摘要
背景:肌母细胞体外培养大多使用添加胎牛血清培养基,存在很多不足,开发无血清培养基,更加稳定、安全、经济,有广泛的应用前景。目的:初步探索适合人肌母细胞培养的无血清培养基。方法:配制含胎牛血清培养基(DMEM/F12 1∶1添加胰岛素样生长因子1、碱性成纤维细胞生长因子和体积分数20%胎牛血清),间充质干细胞无血清培养基组(无血清培养基、2%血清替代物、2 mmol/L L-谷氨酰胺,人脐带间充质干细胞,细胞纯度大于99%,UltraC ULTURETM添加血清替代物,谷氨酰胺)。自制无血清培养基组(GibcoTM添加胰岛素样生长因子1,碱性成纤维细胞生长因子,谷氨酰胺)。3种培养基分别对人肌母细胞分别进行培养,观察肌母细胞的形态、免疫组织化学的鉴定,计算克隆的形成率,绘制细胞生长的曲线,MTT检测肌母细胞的活性,比较3种培养基培养肌母细胞增殖、分化的差异。结果与结论:各组细胞形态上无明显差异;各组免疫组织化学鉴定肌母细胞纯度均达99%;含胎牛血清培养基组和自制无血清培养基组克隆形成率显著高于间充质干细胞无血清培养基组(P<0.01),含胎牛血清培养基组克隆形成率显著高于自制无血清培养基组(P<0.01);含胎牛血清培养基组与自制无血清培养基组细胞数远高于间充质干细胞无血清培养基组;自制无血清培养基组细胞活性显著高于含胎牛血清培养基组和间充质干细胞无血清培养基组(P<0.01)。自制无血清培养基组可有效用于肌母细胞的培养,在促进肌母细胞早期贴壁、增殖上还需进一步优化。
BACKGROUND： Extensive use of fetal bovine serum（FBS） medium to culture myoblasts in vitro has many disadvantages. To develop a more stable, safe, economical serum-free medium that has broad application prospect is indispensable. OBJECTIVE： To preliminarily explore the serum-free medium suitable for the cultivation of human myoblast. METHODS： Three different culture media were made： FBS culture medium（DMEM/F12 containing insulin-like growth factor and basic fibroblast growth factor at a ratio of 1：1 and 20% FBS）, serum-free medium of mesenchymal stem cells（MSCs; serum-free medium, 2% serum replacement, 2 mmol/L L-glutamine, human umbilical cord-derived MSCs with a purity of 〉 99%, Ultra CULTURETM containing serum replacement and glutamine）, and self-made serum-free medium（GibcoTM containing insulin-like growth factor-1, basic fibroblast growth factor, and glutamine）. These three media were used to culture human myoblasts respectively. Human myoblasts were observed morphologically and identified immunohistochemically. Colony forming efficiency wascalculated, growth curve was drawn, and viability of human myoblasts was detected using MTT test. Proliferation and differentiation of human myoblasts cultured in the three media were compared and analyzed. RESULTS AND CONCLUSION： There was no obvious difference in the morphology of the cells among different groups. The purification rate of human myoblasts was up to 99% in each group. The colony forming efficiency of FBS group and self-made serum-free medium group was significantly higher than that of MSCs group（P 〈 0.01）, and the colony forming efficiency of FBS group was significantly higher than that of self-made serum-free medium group（P 〈 0.01）. The viable cell count in the FBS group and self-made serum-free medium group were much higher than that of MSCs group. The cell activity of self-made serum-free medium group was significantly higher than that of FBS group and MSCs group（P 〈 0.01）, while there was no significant d
出处
《中国组织工程研究》
CAS
北大核心
2015年第51期8271-8275,共5页
Chinese Journal of Tissue Engineering Research
