CAJ | 학술논문

为了研究多核酶表达系统在HEK293细胞中对多药耐药相关蛋白表达抑制的作用,我们构建了含有20个可以自身切割的顺式作用核酶和10个靶向MRP1基因特定位点的反式作用核酶的多核酶表达系统.利用RT-PCR、Western blot和MTT分析了多核酶系统分别与MRP1靶基因质粒和MRPI全长基因质粒共转染的HEK293细胞.结果显示,多核酶表达系统能够明显降低荧光融合蛋白在HEK293细胞中的表达.RT-PCR分析表明,MRP1靶mRNA降低程度与多核酶表达系统所含的反式作用核酶数目有关.Western blot分析显示了与RT-PCR相似的结果.MTY分析表明,多核酶表达系统能够逆转由MRP1基因转染HEK293细胞产生的多药耐药性.结果提示,含有多个核酶的表达系统对MRP1基因的抑制效应优于单核酶的表达系统.因此,该策略可能用于基因治疗肿瘤或其他疾病.
위료연구다핵매표체계통재HEK293세포중대다약내약상관단백표체억제적작용,아문구건료함유20개가이자신절할적순식작용핵매화10개파향MRP1기인특정위점적반식작용핵매적다핵매표체계통.이용RT-PCR、Western blot화MTT분석료다핵매계통분별여MRP1파기인질립화MRPI전장기인질립공전염적HEK293세포.결과현시,다핵매표체계통능구명현강저형광융합단백재HEK293세포중적표체.RT-PCR분석표명,MRP1파mRNA강저정도여다핵매표체계통소함적반식작용핵매수목유관.Western blot분석현시료여RT-PCR상사적결과.MTY분석표명,다핵매표체계통능구역전유MRP1기인전염HEK293세포산생적다약내약성.결과제시,함유다개핵매적표체계통대MRP1기인적억제효응우우단핵매적표체계통.인차,해책략가능용우기인치료종류혹기타질병.
To study application of muhi-ribozyme expression system on expression inhibition of multidrug resistance-associated protein(MRP1)in HEK293 cells,the multi-ribozyme expression system containing 20 cis-acting ribozymes for self-cleavage and 10 trans-acting ribozymes for targeting to MRP1 gene specific site were constructed.HEK293 cells cotransfected multi-ribozyme expression system with MRP1 target gene or full length of MRP1 gene respectively were analyzed by RT-PCR,Western blot analysis and MTY assay.The results showed that multi-ribozyme systems were able to dramatically decrease fluorescent fusion protein expression in HEK293 cells.RT-PCR analysis indicated that the extent of MRP1 target mRNA decrease was correlated with the number of trans-acting ribozyme contained in multi-ribozyme expression system.Similar changes have been observed from Western bloL MTT assay showed that muhi-ribozyme systems were able to reverse MDR generated by MRP1 gene in HEK cells.These results suggested that inhibitory effects of multiple copies of ribozymes contained in the system were better than that of single ribozyme contained.There fore,this strategy could be used in treatment of tumor or other diseases via gene therapy.