ASIC1a蛋白和polyQ扩展突变型ataxin-3蛋白表达量之间相互影响的研究
发布时间：2018-09-04 13:34
【摘要】：目的：在SCA3/MJD细胞模型中明确ASIC1a与ataxin-3蛋白表达量之间的相互影响,在细胞水平初步探讨ASIC1a在SCA3/MJD发病机制中的作用,以期为SCA3的发病机制及临床治疗研究提供新的线索。 方法： 1、将质粒pEGFP-N1-ataxin-3-20Q/70Q转染HEK293细胞,构建SCA3/MJD细胞模型。 2、调节细胞培养液分别为DMEM培养基(pH=7.4,以下简称DMEM培养基)、pH=7.4的缓冲液、pH=6.0的缓冲液,Western印迹检测ataxin-3蛋白的表达量,明确细胞培养液pH值改变是否影响ataxin-3蛋白的表达量。 3、在细胞培养液中依次加入不加和加入50μM、100μM、150μM的ASIC1a抑制剂盐酸阿米洛利(5-N,N-dimethyl amiloride, DMA), Western印迹检测ataxin-3蛋白的表达量,明确DMA对ataxin-3蛋白表达量的影响。 4、将质粒pCMV-HA-ASIC1a和pEGFP-N1-ataxin-3-20Q/70Q共转HEK293细胞,构建表达ASIC1a的SCA3/MJD细胞模型。通过调节细胞培养液pH值激活ASIC1a, Western印迹检测ataxin-3蛋白的表达量；在细胞培养液中加入不同浓度DMA, Western印迹检测ataxin-3蛋白的表达量,明确激活或抑制ASIC1a对ataxin-3蛋白表达量的影响。 5、将质粒pCMV-HA-ASIC1a和pEGFP-N1-ataxin-3-20Q/70Q共转HEK293细胞,Western印迹检测ASIC1a蛋白表达量,明确ataxin-3的polyQ扩展突变是否影响ASICla蛋白的表达量。 结果： 1、在单转仅表达ataxin-3蛋白的细胞(无ASICla蛋白表达)中：不同pH处理后的各组细胞之间,ataxin-3-20Q和ataxin-3-70Q的表达量在不同pH值组间均无明显差异；不同浓度DMA处理后的各组细胞之间,ataxin-3-20Q和ataxin-3-70Q的表达量亦无明显的组间差异。 2、在ASIC1a与ataxin-3蛋白共表达的细胞中：经过不同pH处理后的各组细胞之间,ataxin-3-20Q的表达量无明显的组间差异,pH=6.0组其ataxin-3-70Q的表达量明显高于另外两组细胞(P0.01)；pH=6.0时,ataxin-3-70Q的表达量在加50μM的DMA组与不加DMA组间无明显差异,但加100μM和150μMDMA的两组其ataxin-3-70Q表达量均比不加DMA组明显减少(P0.05和P0.01)。 3. ASIC1a与ataxin-3共转细胞经不同pH处理后,ataxin-3-70Q组的ASIC1a表达量均较ataxin-3-20Q组高(P0.05)。 结论： 1、野生型ataxin-3蛋白和ASIC1a蛋白的表达量之间无相互影响。 2、ASIC1a在酸性环境中被激活后,可明显上调扩展突变型ataxin-3蛋白的表达量。 3、ASIC1a抑制剂DMA可逆转激活状态ASIC1a对扩展突变型ataxin-3蛋白表达量的影响。 4、扩展突变型的ataxin-3蛋白可上调ASIC1a的表达量。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
本文编号：2222256
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
【参考文献】
相关期刊论文 前1条
1 沈璐;唐北沙;汤建光;江泓;王成;房海燕;;脊髓小脑型共济失调Ⅲ型ataxin-3相互作用蛋白的筛选[J];中南大学学报(医学版);2006年01期
，本文编号：2222256
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/2222256.html
最近更新
教材专著
