脂多糖诱导体外培养骨骼肌卫星细胞表达肝细胞生长因子的变化

Expression of hepatocyte growth factor in lipopolysaccharide stimulated skeletal muscle satellite cells

目的:研究证实,在众多生长因子中,肝细胞生长因子无论在体外还是体内都可以激活静止状态的肌卫星细胞,修复受损肌肉。采用脂多糖刺激体外培养的骨骼肌卫星细胞,观察肌卫星细胞产生肝细胞生长因子以及肌卫星细胞增殖分化的变化。方法:实验于2006-07/2007-05在华中科技大学同济医学院同济医院创伤外科实验室完成。①实验材料:健康SD成年雄性大鼠,体质量150～200g,由华中科技大学同济医学院实验动物中心提供。实验过程中对动物处置符合实验动物伦理学标准。②实验方法:分离SD大鼠后肢部分股四头肌肉进行骨骼肌卫星细胞的培养。取第2代细胞爬片,待细胞增殖到80%密度时,丙酮固定细胞,常规处理后进行α-sarcometricactin细胞免疫化学染色鉴定,以成纤维细胞作为阴性对照。取第3代骨骼肌卫星细胞,应用0,5,10,20mg/L脂多糖刺激体外培养的大鼠骨骼肌卫星细胞,采用ELISA方法测细胞培养液中的肝细胞生长因子的浓度。取第4代骨骼肌卫星细胞,用含体积分数为0.10胎牛血清的培养基配制成单个细胞悬液,调整浓度为5×107L-1,分成两组,一组加入10mg/L脂多糖,另一组不加任何干预。采用噻唑蓝(MTT)法测定卫星细胞的增殖率。结果:①以未经脂多糖处理的无血清培养基培养的肌肉卫星细胞培养液为对照,将对照组和5,10,20mg/L脂多糖处理组比较,各实验组肝细胞生长因子浓度明显高于对照组(P<0.05)。②5,10,20mg/L脂多糖刺激骨骼肌卫星细胞分泌的肝细胞生长因子水平差异无显著性。③肝细胞生长因子在脂多糖刺激后36h分泌浓度最高。④脂多糖刺激肌卫星细胞的增殖分化明显高于未经刺激的肌卫星细胞。结论:①脂多糖可诱导骨骼肌卫星细胞自分泌肝细胞生长因子。②不同质量浓度脂多糖培养基中肝细胞生长因子水平差异无统计学意义。③脂多糖具有促进骨骼肌卫星细胞增殖分化的效应。

AIM： It is confirmed that many of the growth factor, hepatocyte growth factor either in vitro or in vivo can be activated static muscle satellite cells to repair damaged muscle. Using lipopolysaccharide （LPS） in vitro stimulation of skeletal muscle satellite cells, this article is aimed to observe the changes of muscle satellite cells hepatocyte growth factor and muscle satellite cell proliferation and differentiation. METHODS： The experiment was performed at Laboratory of Department of Traumatic Surgery, Tongji Hospital. Tongji Medical College, Huazhong University of Science and Technology from July 2006 to May 2007. ①SD healthy adult male rats at 150-200 g were offered by Experimental Animal Center of Huazhong Tongji Medical College of Huazhong University of Science and Technology. Experiments on animals met experimental animal ethical standards. ②Quadriceps muscle of thigh from SD rats hind legs was used for satellite cell culture. From the first two cells slide, cell proliferation to be 80% density, acetone was utilized to fix cells. The conventional treatment for α-actin sarcometric immune cells staining was performed and fibroblasts as negative controls. From the first three-generation muscle satellite cells, 0, 5, 10 and 20 mg/L in vitro LPS was applied to stimulate rat skeletal muscle satellite cells. By enzyme-linked immunosorbent assay method, concentration of the hepatocyte growth factor was determined. From the fourth-generation skeletal muscle satellite cells, medium preparation containing the volume fraction of 0.10 fetal calf serum was made into a single cell suspension, adjusting the concentration of 5×10^7 L^-1. The suspension was divided into two groups, cells of one group received the 10 mg/L LPS, and the other group without any intervention. Satellite cell proliferation rate was determined by thiazole. RESULTS： ①Muscle satellite cell culture medium cultured by serum-free culture medium without LPS as the control, hepatocyte growth factor concentrations were significantly higher in the 5, 10 and 20 mg/L LPS treatment group than control group （P 〈 0.05）②Hepatocyte growth factor from 5, 10 and 20 mg/L LPS-stimulated skeletal muscle satellite cells did not show significant difference. ③ Hepatocyte growth factor reached the maximal concentration in LPS-stimulated secretion after 36 hours. ④ LPS-stimulated muscle satellite cell proliferation and differentiation were significantly higher than that of muscle satellite cells without stimulation. CONCLUSION： ①LPS can induce the excretion of hepatocyte growth factor from skeletal muscle satellite cells. ② Hepatocyte growth factor in different concentrations of LPS medium does not show significant difference. ③LPS can promote the proliferation and differentiation of skeletal muscle satellite cells.