CAJ | 학술논문

目的:构建小鼠肿瘤坏死因子相关凋亡诱导配体的死亡受体(Mouse killer,MK)基因干扰RNA(Small interfering RNA,siRNA)重组腺病毒并在小鼠子宫基质细胞中鉴定其干扰作用.方法:人工合成靶向MK的siRNA干扰序列,将其克隆到穿梭载体psES-HUS上,并与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,得到pAdeasy-SES-HUS-MKsiRNA,在HEK293细胞中包装并扩增腺病毒颗粒.用纯化后的腺病毒混合液感染原代培养的小鼠子宫内膜基质细胞,RT-PCR及Western Blot检测基质细胞中MK的mRNA及蛋白表达水平.结果:Adeasy-SES-HUS-MKsiRNA混合液均效价为6.8 X 10~(11)pfu/ml,感染原代培养的小鼠子宫内膜基质细胞48h后,MK的mRNA及蛋白水平均显著下调.结论:构建的MK干扰表达腺病毒能有效地抑制MK基因的表达,为进一步研究MK在小鼠子宫基质细胞的功能提供了有效的工具.
목적:구건소서종류배사인자상관조망유도배체적사망수체(Mouse killer,MK)기인간우RNA(Small interfering RNA,siRNA)중조선병독병재소서자궁기질세포중감정기간우작용.방법:인공합성파향MK적siRNA간우서렬,장기극륭도천사재체psES-HUS상,병여선병독골가질립pAdeasy-1재BJ5183세균중진행동원중조,득도pAdeasy-SES-HUS-MKsiRNA,재HEK293세포중포장병확증선병독과립.용순화후적선병독혼합액감염원대배양적소서자궁내막기질세포,RT-PCR급Western Blot검측기질세포중MK적mRNA급단백표체수평.결과:Adeasy-SES-HUS-MKsiRNA혼합액균효개위6.8 X 10~(11)pfu/ml,감염원대배양적소서자궁내막기질세포48h후,MK적mRNA급단백수평균현저하조.결론:구건적MK간우표체선병독능유효지억제MK기인적표체,위진일보연구MK재소서자궁기질세포적공능제공료유효적공구.
Objective:To construct the recombinant adenovirus carrying a short interfering RNA (siRNA) targeting MK(mouse killer)gene and identify its inhibitive effect in mouse uterine stromal cells.Methods:The siRNA sequence targeting MK gene was synthesized and cloned into the shuttle plasmid pSES-HUS to generate the vector pSES-HUS-MKsiBNA.which was later homogenously recombinanted with the adenovirus backbone plasmid pAdeagy-1 in Ecoli BJ5183.Then the recombinant adenovirus was transfected into HEK293 cells to be packaged and amplified.The primarily cultured mouse stromal cells were infected with this purified adenovirus and the expression levels of MK mRNA and protein were detected by RT-PCR and Western Blot.Results:The recombinant adenoviral vector containing MK siRNA was correctly constructed with an average titer of 6.8 X 10~(11) pfu/mL.Both the MK mRNA and protein levels were signifcanfly decreased in the uterine stromal cells after 48 hours of infection with MK siRNA recombinant adenovirus.Conclusion:The generated recombinant adenovims Adeasy-MK-siRNA was demonstrated to significantly abrogate the expression of MK at mRNA and protein level in mouse uterine stromal cells.