【摘要】 目的:探讨不同培养基组成对皮肤成纤维细胞的增殖、衰老及胶原蛋白分泌的影响。方法:采用两步酶消化法从健康人耳后皮肤中获得成纤维细胞,依据培养基种类不同分为A组(FM无血清培养基)、B组(DMEM+10%FBS)、C组(RPMI-1640+10%FBS)、D组(DMEM+F12(1∶1)+10%FBS),采用MTT法、细胞划痕实验、酶联免疫吸附实验、β-半乳糖苷酶试验、软琼脂克隆形成试验比较不同培养基对细胞增殖、迁移、胶原分泌、老化、变异的影响。结果:采用酶消化法在2d~4d内即可从组织块中获得大量成纤维细胞;其中B、D组较A、C组细胞增殖较快,且趋势与MTT细胞增殖相同;划痕实验显示B、D组较A、C组向缺损部位迁移更为明显;此外,D组胶原蛋白分泌量高于其余三组(P<0.05),且连续传代30次后,细胞无明显衰老及瘤变迹象。结论:两步酶消化法可快速高效的从人组织块中获得成纤维细胞,且采用DMEM+F12(1∶1)完全培养基有利于促进细胞增殖、分泌胶原蛋白及延缓细胞衰老。更多还原

【Abstract】 Objective To investigate the effects of different culture medium on the proliferation, aging and collagen secretion of fibroblasts. Methods Fibroblasts were obtained from healthy human ear skin after using two-step enzyme digestion method. According to different kinds of culture medium, they were divided into A group(serum-free medium), B group(DMEM+10%FBS), C group(RPMI-1640+10%FBS), and D group(DMEM+F12(1∶1) +10%FBS), MTT assay,cell scratch assay,enzyme-linked immunosorbent assay,beta galactosidase assay,and soft agar colony formation test were used to compare the effects of different medium on cell proliferation,migration, collagen secretion,aging and mutation. Results A large number of fibroblasts were obtained from the tissue by enzyme digestion within 2d-4d; group B and group D proliferated more rapidly than group A and group C, which was the same as MTT cells proliferation;the result of scratch test showed that group B and group D migrated to the defect site more obviously than group A and group C; in addition, the collagen secretion of group D was the highest in all the four groups(P <0.05),and after 30 passages,cells had no obvious signs of aging and neoplasia. Conclusion Using two-step enzyme digestion method can obtain fibroblasts from human tissue quickly and efficiently, and the use of DMEM+F12(1∶1) complete culture medium is beneficial to promote cell proliferation and collagen secretion and to delay cell aging.更多还原