金刚烷胺对脊髓小脑共济失调3型ATXN3基因变异表达的抑制作用

Inhibitory Effect of Amantadine on Mutant ATXN3 Expression in Spinocerebellar Ataxia Type 3

目的研究金刚烷胺对于SCA3神经元的保护作用及其机制.方法该研究分为变异型ATXN3组、野生型ATXN3组及空白对照组.建立表达野生型ATXN3-Q14和变异型ATXN3-Q75基因片段的细胞模型,并对其进行鉴定和培养.在上述细胞模型中分别加入不同浓度的盐酸金刚烷胺溶液(1,5,10μg/mL)进行干预,空白对照组加等剂量的去离子水.药物干预48 h后,采用MTT法测定不同组别的细胞活力.采用AnnexinⅤ-FITC测定细胞凋亡,样品用流式细胞仪进行分析.采用Western Blot法检测不同浓度金刚烷胺对ATXN3变异表达的抑制作用.结果(1)在变异型组中,用10μg/mL金刚烷胺干预时,从24 h起HEK293T细胞的增殖率显著高于空白对照组,组间比较差异有统计学意义(P<0.05);而5μg/mL及1μg/mL干预组与空白对照组相比,48 h内细胞增殖率无明显变化.(2)不同浓度的金刚烷胺均能不同程度地减少变异型组HEK293细胞的凋亡,且有剂量依赖性,组间比较差异有统计学意义(P<0.05).而不同浓度金刚烷胺干预野生型的细胞凋亡率和空白对照组相比,差异无统计学意义(P>0.05).(3)与空白对照组相比,金刚烷胺5μg/mL组和10μg/mL组的变异型ATXN3-Q75蛋白表达被明显抑制(P<0.05);而金刚烷胺1μg/mL组对变异型ATXN3-Q75蛋白表达仅轻度抑制(P>0.05);不同浓度的金刚烷胺对野生型ATXN3-Q14蛋白表达仅有轻度抑制作用(P>0.05).结论该研究表明金刚烷胺对ATXN3基因变异表达具有抑制作用,可能成为SCA3治疗的潜在方向.

Objective To observe whether amantadine could suppress the expression of mutant ATXN3 in SCA disease in vitro.Methods This study was divided into mutant ATXN3 group,wild type ATXN3 group and control group.First,we established cell models for the expression of wild-type ATXN3-Q 14 and mutant ATXN3-Q75 gene fragments,and identified and cultured them.Then,different concentrations of amantadine salt(1,5,10μ g/mL)were added to the above cell models for intervention,and the control group was added with deionized water with the same dose.After 48 hours of drug intervention,MTT method was used to measure the cell viability of different groups.Apoptosis was measured by Annexin V-FITC and the samples were analyzed by flow cytometry.Western Blot was used to detect the inhibition of amantadine on ATXN3 expression.Results(1)In the mutant ATXN3 group,the proliferation rate of HEK293T cells was significantly higher than that of the control group(P<0.05)from 24 hours after the intervention with 10μg/mL amantadine,while the proliferation rate of HEK293T cells in the 5μg/mL and 1μg/mL groups had no significant change compared with the control group.(2)Different concentrations of amantadine could reduce the apoptosis of HEK293 cells in varying degrees,and there was a dose-dependent difference among these groups(P<0.05).There was no significant difference in the apoptosis rate of wild-type cells among these above groups(P>0.05).(3)Compared with the control group,the expression of ATXN3-Q75 protein in amantadine 5μg/mL group and 10 μg/mL group was significantly inhibited(P<0.05);while the expression of ATXN3-Q75 protein in amantadine 1(μg/mL group was only slightly inhibited(P>0.05);the expression of wild ATXN3-Q 14 protein in amantadine of different concentrations was only slightly inhibited(P>0.05).Conclusion This study confirmed that amantadine could significantly inhibit the expression of mutant ATXN3 in SCA3 in vitro,indicating that amantadine may be the potential direction in SCA3 treatment.