CAJ | 학술논문

目的研究低强度脉冲超声波(LIPUS)对培养成肌细胞增殖分化的影响,探讨其治疗效应的细胞分子机制.方法建立小鼠骨骼肌成肌细胞体外培养模型,将培养成肌细胞随机分增殖超声组、分化超声组、增殖对照组和分化对照组.增殖超声组和分化超声组均采用超声频率为1.5 MHz,平均强度为30 mW/cm2的低强度脉冲超声波辐射,每次20 min,每日1次,增殖超声组连续处理6d,分化超声组连续处理4d.增殖超声组与增殖对照组采用流式细胞仪进行细胞增殖动力学检测,免疫荧光染色检测成肌细胞生长因子MyoD、血红素加氧酶-1(HO-1)的表达,并对分化超声组与分化对照组进行肌球蛋白重链(MHC)染色和成肌细胞融合指数分析.结果增殖超声组成肌细胞进入活性细胞周期G2/M与S期细胞比例和增殖指数(PI)分别为(19.30±5.14)％,(37.00±8.72)％和(47.93±0.87)％,与增殖对照组的(10.33±1.53)％,(25.00±4.36)％和(38.66 ±0.67)％比较,差异具有统计学意义(P＜0.05)；增殖超声组HO-1荧光染色阳性比例和平均荧光强度分别为(82.03 ±5.14)％和(152.02±4.76)％,与对照组的(60.01±3.22)％和(138.70±5.08)％比较,差异具有统计学意义(P＜0.05)；分化超声组成肌细胞融合指数(18.73 ±6.81)％,与分化对照组的(37.52±11.23)％比较,差异有统计学意义(P＜0.05).结论低强度脉冲超声波促进成肌细胞的增殖,抑制其分化,且不改变其肌原属性,血红素加氧酶-1参与该调控过程.
목적 연구저강도맥충초성파(LIPUS)대배양성기세포증식분화적영향,탐토기치료효응적세포분자궤제.방법 건립소서골격기성기세포체외배양모형,장배양성기세포수궤분증식초성조、분화초성조、증식대조조화분화대조조.증식초성조화분화초성조균채용초성빈솔위1.5 MHz,평균강도위30 mW/cm2적저강도맥충초성파복사,매차20 min,매일1차,증식초성조련속처리6d,분화초성조련속처리4d.증식초성조여증식대조조채용류식세포의진행세포증식동역학검측,면역형광염색검측성기세포생장인자MyoD、혈홍소가양매-1(HO-1)적표체,병대분화초성조여분화대조조진행기구단백중련(MHC)염색화성기세포융합지수분석.결과 증식초성조성기세포진입활성세포주기G2/M여S기세포비례화증식지수(PI)분별위(19.30±5.14)％,(37.00±8.72)％화(47.93±0.87)％,여증식대조조적(10.33±1.53)％,(25.00±4.36)％화(38.66 ±0.67)％비교,차이구유통계학의의(P＜0.05)；증식초성조HO-1형광염색양성비례화평균형광강도분별위(82.03 ±5.14)％화(152.02±4.76)％,여대조조적(60.01±3.22)％화(138.70±5.08)％비교,차이구유통계학의의(P＜0.05)；분화초성조성기세포융합지수(18.73 ±6.81)％,여분화대조조적(37.52±11.23)％비교,차이유통계학의의(P＜0.05).결론 저강도맥충초성파촉진성기세포적증식,억제기분화,차불개변기기원속성,혈홍소가양매-1삼여해조공과정.
Objective To study in vitro the effects of low-intensity pulsed ultrasound (LIPUS) on the proliferation and differentiation of cultured myoblasts,and to explore the cellular and molecular mechanisms behind any therapeutic effect of LIPUS.Methods Myoblasts were isolated from the skeletal muscles of mice and cultured in vitro.Treatment and control groups of proliferating and differentiating myoblasts were defined.The treatment groups were exposed to LIPUS at 1.5 MHz and a spatial and temporal average intensity of 30 mW/cm2,for 20 min daily,the proliferation group for 6 consecutive days and the differentiation group for 4 consecutive days.The cell proliferation kinetics of the proliferation group were analyzed using flow cytometry.The expression of myogenic regulation factor MyoD and heme oxygenase-1 (HO-1) in the proliferation group,and of myosin heavy chain (MHC) in the differentiation group were examined by immunofluorescent staining.Myoblast fusion indexes were analyzed.Results In the LIPUS treatment groups the proliferating myoblasts had a higher ratio of active cells in the G2 and S phases (19.30％ ±5.14％,37.00％ ±8.72％),compared with the controls (10.33％ ± 1.53％,25.00％ ±4.36％),and the proliferation index increased significantly.The expression of HO-1 was up-regulated,while MyoD staining was unchanged.During the induction of differentiation,the myoblasts of the treatment group fused into smaller myotubes and the myoblast fusion index (18.73％ ± 6.81％) was significantly lower than that of the control group (37.52％ ± 11.23％),while MHC expression did not change markedly.Conclusion LIPUS can promote myoblast proliferation while inhibiting their differentiation,but it does not affect the cells' myogenic properties.HO-1 may be involved in the regulation process.