CAJ | 학술논문

目的：研究髓样细胞白血病-1(myeloid cell leukemia-1,Mcl-1)基因在小鼠巨噬细胞 Raw264.7和人巨噬细胞THP-1中的表达情况,筛选出表达含量高的细胞株作为实验用细胞,根据筛选的结果构建靶向小鼠 Mcl-1基因的短发夹 RNA(shRNA)真核表达质粒进行转染,最后筛选出沉默 Mcl-1基因效果最明显的 shRNA 表达质粒。方法利用半定量 RT-PCR 和蛋白质免疫印迹(Western blotting)分别检测两种巨噬细胞中 Mcl-1mRNA 和蛋白表达情况。采用小分子干扰 RNA(siRNA)软件设计3个针对 Mcl-1基因不同位点的 shRNA 片段,由公司构建携带此 shRNA 片段的真核表达质粒(Mcl-1 shRNA1-3),然后通过脂质体将真核表达质粒载体转染到小鼠巨噬细胞株 Raw264.7中,24、48 h 后通过倒置荧光显微镜观察转染效果,并分别采用实时定量 PCR 和 Western blot 检测 Mcl-1 mRNA 和蛋白表达情况。结果小鼠巨噬细胞 Raw264.7中 Mcl-1的 mRNA 及蛋白质的表达显著高于人巨噬细胞,差异有统计学意义(P <0.05)；构建的 shRNA 表达载体在24、48 h 均能降低 Raw264.7细胞内 mcl-1 mRNA 和蛋白水平,尤其在48 h 沉默效果最为明显；转染48 h 后与正常组、脂质体组和阴性对照组相比,差异有统计学意义(P <0.05)；与 Mcl-1 shR-NA1和 Mcl-1 shRNA2相比,Mcl-1 shRNA3对 Mcl-1 mRNA 和蛋白的沉默作用最强。结论成功筛选出了实验所用细胞 Raw264.7及对小鼠巨噬细胞 Raw264.7内 Mcl-1具有明显沉默效果的靶向 Mcl-1 shRNA3真核表达质粒。
목적：연구수양세포백혈병-1(myeloid cell leukemia-1,Mcl-1)기인재소서거서세포 Raw264.7화인거서세포THP-1중적표체정황,사선출표체함량고적세포주작위실험용세포,근거사선적결과구건파향소서 Mcl-1기인적단발협 RNA(shRNA)진핵표체질립진행전염,최후사선출침묵 Mcl-1기인효과최명현적 shRNA 표체질립。방법이용반정량 RT-PCR 화단백질면역인적(Western blotting)분별검측량충거서세포중 Mcl-1mRNA 화단백표체정황。채용소분자간우 RNA(siRNA)연건설계3개침대 Mcl-1기인불동위점적 shRNA 편단,유공사구건휴대차 shRNA 편단적진핵표체질립(Mcl-1 shRNA1-3),연후통과지질체장진핵표체질립재체전염도소서거서세포주 Raw264.7중,24、48 h 후통과도치형광현미경관찰전염효과,병분별채용실시정량 PCR 화 Western blot 검측 Mcl-1 mRNA 화단백표체정황。결과소서거서세포 Raw264.7중 Mcl-1적 mRNA 급단백질적표체현저고우인거서세포,차이유통계학의의(P <0.05)；구건적 shRNA 표체재체재24、48 h 균능강저 Raw264.7세포내 mcl-1 mRNA 화단백수평,우기재48 h 침묵효과최위명현；전염48 h 후여정상조、지질체조화음성대조조상비,차이유통계학의의(P <0.05)；여 Mcl-1 shR-NA1화 Mcl-1 shRNA2상비,Mcl-1 shRNA3대 Mcl-1 mRNA 화단백적침묵작용최강。결론성공사선출료실험소용세포 Raw264.7급대소서거서세포 Raw264.7내 Mcl-1구유명현침묵효과적파향 Mcl-1 shRNA3진핵표체질립。
Objective To study the expressions of myeloid cell leukemia-1 (Mcl-1 ) gene in mouse macrophages Raw264.7 and human macrophages THP-1,to screen out the cell lines with high levels of expression as the experimental cells,and based on the screening results to construct the short hairpin RNA(shRNA)eukaryotic expression plasmid targeting mice Mcl-1 gene for transfection and further screen out the shRNA expression plasmid with the most obvious effect in silencing Mcl-1 gene.Methods Semi-quantitative PCR method was used to detect the expression of Mcl-1 mRNA in the two kinds of macrophages.Western blotting was adopted to detect the expressions of Mcl-1 proteins in the two kinds of macrophages.Three different gene loci for Mcl-1 shRNA fragments were designed with small molecules interfering RNA (siRNA)software.Eukaryotic expression plasmid Mcl-1 shRNA 1-3 carrying the shRNA fragments was constructed by a company.And the eukaryotic expression plasmid vector was transfected into scavenger cells Raw264.7 mice via through the liposome.The transfection results 24 h and 48 h after transfection were observed under the inverted fluorescence microscope;Mcl-1 mRNA and protein expression were detected by real time quantitative PCR and Western blot,respectively.Results The relative expression levels of Mcl-1 mRNA and protein in mouse macrophages Raw264.7 were significantly higher than those of human macrophages (P <0.05).The shRNA expression vector constructed within 24 h and 48 h could decrease Mcl-1 mRNA and protein levels in Raw264.7 cells,especially with the most obvious silencing effect at 48 h.The 48-h transfection group differed significantly from normal group,liposome group and negative control group (P <0.05).Compared with Mcl-1shRNA1and Mcl-1shRNA2,Mcl-1shRNA3 had the strongest effect in silencing Mcl-1mRNA and protein.Conclusion We have successfully screened out experimental Raw264.7 cells and Mcl-1 shRNA eukaryotic expression plasmid which has an obvious silencing effect targeting on Mcl-1 in mice macrophages Raw264.7.