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

吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (05): 848-854.doi: 10.13481/j.1671-587x.20160502

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

方滕姣子, 刘杰, 顾中一, 宫海环, 布文奂, 董悦, 孙宏晨

吉林大学口腔医院病理科, 吉林长春 130021

收稿日期:2016-01-10 出版日期:2016-09-28 发布日期:2016-09-29
通讯作者: 孙宏晨,教授,博士研究生导师(Tel:0431-88796010,E-mail:hcsun@mail.jlu.edu.cn) E-mail:hcsun@mail.jlu.edu.cn
作者简介:方滕姣子(1989-),女,北京市人,在读医学硕士,主要从事颌骨重塑机制和纳米材料与转基因方面的研究。
基金资助:
国家自然科学基金资助课题（81271111）；国家自然科学基金青年基金资助课题（81400488）；国家自然科学基金重大国际合作项目资助课题（81320108011）；吉林大学研究生创新基金资助课题（2015072）

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

FANG Tengjiaozi, LIU Jie, GU Zhongyi, GONG Haihuan, BU Wenhuan, DONG Yue, SUN Hongchen

Department of Pathology, Stomatology Hospital, Jilin University, Changchun 130021, China

Received:2016-01-10 Online:2016-09-28 Published:2016-09-29

摘要/Abstract

摘要：

目的：通过非病毒载体聚乙烯亚胺（PEI）介导miRNA-2861（miR-2861）模拟物转染MC3T3-E1细胞系，探讨miR-2861/PEI复合物在前成骨细胞中的转染效率及其对细胞增殖和成骨向分化的影响。方法：将适量的PEI分别与miR-2861和阴性对照（NC）以元素N/P=10的比例混合形成基因/载体复合物。将miR-2861/PEI复合物作为实验组，NC/PEI复合物作为阴性对照组以排除人工合成的双链基因对成骨作用的干扰。采用MTT法筛选PEI复合miR-2861模拟物的最佳使用浓度；应用荧光成像和茎环法RT-PCR技术分别检测10、30、50和100 nmol·L^-1 miR/PEI复合物对MC3T3细胞的瞬时转染效率和miR-2861的表达情况；采用qRT-PCR技术和茜素红染色检测用选定浓度瞬时转染miR-2861/PEI复合物作用下MC3T3细胞的成骨能力。结果：与空白对照组比较，100 nmol·L^-1 miR-2861/PEI复合物作用72h时MC3T3细胞增殖率明显下降（P<0.05）。随转染浓度增加，miR-2861/PEI复合物在MC3T3细胞中的转染效率逐渐升高。茜素红染色及定量分析，实验组诱导21 d后出现较多的钙盐沉积结节，而空白对照组和阴性对照组均较少。结论：以PEI作为载体可使miR-2861模拟物有效转染MC3T3-E1细胞并在细胞中高表达，miR-2861模拟物具有一定的促MC3T3-E1细胞成骨向分化的作用。

关键词: miRNA-2861模拟物, 聚乙烯亚胺, MC3T3-E1细胞系, 成骨分化, 基因治疗

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

中图分类号:

R336

方滕姣子, 刘杰, 顾中一, 宫海环, 布文奂, 董悦, 孙宏晨. 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[J]. 吉林大学学报(医学版), 2016, 42(05): 848-854.

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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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

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

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