L6细胞胰岛素抵抗的骨骼肌细胞模型  

Skeletal muscle models of insulin resistance induced by L6 cell line

作　　者：杨亮[1] 迟戈 张俊[3] 王良君[1] 刘丽娜[1] 樊淼[1] 刘海东[1] 李伟伟[1] 张健飞[3] 杨菁[4] 隋鸿锦[3] 

机构地区：[1]辽宁医学院基础学院解剖教研室,辽宁省锦州市121000 [2]辽宁省食品药品监督管理局技术审评中心,辽宁省沈阳市110001 [3]大连医科大学解剖教研室,辽宁省大连市116044 [4]辽宁医学院基础学院生物化学与分子生物学教研室,辽宁省锦州市121000

出　　处：《中国组织工程研究与临床康复》2009年第2期248-251,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：辽宁省教育厅资助课题(20072202)~~

摘　　要：背景:在胰岛素抵抗的细胞模型中,以游离脂肪酸诱导3T3-L1脂肪细胞和高胰岛素诱导的HepG2肝癌细胞最为常见,对分布最为广泛靶组织骨骼肌的胰岛素抵抗模型报道较少。目的:拟使用L6细胞株建立胰岛素抵抗的骨骼肌细胞模型。设计、时间及地点:细胞学体外观察,于2007-06/2008-05在辽宁医学院生物化学与分子生物学教研室完成。材料:L6细胞株由中国协和医科大学细胞中心提供。α-MEM培养基为美国Gibco公司产品。方法:复苏后的L6细胞置于α-MEM培养基中诱导培养14d,以考马斯亮蓝染色观察肌管出现来确定L6成肌细胞株分化完成。将分化后的细胞建立胰岛素抵抗模型,按1×106/孔接种于24孔板内,用含体积分数为0.02胎牛血清的α-MEM培养,以L6细胞贴满24孔板的板底为宜。换液后,向各孔内分别加入含体积分数为0.02牛血清白蛋白的α-MEM无血清培养基饥饿培养5h。设立2组,实验组的板孔内加入含1×10-7mol/L胰岛素的HEPES缓冲液(136mmol/LNaCl,4.7mmol/LKCl,1.25mmol/LMgSO4,1.2mmol/LCaCl2,0.2%牛血清白蛋白,20mmol/LHEPES,pH7.4)孵育1h;对照组的板孔内加入不含胰岛素的HEPES缓冲液孵育1h。弃上清后用HBS缓冲液冲洗,每孔内分别加入含0.1mol/L葡萄糖的HBS缓冲液100μL,孵育10min后取出置于冰板上快速回收上清。主要观察指标:考马斯亮蓝染色观察L6细胞出现肌管结构情况,葡萄糖氧化酶法检测上清中葡萄糖浓度。结果:诱导前L6细胞未见肌性结构出现,诱导分化后L6细胞内出现肌管结构。使用α-MEM无血清培养基饥饿培养1,3h,两组上清中葡萄糖浓度基本相似(P>0.05);使用α-MEM无血清培养基饥饿培养5h后,实验组上清中葡萄糖浓度明显高于对照组(P<0.05)。结论:诱导分化成肌后的L6骨骼肌细胞经α-MEM无血清培养基饥饿培养5h,在胰岛素浓度为1×10-7mol/L的刺激下可形成胰岛素抵抗的骨骼肌细胞模型。BACKGROUND： In insulin resistance models, 3T3-L1 adipocyte induced by free fatty acids or HepG2 hepatoma carcinoma cell induced by hyperinsulinism are common. However, the reports on target tissue of myoblast in insulin resistance skeletal muscle cell models are few. OBJECTIVE： To establish a skeletal muscle model of insulin resistance by using L6 cell line induced with high insulin method. DESIGN, TIME AND SETTING： The in vitro cytology observation was performed at the Department of Biochemistry and Molecular Biology, Liaoning Medical University between June 2007 and May 2008. MATERIALS： L6 cell line was supplied by the Cell Center of Peking Union Medical College. And the a -MEM medium was purchased from America Gibco Company. METHODS： L6 cell line was cultured with α -MEM medium for 14 days, until myotube could be observed by coomassie brilliant blue staining. The differentiated cells were inoculated in the 24-well board as 1×10^6/well and were cultivated by α -MEM with 2% bovine serum. After L6 cells covered the board, α -MEM serum-free medium with 2% bovine serum albumin （BSA） was added in each aperture for another 5 hours culture under starved condition. In the experimental group, HEPES buffer solution （Contains： 136 mmol/L NaCl, 4.7 mmol/L KCl, 1.25 mmol/L MgSO4, 1.2 mmol/L CaCl2, 2% bovine serum albumin, and 20 mmol/L HEPES, pH=7.4） with 1×10^-7 mol/L insulin was added into apertures of the board for 1 hour culture; in the control group, HEPES buffer solution without insulin were added into apertures, cultured for 1 hour. After that, eliminated the supernatant and washed with HBS buffer solution, 100μL HBS buffer solution with 0.1 mmol/mL glucose was added to each apertures, placed on the ice board to extract the supernatant after cultivating for 10 minutes. MAIN OUTCOME MEASURES： The appearance of myotube like structure was observed by coomassie brilliant blue staining, and the content of glucose in the supernatant was detected by using glucose oxidase method. RESULTS： The my

关 键 词：L6骨骼肌细胞 模型 α-MEM 胰岛素抵抗 

分 类 号：R587.1[医药卫生—内分泌]