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

发布时间：2018-01-06 14:27

  本文关键词：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　出处：《吉林大学学报(医学版)》2016年05期 　论文类型：期刊论文

  更多相关文章： miRNA-模拟物 聚乙烯亚胺 MCT-E细胞系 成骨分化 基因治疗

【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100nmol·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,100nmol·L~(-1) miR-2861/PEI复合物作用72h时MC3T3细胞增殖率明显下降(P0.05)。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21d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。
[Abstract]:Objective: to transfect miRNA-2861 (miR-2861) mimetic into MC3T3-E1 cell line by non-viral vector polyethylene imide (PEI). To investigate the transfection efficiency of miR-2861/PEI complex in preosteoblasts and its effect on cell proliferation and osteogenic differentiation. Appropriate amounts of PEI were compared with miR-2861 and negative controls (. The gene / vector complex was formed by the proportion of element N / P 10. The miR-2861/PEI complex was used as the experimental group. NC/PEI complex was used as negative control group to eliminate interference of synthetic double-stranded gene on osteogenesis. MTT method was used to screen the optimal concentration of PEI complex miR-2861 mimics. Fluorescence imaging and stem ring RT-PCR were used to detect 1030. The transient transfection efficiency and miR-2861 expression of 50 and 100 nmol 路L ~ (-1) miR/PEI complexes in MC3T3 cells; QRT-PCR technique and alizarin red staining were used to detect the osteogenic ability of MC3T3 cells under transient transfection of miR-2861/PEI complex with selected concentration. Compared with the blank control group. The proliferation rate of MC3T3 cells decreased significantly at 72 h after treatment with 100nmol 路L ~ (-1) miR-2861/PEI complex, and increased with the increase of transfection concentration. The transfection efficiency of miR-2861/PEI complex in MC3T3 cells increased gradually. Alizarin red staining and quantitative analysis showed that more calcium-deposited nodules appeared in the experimental group after 21 days of induction. Conclusion: miR-2861 mimics can be effectively transfected into MC3T3-E1 cells and overexpressed in MC3T3-E1 cells by using PEI as vector. MiR-2861 mimics can promote the osteogenic differentiation of MC3T3-E1 cells.
【作者单位】： 吉林大学口腔医院病理科;
【基金】：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)
【分类号】：R781
【正文快照】： 微小RNA(microRNA,miRNA)是近年来发现的一类长约22nt的非编码单链RNA,可沉默转录后水平的基因表达从而发挥调节功能。作为内源性的RNA干涉(RNA interference,RNAi)途径,miRNA几乎参与了体内所有生物调控过程。在基因治疗中,由于miRNA能通过多个信号通路有效调控成骨细胞和破，

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