聚乙烯亚胺介导miR-2861模拟物转染MC3T3-E1细胞的体外成骨分化

doi:10.13481/j.1671-587x.20160502

Abstract

Abstract:

Objective: To transfect the non-viral vector polyethylenimine (PEI) mediated miR-2861 mimic into the MC3T3-E1 cell line,and to explore the transfection efficiency of PEI/miR-2861 complex and its effects on the proliferation and osteogenesis differentiation in pre-osteoblasts. Methods: The proper amount of PEI was blended with miR-2861 mimic and negative control (NC) separately in a ratio of N:P=10:1, and they were divided into experiment group and NC group. The NC/PEI complex acted as the NC group was used to eliminate the interference of osteogenesis from the addition of double-stranded RNA mimic. MTT assay was used to determine the optimal concentration of PEI/miR-2861 mimic complex. The fluorescence imaging technique and bulge-loop RT-PCR were used to detect the transfection efficiency and mRNA expression of miRNA-2861 in the cells with different concentrations(10,30,50, and 100 nmol·L^-1),separately. The osteogenesis ability of MC3T3 cells was identified with RT-PCR and Alizarin red staining with the selected concentration of PEI/miR-2861 by transient transfection. Results: Compared with blank control group, the proliferation rates of MC3T3 cells in 100 nmol·L^-1 PEI/miR-2861 group was decreased significantly at 72 h(P<0.05). With the increasing of transfected concentration the transfection efficiency of miRNA/PEI complex was increased gradually. The results of Alizarin red staining and quantitative analysis showed that calcium deposits were more and bigger in experiment group after induced for 21 d, while both in blank control group and NC group they were less. Conclusion: The miRNA-2861 mimic can be effectively transfected into the MC3T3-E1 cell line and expresses with a high level, which is mediated by PEI as the gene vector. miR-2861 mimic has a certain ability of promoting osteogenesis differentiation of MC3T3-E1 cells.

Key words: miRNA-2861 mimic, polyethylenimine, MC3T3-E1 cells, osteogenesis differentiation, gene therapy

CLC Number:

R336

FANG Tengjiaozi, LIU Jie, GU Zhongyi, GONG Haihuan, BU Wenhuan, DONG Yue, SUN Hongchen. Osteogenesis differentiation of MC3T3-E1 cells induced by miRNA-2861 mimic transfection mediated by polyethylenimine[J].Journal of Jilin University Medicine Edition, 2016, 42(05): 848-854.

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Osteogenesis differentiation of MC3T3-E1 cells induced by miRNA-2861 mimic transfection mediated by polyethylenimine

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