摘要
目的探讨乙酰辅酶A羧化酶2(ACC2)表达下调对高糖培养的人肾小管上皮细胞(HKC)脂质沉积及小管上皮细胞间充质转化(EMT)的影响及可能机制。方法构建ACC2基因特异性的短发夹双链RNA(shRNA)慢病毒感染HKC,将其分为5组:正常糖组(5.5 mmol/L葡萄糖,NG组)、高糖组(30 mmol/L葡萄糖,HG组)、高渗对照组(5 mmol/L葡萄糖+25 mmol/L甘露醇,HO组)、ACC2干扰组(感染含ACC2干扰序列慢病毒,ACC2-shRNA组)、干扰对照组(感染携带绿色萤光蛋白的阴性对照慢病毒,NC-shRNA组),分别处理96 h,Western blotting检测ACC2干扰效率及E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)表达,油红O染色检测细胞内脂质沉积,光镜下观察细胞形态变化。结果与HO组相比,HG组HKC细胞内脂质沉积增加,细胞拉长,E-cadherin表达减少,α-SMA表达增多(P<0.05);干扰ACC2基因后,高糖诱导的细胞内脂质沉积减少,细胞形态改善,E-cadherin和α-SMA表达趋于正常。结论下调ACC2表达可抑制高糖诱导的HKC脂质沉积和小管上皮细胞EMT。
Objective To explore the effects and mechanisms of RNA interference mediated by lentivirus down-regulating acetyl CoA carboxylase 2（ ACC2） on intracellular lipid accumulation and epithelial mesenchymal transition（ EMT） in high glucose cultured human renal proximal tubular epithelial cells（ HKC）. Methods The lentivirus vector based on small hairpin RNA（ shRNA） targeting ACC2 was constructed and delivered to HKC cells cultured in high glucose. Then the cells were divided into 5 groups： normal glucose group（ 5. 5 mmol /L glucose,NG group）,high glucose group（ 30 mmol /L glucose,HG group）,high osmotic pressure control group（ 5. 5mmol /L glucose plus 24. 5mmol /L mannitol,HO group）,ACC2-shRNA group（ 30 mmol /L glucose plus ACC2-shRNA lentivirous）,and NC-shRNA group（ 30 mmol /L glucose plus empty lentivirous carrying the green fluorescent protein as negative control）. After treated for 96 h,morphology change of HKC cells was observed under microscope,intracellular lipid accumulation was obtained by Oil Red O staining,and E-cadherin and α-SMA protein levels were detected by Western blotting. Results High glucose induced morphology change and intracellular lipid accumulation of cultured HKC cells.Protein level of E-cadherin was decreased,while α-SMA was increased（ P〈 0. 05）. Interference of ACC2 successfully restored high glucose stimulated morphology change and cellular lipid accumulation; the expression level of E-cadherin protein was increased and α-SMA protein was reduced. Conclusion Interference of ACC2 can ameliorate high glucosestimulated cellular lipid accumulation and epithelial mesenchymal transition,then improve renal fibrosis.
出处
《山东大学学报（医学版）》
CAS
北大核心
2014年第7期16-21,共6页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金(81200610)
山东省自然科学基金(ZR2009CM043)
