本文關(guān)鍵詞：聚乙烯亞胺介導(dǎo)miR-2861模擬物轉(zhuǎn)染MC3T3-E1細(xì)胞的體外成骨分化　出處：《吉林大學(xué)學(xué)報(醫(yī)學(xué)版)》2016年05期 　論文類型：期刊論文

  更多相關(guān)文章： miRNA-模擬物 聚乙烯亞胺 MCT-E細(xì)胞系 成骨分化 基因治療

【摘要】：目的:通過非病毒載體聚乙烯亞胺(PEI)介導(dǎo)miRNA-2861(miR-2861)模擬物轉(zhuǎn)染MC3T3-E1細(xì)胞系,探討miR-2861/PEI復(fù)合物在前成骨細(xì)胞中的轉(zhuǎn)染效率及其對細(xì)胞增殖和成骨向分化的影響。方法:將適量的PEI分別與miR-2861和陰性對照(NC)以元素N/P=10的比例混合形成基因/載體復(fù)合物。將miR-2861/PEI復(fù)合物作為實驗組,NC/PEI復(fù)合物作為陰性對照組以排除人工合成的雙鏈基因?qū)Τ晒亲饔玫母蓴_。采用MTT法篩選PEI復(fù)合miR-2861模擬物的最佳使用濃度;應(yīng)用熒光成像和莖環(huán)法RT-PCR技術(shù)分別檢測10、30、50和100nmol·L~(-1) miR/PEI復(fù)合物對MC3T3細(xì)胞的瞬時轉(zhuǎn)染效率和miR-2861的表達情況;采用qRT-PCR技術(shù)和茜素紅染色檢測用選定濃度瞬時轉(zhuǎn)染miR-2861/PEI復(fù)合物作用下MC3T3細(xì)胞的成骨能力。結(jié)果:與空白對照組比較,100nmol·L~(-1) miR-2861/PEI復(fù)合物作用72h時MC3T3細(xì)胞增殖率明顯下降(P0.05)。隨轉(zhuǎn)染濃度增加,miR-2861/PEI復(fù)合物在MC3T3細(xì)胞中的轉(zhuǎn)染效率逐漸升高。茜素紅染色及定量分析,實驗組誘導(dǎo)21d后出現(xiàn)較多的鈣鹽沉積結(jié)節(jié),而空白對照組和陰性對照組均較少。結(jié)論:以PEI作為載體可使miR-2861模擬物有效轉(zhuǎn)染MC3T3-E1細(xì)胞并在細(xì)胞中高表達,miR-2861模擬物具有一定的促MC3T3-E1細(xì)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.
【作者單位】： 吉林大學(xué)口腔醫(yī)院病理科;
【基金】：國家自然科學(xué)基金資助課題(81271111);國家自然科學(xué)基金青年基金資助課題(81400488);國家自然科學(xué)基金重大國際合作項目資助課題(81320108011) 吉林大學(xué)研究生創(chuàng)新基金資助課題(2015072)
【分類號】：R781
【正文快照】： 微小RNA(microRNA,miRNA)是近年來發(fā)現(xiàn)的一類長約22nt的非編碼單鏈RNA,可沉默轉(zhuǎn)錄后水平的基因表達從而發(fā)揮調(diào)節(jié)功能。作為內(nèi)源性的RNA干涉(RNA interference,RNAi)途徑,miRNA幾乎參與了體內(nèi)所有生物調(diào)控過程。在基因治療中,由于miRNA能通過多個信號通路有效調(diào)控成骨細(xì)胞和破，

本文編號：1388265