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

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