20Q group (P 0.05).
Conclusion:
1, there was no interaction between wild-type ataxin-3 protein and ASIC1a protein expression.
2, when ASIC1a was activated in acidic environment, the expression of extended mutant ataxin-3 protein was significantly up-regulated.
3, ASIC1a inhibitor DMA can reverse the effect of activated ASIC1a on the expression of extended mutant ataxin-3 protein.
4, the extended mutant ataxin-3 protein can upregulate the expression of ASIC1a.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R741

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相關(guān)期刊論文 前1條

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