中国医药导报
中國醫藥導報
중국의약도보
CHINA MEDICAL HERALD
2014年
24期
16-19
,共4页
欧阳嶷%何志义%刘嘉晖%朱华倩
歐暘嶷%何誌義%劉嘉暉%硃華倩
구양억%하지의%류가휘%주화천
Machado-Joseph病%基因沉默%RNA干扰%小干扰RNA%慢病毒载体
Machado-Joseph病%基因沉默%RNA榦擾%小榦擾RNA%慢病毒載體
Machado-Joseph병%기인침묵%RNA간우%소간우RNA%만병독재체
Machado-Joseph disease%Gene silencing%siRNA%RNA interference(RNAi)%Lentivirus vector
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。
目的：利用RNAi技術，觀察不同siRNA在體外對Machado-Joseph病變異基因錶達的抑製作用，為其應用于臨床打下基礎。方法本研究分為變異型ATXN3組、野生型ATXN3組及空病毒載體組(對照組)。通過設計針對ATXN3變異基因的特異性siRNA，構建重組型慢病毒載體轉染HEK293T細胞，採用Real-time PCR及West-ern blot檢測ATXN3 mRNA和蛋白的錶達水平，從而對siRNA體外抑製Machado-Joseph病變異基因的效果進行評估。結果 Real-time PCR分析錶明，在共轉染錶達人變異型ATXN3基因和siRNA ATXN3 Mut載體的細胞中，變異型ATXN3組中ATXN3 mRNA的錶達較對照組明顯下降，差異有統計學意義(P<0.05)，而野生型ATXN3組中ATXN3 mRNA的錶達較對照組僅輕微下降，差異無統計學意義(P>0.05)。Western blot結果錶明，與對照組比較，變異型ATXN3組ATXN3蛋白錶達被siRNA ATXN3 Mut 1~4明顯抑製，差異有統計學意義(P<0.05)，野生型ATXN3組中ATXN3蛋白錶達僅被siRNA ATXN3 Mut 1~4輕度抑製。結論特異性siRNA可以選擇性沉默變異型ATXN3，說明RNAi是Machado-Joseph病治療的潛在方嚮。
목적：이용RNAi기술，관찰불동siRNA재체외대Machado-Joseph병변이기인표체적억제작용，위기응용우림상타하기출。방법본연구분위변이형ATXN3조、야생형ATXN3조급공병독재체조(대조조)。통과설계침대ATXN3변이기인적특이성siRNA，구건중조형만병독재체전염HEK293T세포，채용Real-time PCR급West-ern blot검측ATXN3 mRNA화단백적표체수평，종이대siRNA체외억제Machado-Joseph병변이기인적효과진행평고。결과 Real-time PCR분석표명，재공전염표체인변이형ATXN3기인화siRNA ATXN3 Mut재체적세포중，변이형ATXN3조중ATXN3 mRNA적표체교대조조명현하강，차이유통계학의의(P<0.05)，이야생형ATXN3조중ATXN3 mRNA적표체교대조조부경미하강，차이무통계학의의(P>0.05)。Western blot결과표명，여대조조비교，변이형ATXN3조ATXN3단백표체피siRNA ATXN3 Mut 1~4명현억제，차이유통계학의의(P<0.05)，야생형ATXN3조중ATXN3단백표체부피siRNA ATXN3 Mut 1~4경도억제。결론특이성siRNA가이선택성침묵변이형ATXN3，설명RNAi시Machado-Joseph병치료적잠재방향。
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.
