【摘要】：目的:通過N-Ac-L-Leu-PEI介導(dǎo)miR-34a轉(zhuǎn)染MG63細(xì)胞,檢測N-Ac-L-Leu-PEI/miR-34a復(fù)合物表征,N-Ac-L-Leu-PEI/miR-34a復(fù)合物對MG63生物學(xué)性能的影響,N-Ac-L-Leu-PEI/miR-34a復(fù)合物對MG63成骨向分化的影響。方法:首先,為篩選出N-Ac-L-Leu-PEI/miR-34a最佳轉(zhuǎn)染質(zhì)量比,以N-Ac-L-Leu-PEI為載體按不同質(zhì)量比靜電裝載miR-34a形成N-Ac-L-Leu-PEI/miR-34a復(fù)合物。采用馬爾文粒度電位儀檢測N-Ac-L-Leu-PEI/miR-34a復(fù)合物粒徑、電位;瓊脂糖凝膠電泳檢測N-Ac-L-Leu-PEI裝載miR-34a能力;應(yīng)用熒光成像、流式細(xì)胞術(shù)及RT-PCR技術(shù)檢測復(fù)合物對MG63細(xì)胞轉(zhuǎn)染效率及miR-34a基因表達(dá)情況,篩選最佳轉(zhuǎn)染質(zhì)量比;其次,為評價N-Ac-L-Leu-PEI/miR-34a對MG63細(xì)胞生物學(xué)性能的影響,采用MTT檢測復(fù)合物對MG63細(xì)胞增殖的影響;通過流式細(xì)胞術(shù)檢測復(fù)合物對MG63細(xì)胞周期、凋亡的影響;最后,通過RT-PCR技術(shù)及Western blot技術(shù)檢測復(fù)合物對MG63細(xì)胞內(nèi)成骨相關(guān)因子在基因及蛋白表達(dá)水平的改變,探討N-Ac-L-Leu-PEI/miR-34a對成骨細(xì)胞成骨向分化的調(diào)控。N-Ac-L-Leu-PEI/miR-34a復(fù)合物隨質(zhì)量比增加,粒徑呈減小趨勢,電位呈增大趨勢,且質(zhì)量比≥2:1時,miR-34a被N-Ac-L-Leu-PEI完全裝載,形成穩(wěn)定復(fù)合物。當(dāng)質(zhì)量比為4:1時,N-Ac-L-Leu-PEI/miR-34a復(fù)合物在MG63細(xì)胞中轉(zhuǎn)染效率最高。與空白對照組比較,N-Ac-L-Leu-PEI/miR-34a復(fù)合物抑制細(xì)胞增殖,抑制細(xì)胞周期,對細(xì)胞凋亡無明顯影響。RT-PCR和Western blot分析,與空白對照組比較,N-Ac-L-Leu-PEI/miR-34a組促進(jìn)Runx2、SP7和ColⅠ成骨基因表達(dá)。結(jié)果:結(jié)論:以N-Ac-L-Leu-PEI作為載體可使miR-34a有效轉(zhuǎn)染至MG63細(xì)胞并在細(xì)胞中高表達(dá),miR-34a具有一定的抑制MG63細(xì)胞增殖、促進(jìn)MG63細(xì)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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