摘要
目的观察siRNA-pool对大鼠脊髓背角神经细胞(rat neurons-dorsal spinal cord cells,RNdsc)上T细胞死亡偶联基因8(T-cell death-associated gene 8,TDAG8)表达的影响。方法设计并合成针对TDAG8的siRNA 3对(siRNA63、siRNA341、siRNA897)及一条带绿色荧光标记的阴性对照siRNA。在阳离子聚合物纳米粒jetPEITM介导下转染RNdsc。RNdsc细胞随机分为正常(normal)组,载体(vehicle)组,阴性对照(mismatch)组,siRNA 50、100、200 pmol组。转染24 h后,荧光显微镜下计算转染复合物的转染效率,用MTT法检测转染复合物的细胞毒性,采用real-time PCR及Western blot测定siRNA-pool的干扰效率。结果采用jetPEITM作为转染介质转染效率高达98.9%。转染复合物的细胞毒性低,各组细胞成活率差异无统计学意义。siRNA 50、100、200 pmol可剂量依赖性地抑制TDAG8的表达,siRNA200 pmol组对TDAG8 mRNA表达的抑制率达88%,对TDAG8蛋白的表达抑制率达73%。结论 siRNA-pool能高效地抑制RNdsc上TDAG8的表达。
Aim To investigate the ability of siRNA- pool to knockdown TDAG8 in rat neurons-dorsal spinal cord （RNdsc） ceils. Methods Four siRNAs were chemically synthesized ： three of them were used to in- hibit TDAG8 expression in RNdsc cells, and the rest was fluorescence-labeled mismatch siRNA as a negative control. They were all transfected into RNdsc cells with jetPEITM, respectively. RNdsc cells were randomly di- vided into 5 groups： normal, vehicle, mismatch, and siRNA 50, 100, 200 pmol group. After transfection with siRNA for 24 h, the rate of transfection was calcu- lated under fluorescence microscope,and the cytotoxic- ity of complex was detected using MTT. The expressionof TDAG8 was detected blot analysis. Results by real-time PCR and Western The transfection rate was high enough to reach 98.9%, and the cytotoxicity of com- plex was very low. Meanwhile, TDAG8-siRNA 50, 100, 200 pmol could dose-dependently inhibit the ex- pression of TDAG8 mRNA and protein levels. The TD- AG8 siRNA 200 pmol reduced the expression of TD- AG8 mRNA and protein up to 88% and 73%, individ- ually. Conclusion SiRNA-pool can effectively inhibit the expression of TDAG8 in RNdsc cells.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2013年第12期1699-1701,共3页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81171057
81000479)
镇江市社会发展基金资助项目(No SH2011036)
