發(fā)布時(shí)間：2018-04-01 19:08

  本文選題：腎疾病　切入點(diǎn)：萎縮性肌疾病　出處：《第二軍醫(yī)大學(xué)學(xué)報(bào)》2017年06期

【摘要】：目的明確法舒地爾(fasudil)能否阻斷Rho相關(guān)卷曲螺旋形成蛋白激酶1(ROCK1)激活導(dǎo)致的C2C12成肌細(xì)胞呼吸功能異常介導(dǎo)的肌肉萎縮。方法體外培養(yǎng)C2C12成肌細(xì)胞,給予2%的馬血清誘導(dǎo)分化為成熟肌小管細(xì)胞。然后根據(jù)給予不同處理將其分成4組:Ad-GFP組,僅感染GFP腺病毒載體;Ad-ROCK1組,感染ROCK1腺病毒載體誘導(dǎo)細(xì)胞過(guò)表達(dá)ROCK1;Ad-GFPF組,感染GFP腺病毒載體,并給予10μmol/L法舒地爾刺激;Ad-ROCK1F組,感染ROCK1腺病毒載體誘導(dǎo)細(xì)胞過(guò)表達(dá)ROCK1,并給予10μmol/L法舒地爾刺激。通過(guò)Seahorse能量代謝分析儀評(píng)估不同刺激條件下C2C12成肌細(xì)胞的細(xì)胞耗氧率(OCR)及胞外酸化率(ECAR),以明確ROCK1過(guò)表達(dá)和法舒地爾刺激對(duì)C2C12成肌細(xì)胞呼吸功能的影響。采用MitoTracker○R紅色熒光探針測(cè)定線粒體裂變情況。用蛋白質(zhì)印跡法檢測(cè)ROCK1、線粒體動(dòng)力相關(guān)蛋白1(Drp1)及其磷酸化蛋白p-Drp1、肌肉萎縮相關(guān)蛋白E3泛素連接酶肌肉環(huán)指蛋白1(MuRF1)和肌肉萎縮F-box(MAFbx,又稱Atrogin1)的表達(dá)。結(jié)果 Seahorse分析結(jié)果顯示,與Ad-GFP組比較,AdROCK1組C2C12成肌細(xì)胞OCR及ECAR明顯增加,細(xì)胞的基礎(chǔ)呼吸、最大呼吸及偶聯(lián)ATP產(chǎn)生所需的呼吸增加,差異均有統(tǒng)計(jì)學(xué)意義(P0.01);MitoTracker○R熒光探針結(jié)果顯示Ad-ROCK1組細(xì)胞線粒體裂變?cè)黾?線粒體大小頻數(shù)分布左移。Ad-ROCK1F組C2C12成肌細(xì)胞OCR和ECAR較Ad-ROCK1組明顯減少(P0.05),基礎(chǔ)呼吸和最大呼吸也降低(P0.05)。Ad-ROCK1F組p-Drp1/Drp1比例、ROCK1、MuRF1和Atrogin1的表達(dá)均較Ad-ROCK1組減少(P0.05)。結(jié)論法舒地爾作為ROCK1的抑制劑,可阻斷體外培養(yǎng)C2C12成肌細(xì)胞ROCK1過(guò)表達(dá)導(dǎo)致的細(xì)胞呼吸異常,并減少線粒體動(dòng)力相關(guān)蛋白的活性和肌肉萎縮相關(guān)蛋白的表達(dá)。
[Abstract]:Aim to determine whether fasudil can block the respiratory dysfunction of C2C12 myoblasts induced by the activation of Rho-associated crimp-forming protein kinase 1-ROCK1.Methods C2C12 myoblasts were cultured in vitro and differentiated into mature myotubule cells with 2% horse serum.Then they were divided into 4 groups according to the different treatments, only infected with GFP adenovirus vector Ad-ROCK1, infected with ROCK1 adenovirus vector ROCK1, infected with GFP adenovirus vector, and treated with 10 渭 mol/L fastidil to stimulate Ad-ROCK1F group.Adenovirus vector infected with ROCK1 induced overexpression of Rock1 and was stimulated by 10 渭 mol/L faridil.The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of C2C12 myoblasts under different stimulation conditions were evaluated by Seahorse Energy Metabolism Analyzer to determine the effects of ROCK1 overexpression and fasudil stimulation on the respiratory function of C2C12 myoblasts.Mitochondrial fission was determined by MitoTracker0R red fluorescence probe.The expressions of rock 1, mitochondrial motility associated protein 1 (mtp1) and its phosphorylated protein p-Drp1, muscle dystrophy associated protein E3 ubiquitin ligase muscle ring ligase protein 1 MuRF1) and muscle atrophy F-box MAFbx (also known as Atrogin1) were detected by Western blotting.Results compared with Ad-GFP group, OCR and ECAR of C2C12 myoblast in AdROCK1 group were significantly increased, and the basic respiration, maximum respiration and the respiration required by coupling ATP were increased in AdROCK1 group.The difference was statistically significant (P 0.01). The results of Mito Tracker0R fluorescence probe showed that mitochondrial fission increased in Ad-ROCK1 group.
【作者單位】： 第二軍醫(yī)大學(xué)長(zhǎng)海醫(yī)院腎內(nèi)科;
【基金】：國(guó)家自然科學(xué)基金(81470970)~~
【分類號(hào)】：R692

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