本文選題：低氧 + C2C12成肌細(xì)胞��； 參考：《皖南醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:機(jī)械通氣是急慢性呼吸衰竭的主要治療手段,機(jī)械通氣過程中可能由于多種因素引起膈肌收縮力下降并進(jìn)行性加重,臨床主要表現(xiàn)為脫機(jī)困難,即呼吸機(jī)相關(guān)膈肌功能障礙(ventilator-induced diaphragmatic dysfunction,VIDD)。研究表明機(jī)械通氣所致脂質(zhì)代謝異常及其觸發(fā)的線粒體損傷是導(dǎo)致VIDD的重要因素,但其啟動調(diào)控機(jī)制尚不明確。雷帕霉素靶蛋白(mTOR)激活在動脈粥樣硬化脂質(zhì)代謝中起重要調(diào)控作用,但其在VIDD中的作用尚不清楚。機(jī)械通氣導(dǎo)致膈肌氧供減少所引起的低氧環(huán)境可能是膈肌纖維脂質(zhì)沉積的刺激因素,從而導(dǎo)致膈肌氧化應(yīng)激增強(qiáng)及膈肌功能障礙。推測機(jī)械通氣過程中脂質(zhì)代謝紊亂引起mTOR信號通路的激活,導(dǎo)致膈肌脂質(zhì)沉積的加重,加速機(jī)械通氣早期膈肌功能障礙的發(fā)生發(fā)展。本研究擬通過建立C2C12小鼠成肌細(xì)胞低氧環(huán)境模型,從細(xì)胞水平觀察低氧環(huán)境下,mTOR通路激活對細(xì)胞脂質(zhì)沉積的作用,揭示機(jī)械通氣條件下膈肌萎縮的啟動和調(diào)節(jié)機(jī)制,為臨床VIDD的預(yù)防和治療提供理論基礎(chǔ)。方法:C2C12小鼠成肌細(xì)胞在10%胎牛血清DMEM高糖培養(yǎng)基培養(yǎng)傳代后,在2%馬血清高糖DMEM高糖培養(yǎng)基誘導(dǎo)分化成具有肌管后,顯微鏡拍照觀察細(xì)胞形態(tài)變化,并分為四個實(shí)驗(yàn)組:1)對照組:常氧(37℃、5%CO2)二氧化碳培養(yǎng)箱中的分化成具有肌管的C2C12成肌細(xì)胞。2)低氧組:在低氧條件(5%二氧化碳、1%氧氣、94%氮?dú)?下分別處理分化成肌管的C2C12成肌細(xì)胞12小時,24小時。3)雷帕霉素組:在分化成肌管的C2C12成肌細(xì)胞培養(yǎng)基中加入終濃度為10ng/ml雷帕霉素分別處理12小時,24小時。4)雷帕霉素加低氧組:終濃度為10ng/ml雷帕霉素的分化成肌管的C2C12成肌細(xì)胞在低氧條件下分別處理12小時,24小時。油紅染色用顯微鏡拍照觀察各組各時間點(diǎn)細(xì)胞脂質(zhì)沉積情況,另收集細(xì)胞,提取蛋白行Western blotting檢測各組各時間點(diǎn)p-mTOR、p-4EBP1、p-S6K1、SCAP、SREBP-2、LDLR等蛋白表達(dá)水平,并用細(xì)胞實(shí)時熒光法檢測mTOR、4EBP1、S6K1、SCAP、SREBP-2、LDLR mRNA表達(dá)情況。結(jié)果:(1)用2%馬血清高糖培養(yǎng)基C2C12成肌細(xì)胞,7天左右細(xì)胞分化成具有肌管形態(tài)。(2)油紅染色,結(jié)果顯示各低氧組細(xì)胞有脂質(zhì)沉積,雷帕霉素可以減輕細(xì)胞內(nèi)脂質(zhì)沉積。(3)低氧環(huán)境下,細(xì)胞內(nèi)mTOR信號通路和脂質(zhì)代謝通路相關(guān)因子蛋白和mRNA表達(dá)增加,并伴隨者細(xì)胞內(nèi)脂質(zhì)的沉積。(4)隨著低氧刺激時間的延長,mTOR信號通路和脂質(zhì)代謝通路相關(guān)因子蛋白和mRNA表達(dá)增加,雷帕霉素能夠減輕細(xì)胞mTOR信號通路和脂質(zhì)代謝通路蛋白和mRNA表達(dá)增加,并且減輕脂質(zhì)沉積。結(jié)論:(1)脂質(zhì)代謝異常可能是mTOR信號通路激活的刺激因素(2)mTOR信號通路激活加重細(xì)胞脂質(zhì)的沉積,可能是調(diào)控細(xì)胞脂質(zhì)代謝的一個靶點(diǎn)
[Abstract]:Objective: mechanical ventilation is the main treatment for acute and chronic respiratory failure. That is ventilator-induced diaphragmatic dysfunction VIDD. The results showed that the abnormal lipid metabolism induced by mechanical ventilation and mitochondrial damage triggered by mechanical ventilation were important factors leading to VIDD, but the mechanism of its priming regulation was not clear. The activation of rapamycin target protein mTORplays an important role in the lipid metabolism of atherosclerosis, but its role in VIDD is unclear. The hypoxic environment induced by mechanical ventilation may be a stimulating factor for the deposition of lipid in the diaphragm, which may lead to the increase of oxidative stress and the dysfunction of the diaphragm. It is speculated that lipid metabolism disorder during mechanical ventilation causes activation of mTOR signaling pathway, exacerbates lipid deposition of diaphragm, and accelerates the occurrence and development of diaphragm dysfunction in the early stage of mechanical ventilation. The aim of this study was to investigate the effects of activation of mTOR pathway on lipid deposition in C2C12 mouse myoblast under hypoxic environment, and to reveal the mechanism of initiation and regulation of diaphragmatic atrophy under mechanical ventilation. To provide a theoretical basis for the prevention and treatment of clinical VIDD. Methods the myoblasts were cultured in 10% fetal bovine serum DMEM high sugar medium, and then differentiated into myotubes on 2% equine serum high glucose DMEM medium. The morphologic changes of the cells were observed by microscope. And was divided into four experimental groups: control group: normal oxygen 37 鈩，

本文編號：1992465