miR-378对高糖作用下成骨细胞分化的调控作用及机制探讨
发布时间：2019-06-28 11:07
【摘要】：目的探讨miR-378对高糖作用下成骨细胞增殖分化的影响及可能的机制。方法1)采用慢病毒表达载体进行细胞转染;2)采用MTT法建立高糖模型;3)观察高糖环境对成骨细胞增殖分化的影响:通过茜素红染色法了解对成骨细胞矿化的影响;分别通过MTT法和流式细胞仪判断成骨细胞增殖和凋亡情况;通过对硝基苯酚法检测成骨特异标记物碱性磷酸酶(ALP)和通过Real time PCR方法检测成骨相关基因表达;4)确定miR-378靶基因:采用生物信息学预测结合荧光素酶报告检测和Real time PCR、Western Blot检测;5)采用RNA干扰技术构建靶基因shRNA质粒,并通过慢病毒转染的方式沉没靶基因在MC3T3-E1细胞中的表达;6)miR-378对PI3K/Akt信号通路的影响以及加入PI3K/Akt信号通路阻断剂后目的蛋白的变化:采用Western Blot方法检测目的蛋白(p-PI3K,PI3K;p-Akt,Akt;CytC,Apaf-1,Bax)。结果1)确定25.5 mM高糖浓度为后续实验的高糖模型;2)高糖可抑制MC3T3-E1细胞的生长和ALP活性以及成骨相关蛋白的表达;高糖可使茜素红染色区域和钙化减少,抑制MC3T3-E1细胞的矿化;高糖可促进MC3T3-E1细胞凋亡;高糖条件下MC3T3-E1细胞内miR-378表达下调;3)与对照组(HG组和HG+miR-con组)相比,过表达miR-378(HG+miR-378组)可提高高糖作用下MC3T3-E1细胞内miR-378的含量;可减轻高糖对MC3T3-E1细胞生长的抑制以及高糖引起的MC3T3-E1细胞凋亡,促进高糖下MC3T3-E1的矿化,提高ALP活性以及促进成骨相关蛋白的表达;4)生物信息学预测和双荧光素酶报告基因实验以及real time PCR和Western blot检测证实miR-378的靶基因是caspase-3;5)过表达mi R-378可下调成骨细胞caspase-3 mRNA和蛋白质水平表达;干扰CASP3可提高被高糖抑制的ALP活性以及促进成骨相关基因的表达;6)mi R-378过表达显著促进了PI3K/Akt信号通路p-PI3K和p-AKt蛋白的表达,而加入PI3K/Akt信号通路阻断剂LY294002后,p-PI3K和p-AKt蛋白表达受阻,表明miR-378是通过PI3K/Akt通路发挥作用。miR-378过表达可抑制凋亡相关蛋白CytC、Apaf-1和Bax的表达;而加入PI3K/Akt信号通路阻断剂LY294002后,凋亡相关蛋白CytC、Apaf-1和Bax的蛋白表达又重新恢复,促进MC3T3-E1细胞细胞凋亡。以上结果提示,mi R-378是通过PI3K/Akt信号通路调节凋亡相关蛋白的表达,从而影响成骨细胞生长、分化。结论过表达miR-378可通过下调靶基因caspase-3改善高糖抑制的成骨细胞分化。作用机制可能包括:通过直接靶向调控caspase-3,抑制通过caspase-3依赖方式引起的细胞凋亡;通过PI3K/Akt信号通路调控凋亡相关蛋白的表达,减少成骨细胞凋亡。
[Abstract]:Objective to investigate the effect of miR-378 on the proliferation and differentiation of osteoblasts induced by high glucose and its possible mechanism. Methods 1) lentivirus expression vector was used to transfer cells, 2) high glucose model was established by MTT method, 3) the effect of high glucose environment on the proliferation and differentiation of osteoblasts was observed: the effect on osteoblast mineralization was investigated by alizalin red staining, and the proliferation and apoptosis of osteoblasts were judged by MTT assay and flow cytometry, respectively. Alkaline phosphatase (ALP) was detected by p-nitrophenol method and osteogenic related gene expression was detected by Real time PCR. 4) miR-378 target gene was determined: bioinformatics prediction combined with luciferase report detection and Real time PCR,Western Blot detection; 5) target gene shRNA plasmid was constructed by RNA interference technique, and the expression of target gene in MC3T3-E1 cells was sunk by lentivirus transfer. 6) the effect of miR-378 on PI3K/Akt signaling pathway and the changes of target protein after adding PI3K/Akt signal pathway blocker: the target protein (p 鈮，
本文编号：2507252
[Abstract]:Objective to investigate the effect of miR-378 on the proliferation and differentiation of osteoblasts induced by high glucose and its possible mechanism. Methods 1) lentivirus expression vector was used to transfer cells, 2) high glucose model was established by MTT method, 3) the effect of high glucose environment on the proliferation and differentiation of osteoblasts was observed: the effect on osteoblast mineralization was investigated by alizalin red staining, and the proliferation and apoptosis of osteoblasts were judged by MTT assay and flow cytometry, respectively. Alkaline phosphatase (ALP) was detected by p-nitrophenol method and osteogenic related gene expression was detected by Real time PCR. 4) miR-378 target gene was determined: bioinformatics prediction combined with luciferase report detection and Real time PCR,Western Blot detection; 5) target gene shRNA plasmid was constructed by RNA interference technique, and the expression of target gene in MC3T3-E1 cells was sunk by lentivirus transfer. 6) the effect of miR-378 on PI3K/Akt signaling pathway and the changes of target protein after adding PI3K/Akt signal pathway blocker: the target protein (p 鈮，
本文编号：2507252
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