中文摘要：

目的：利用RNAi技术，观察不同siRNA在体外对Machado-Joseph病变异基因表达的抑制作用，为其应用于临床打下基础。方法本研究分为变异型ATXN3组、野生型ATXN3组及空病毒载体组（对照组）。通过设计针对ATXN3变异基因的特异性siRNA，构建重组型慢病毒载体转染HEK293T细胞，采用Real-time PCR及West-ern blot检测ATXN3 mRNA和蛋白的表达水平，从而对siRNA体外抑制Machado-Joseph病变异基因的效果进行评估。结果 Real-time PCR分析表明，在共转染表达人变异型ATXN3基因和siRNA ATXN3 Mut载体的细胞中，变异型ATXN3组中ATXN3 mRNA的表达较对照组明显下降，差异有统计学意义（P〈0.05），而野生型ATXN3组中ATXN3 mRNA的表达较对照组仅轻微下降，差异无统计学意义（P〉0.05）。Western blot结果表明，与对照组比较，变异型ATXN3组ATXN3蛋白表达被siRNA ATXN3 Mut 1-4明显抑制，差异有统计学意义（P〈0.05），野生型ATXN3组中ATXN3蛋白表达仅被siRNA ATXN3 Mut 1-4轻度抑制。结论特异性siRNA可以选择性沉默变异型ATXN3，说明RNAi是Machado-Joseph病治疗的潜在方向。

英文摘要：

Objective To observe the inhibitory effect of specific different siRNAs on the expression of mutant ATXN3 in Machado-Joseph disease in v itro by RNA interfere, and lay the foundation for its application in clinical. Methods This study was divided into mutant ATXN3 group, wild type ATXN3 group and empty vector group （control group）. The siRNAs interfering sequence targeting to mutant ATXN3 gene were designed and synthesized. Then the recombinant lentivirus vector were constructed and used to transfect HEK293T cells.The expression level of ATXN3 mRNA and protein was detected by Real-time PCR and Western blot, and the inhibitory effect of siRNAs on the expression of mutant ATXN3 in Machado-Joseph disease in v itro were evaluated. Results Real-time PCR analysis showed that, ATXN3 mRNA of mutant ATXN3 group was decreased significantly compared with the control group, the difference was statistically significant （P〈0.05）, but ATXN3 mRNA in wild type ATXN3 group was only slightly decline, had no statistically significant difference （P〉0.05）. Western blot analysis confirmed that, compared with the control group, the expression of ATXN3 protein in mutant ATXN3 group was significantly inhibited, the difference was statistically signifi-cant （P〈0.05）, and that of wild type ATXN3 group was only slight inhibited by siRNA ATXN3 Mut 1-4. Conclusion The specific silencing of ATXN3 significantly decreases the expression of mutant ATXN3 in Machado-Joseph disease in vitro, these data demonstrate that RNAi has potential for use in Machado-Joseph treatment.