Wang C.,Shihezi University |
Wang C.,Key Laboratory of Endemic and Ethnic Diseases in Xinjiang of Education Ministry |
Wang X.-M.,Shihezi University |
Wang F.-Y.,Shihezi University |
And 13 more authors.
Journal of Xi'an Jiaotong University (Medical Sciences) | Year: 2015
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. ©, 2015, Xi'an Medical University. All right reserved.