电针血清对饥饿条件下肌卫星细胞成肌分化抗原及自噬蛋白Beclin 1表达的影响  

Effect of electroacupuncture serum on expression of myogenic differentiation antigen and autophagy-related protein Beclin 1 in cultured muscle satellite cells under starvation

作　　者：刘通[1] 于佳妮[2] 刘悦[1] 邝伟川[1] 陈欢[3] 王小寅[1] 文希[1] 江烨[1] 邱晓佳[1] LIU Tong;YU Jia-ni;LIU Yue;KUANG Wei-chuan;CHEN Huan;WANG Xiao-yin;WEN Xi;JIANG Ye;QIU Xiao-jia(Department of Acupuncture and Rehabilitation,Guangdong Second Hospital of Traditional Chinese Medicine,Guangzhou 510095,China;Department of Rehabilitation,Guangdong Hospital of Traditional Chinese Medicine,Guangzhou 510120;Department of Acupunc mre-moxibustion,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029)

机构地区：[1]广东省第二中医院针灸康复科,广州510095 [2]广东省中医院康复科,广州510120 [3]南京医科大学第一附属医院针灸科,南京210029

出　　处：《针刺研究》2019年第11期799-804,共6页Acupuncture Research

基　　金：国家自然科学基金青年项目(No.81704179、81503641)

摘　　要：目的:观察电针血清对饥饿条件下肌卫星细胞成肌分化抗原(Myod)及自噬相关蛋白Beclin 1表达的影响,探讨电针调控肌卫星细胞增殖及自噬的机制。方法:电针"委中"穴7 d后获取并制备不同浓度电针血清。分别以不同浓度(10%、20%、30%)的电针血清及10%胎牛血清对体外培养的原代肌卫星细胞干预12 h及24 h,CCK-8法检测各组细胞不同时间点的增殖情况,确定促进细胞增殖的最佳血清浓度。以无血清培养基培养原代肌卫星细胞12 h后,将细胞随机分为无血清组、10%胎牛血清组、最佳浓度电针血清组,分别加入相应浓度的血清。Western blot法检测不同血清干预12 h和24 h后原代肌卫星细胞中细胞增殖蛋白Myod及自噬蛋白Beclin 1的表达水平。结果:10%电针血清、20%电针血清、30%电针血清干预24 h后,肌卫星细胞增殖能力均优于10%胎牛血清(P<0. 01),但3个浓度电针血清之间的肌卫星细胞增殖能力无明显差异(P>0. 05),故取10%电针血清作为最佳浓度。Western blot结果显示:血清干预12 h后,无血清组肌卫星细胞中Myod及Beclin 1表达水平与干预前比较差异无统计学意义(P>0. 05),10%胎牛血清组、10%电针血清组Myod表达水平较干预前升高(P<0. 05)、Beclin 1表达水平较干预前降低(P<0. 05);与同时点无血清组比较,10%胎牛血清组、10%电针血清组Myod表达水平升高(P<0. 05)、Beclin 1表达水平降低(P<0. 01)。血清干预24 h后,无血清组Myod及Beclin 1表达水平较12 h时明显降低(P<0. 01),10%胎牛血清组、10%电针血清组Myod表达水平较12 h时升高(P<0. 05)、Beclin 1表达水平较12 h时降低(P<0. 05);与同时点无血清组比较,10%胎牛血清组、10%电针血清组Myod及Beclin 1表达水平均升高(P<0. 01,P<0. 05)。结论:饥饿条件下电针血清可改善肌卫星细胞的营养不良环境,抑制其凋亡趋势,促进肌卫星细胞增殖,这种作用可能是通过改善细胞的过度自噬实现的。Objective To explore the effect of electroacupuncture(EA)serum on expression of myogenic differentiation antigen(Myod)and autophagy-related protein Beclin 1 in cultured muscle satellite cells of rats under starvation conditions.MethodsThe primary multifidus muscle satellite cells of one male SD rat were isolated and cultured to obtain the 3rd generation of cells. The EA serum was got from the rat received EA stimulation of bilateral"Weizhong"(BL40,2 Hz/10 Hz,1 mA,duration of 20 min,once daily for 7 days). The cell suspension(2×104/well)of the 3rdgeneration of cultured cells was transferred to each well of a 96-well plate in medium containing 10% fetal bovine serum(FBS).Twelve duplicate wells were set up for the blank control serum(without FBS),10% FBS,10% EA serum,20% EA serum and 30% EA serum groups and incubated for 12 h and 24 h,respectively. Each well was supplemented with 10 μL CCK-8 reagent to be incubated for 1 h again for observing the state of cell proliferation. After culturing the primary muscle satellite cells in serum-free medium for 12 h,the cells were randomly divided into serum-free group,10% fetal bovine serum group and optimal concentration electroacupuncture serum group,and serum of corresponding concentration was added respectively. The expression levels of Beclin 1 and cell-proliferation-related protein Myod were detected by Western blot.ResultsCCK-8 assay displayed that the proliferation levels were significantly higher at 12 h and 24 h after serum intervention in the 10% FBS,10% EA serum,20% EA serum and 30% EA serum groups than that in the blank control serum group(P<0. 01),and at 24 h in the 3 EA serum groups than in the 10% FBS group(P<0. 01),but without significant difference among the three EA serum groups(P>0. 05). As a result,10% EA serum was selected as the optimal concentration for Western blot tests. No significant difference was found in the expression levels of Myod and Beclin 1 proteins among the serum-free,10% FBS and 10% EA serum groups before intervention(P>0. 05),and ther

关 键 词：电针血清 原代肌卫星细胞 饥饿 自噬 成肌分化抗原 BECLIN 1 

分 类 号：R245.9+7[医药卫生—针灸推拿学]