N-Ac-L-Leu-PEI介导miR-34a复合物对MG63细胞生物学性能及成骨向分化的影响

发布时间：2018-10-15 16:13

【摘要】：目的:通过N-Ac-L-Leu-PEI介导miR-34a转染MG63细胞,检测N-Ac-L-Leu-PEI/miR-34a复合物表征,N-Ac-L-Leu-PEI/miR-34a复合物对MG63生物学性能的影响,N-Ac-L-Leu-PEI/miR-34a复合物对MG63成骨向分化的影响。方法:首先,为筛选出N-Ac-L-Leu-PEI/miR-34a最佳转染质量比,以N-Ac-L-Leu-PEI为载体按不同质量比静电装载miR-34a形成N-Ac-L-Leu-PEI/miR-34a复合物。采用马尔文粒度电位仪检测N-Ac-L-Leu-PEI/miR-34a复合物粒径、电位;琼脂糖凝胶电泳检测N-Ac-L-Leu-PEI装载miR-34a能力;应用荧光成像、流式细胞术及RT-PCR技术检测复合物对MG63细胞转染效率及miR-34a基因表达情况,筛选最佳转染质量比;其次,为评价N-Ac-L-Leu-PEI/miR-34a对MG63细胞生物学性能的影响,采用MTT检测复合物对MG63细胞增殖的影响;通过流式细胞术检测复合物对MG63细胞周期、凋亡的影响;最后,通过RT-PCR技术及Western blot技术检测复合物对MG63细胞内成骨相关因子在基因及蛋白表达水平的改变,探讨N-Ac-L-Leu-PEI/miR-34a对成骨细胞成骨向分化的调控。N-Ac-L-Leu-PEI/miR-34a复合物随质量比增加,粒径呈减小趋势,电位呈增大趋势,且质量比≥2:1时,miR-34a被N-Ac-L-Leu-PEI完全装载,形成稳定复合物。当质量比为4:1时,N-Ac-L-Leu-PEI/miR-34a复合物在MG63细胞中转染效率最高。与空白对照组比较,N-Ac-L-Leu-PEI/miR-34a复合物抑制细胞增殖,抑制细胞周期,对细胞凋亡无明显影响。RT-PCR和Western blot分析,与空白对照组比较,N-Ac-L-Leu-PEI/miR-34a组促进Runx2、SP7和ColⅠ成骨基因表达。结果:结论:以N-Ac-L-Leu-PEI作为载体可使miR-34a有效转染至MG63细胞并在细胞中高表达,miR-34a具有一定的抑制MG63细胞增殖、促进MG63细胞成骨向分化的作用。
[Abstract]:Aim: to investigate the characterization of N-Ac-L-Leu-PEI/miR-34a complexes, the effects of N-Ac-L-Leu-PEI/miR-34a complexes on the biological properties of MG63, and the effects of N-Ac-L-Leu-PEI/miR-34a complexes on the osteogenic differentiation of MG63 by N-Ac-L-Leu-PEI mediated miR-34a transfection into MG63 cells. Methods: firstly, in order to select the best transfection mass ratio of N-Ac-L-Leu-PEI/miR-34a, N-Ac-L-Leu-PEI was used as carrier and miR-34a was loaded with different mass ratios to form N-Ac-L-Leu-PEI/miR-34a complex. Ma Erwen granularity potentiometer was used to detect the particle size and potential of N-Ac-L-Leu-PEI/miR-34a complex; agarose gel electrophoresis was used to detect the ability of N-Ac-L-Leu-PEI to load miR-34a; fluorescence imaging, flow cytometry and RT-PCR techniques were used to detect the transfection efficiency and miR-34a gene expression of MG63 cells. Secondly, in order to evaluate the effect of N-Ac-L-Leu-PEI/miR-34a on the biological performance of MG63 cells, MTT was used to detect the effects of the complexes on the proliferation of MG63 cells; flow cytometry was used to detect the effects of the complexes on the cell cycle and apoptosis of MG63 cells. RT-PCR and Western blot techniques were used to detect the expression level of osteoblast-associated factors in MG63 cells. The regulation of N-Ac-L-Leu-PEI/miR-34a on osteoblast osteogenesis differentiation was studied. The size of N-Ac-L-Leu-PEI/miR-34a complex decreased with the increase of mass ratio. The potential showed an increasing trend, and when the mass ratio was greater than 2:1, miR-34a was completely loaded by N-Ac-L-Leu-PEI to form a stable complex. When the mass ratio was 4:1, the transfection efficiency of N-Ac-L-Leu-PEI/miR-34a complex was the highest in MG63 cells. Compared with the blank control group, N-Ac-L-Leu-PEI/miR-34a complex inhibited cell proliferation and cell cycle, but had no obvious effect on apoptosis. RT-PCR and Western blot analysis showed that N-Ac-L-Leu-PEI/miR-34a group promoted the expression of Runx2,SP7 and Col 鈪，

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