【摘要】：目的探討miR-378對(duì)高糖作用下成骨細(xì)胞增殖分化的影響及可能的機(jī)制。方法1)采用慢病毒表達(dá)載體進(jìn)行細(xì)胞轉(zhuǎn)染;2)采用MTT法建立高糖模型;3)觀察高糖環(huán)境對(duì)成骨細(xì)胞增殖分化的影響:通過茜素紅染色法了解對(duì)成骨細(xì)胞礦化的影響;分別通過MTT法和流式細(xì)胞儀判斷成骨細(xì)胞增殖和凋亡情況;通過對(duì)硝基苯酚法檢測成骨特異標(biāo)記物堿性磷酸酶(ALP)和通過Real time PCR方法檢測成骨相關(guān)基因表達(dá);4)確定miR-378靶基因:采用生物信息學(xué)預(yù)測結(jié)合熒光素酶報(bào)告檢測和Real time PCR、Western Blot檢測;5)采用RNA干擾技術(shù)構(gòu)建靶基因shRNA質(zhì)粒,并通過慢病毒轉(zhuǎn)染的方式沉沒靶基因在MC3T3-E1細(xì)胞中的表達(dá);6)miR-378對(duì)PI3K/Akt信號(hào)通路的影響以及加入PI3K/Akt信號(hào)通路阻斷劑后目的蛋白的變化:采用Western Blot方法檢測目的蛋白(p-PI3K,PI3K;p-Akt,Akt;CytC,Apaf-1,Bax)。結(jié)果1)確定25.5 mM高糖濃度為后續(xù)實(shí)驗(yàn)的高糖模型;2)高糖可抑制MC3T3-E1細(xì)胞的生長和ALP活性以及成骨相關(guān)蛋白的表達(dá);高糖可使茜素紅染色區(qū)域和鈣化減少,抑制MC3T3-E1細(xì)胞的礦化;高糖可促進(jìn)MC3T3-E1細(xì)胞凋亡;高糖條件下MC3T3-E1細(xì)胞內(nèi)miR-378表達(dá)下調(diào);3)與對(duì)照組(HG組和HG+miR-con組)相比,過表達(dá)miR-378(HG+miR-378組)可提高高糖作用下MC3T3-E1細(xì)胞內(nèi)miR-378的含量;可減輕高糖對(duì)MC3T3-E1細(xì)胞生長的抑制以及高糖引起的MC3T3-E1細(xì)胞凋亡,促進(jìn)高糖下MC3T3-E1的礦化,提高ALP活性以及促進(jìn)成骨相關(guān)蛋白的表達(dá);4)生物信息學(xué)預(yù)測和雙熒光素酶報(bào)告基因?qū)嶒?yàn)以及real time PCR和Western blot檢測證實(shí)miR-378的靶基因是caspase-3;5)過表達(dá)mi R-378可下調(diào)成骨細(xì)胞caspase-3 mRNA和蛋白質(zhì)水平表達(dá);干擾CASP3可提高被高糖抑制的ALP活性以及促進(jìn)成骨相關(guān)基因的表達(dá);6)mi R-378過表達(dá)顯著促進(jìn)了PI3K/Akt信號(hào)通路p-PI3K和p-AKt蛋白的表達(dá),而加入PI3K/Akt信號(hào)通路阻斷劑LY294002后,p-PI3K和p-AKt蛋白表達(dá)受阻,表明miR-378是通過PI3K/Akt通路發(fā)揮作用。miR-378過表達(dá)可抑制凋亡相關(guān)蛋白CytC、Apaf-1和Bax的表達(dá);而加入PI3K/Akt信號(hào)通路阻斷劑LY294002后,凋亡相關(guān)蛋白CytC、Apaf-1和Bax的蛋白表達(dá)又重新恢復(fù),促進(jìn)MC3T3-E1細(xì)胞細(xì)胞凋亡。以上結(jié)果提示,mi R-378是通過PI3K/Akt信號(hào)通路調(diào)節(jié)凋亡相關(guān)蛋白的表達(dá),從而影響成骨細(xì)胞生長、分化。結(jié)論過表達(dá)miR-378可通過下調(diào)靶基因caspase-3改善高糖抑制的成骨細(xì)胞分化。作用機(jī)制可能包括:通過直接靶向調(diào)控caspase-3,抑制通過caspase-3依賴方式引起的細(xì)胞凋亡;通過PI3K/Akt信號(hào)通路調(diào)控凋亡相關(guān)蛋白的表達(dá),減少成骨細(xì)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，

本文編號(hào)：2507252