人网膜脂肪干细胞与小鼠胚胎成纤维细胞3T3-L1体外增殖分化对比观察

Comparison of in vitro proliferation and differentiation between human omental adipose stem cells and mouse embryonic fibroblasts 3T3-L1

目的比较人网膜脂肪干细胞与小鼠胚胎成纤维细胞(3T3-L1)体外增殖分化情况。方法使用胶原酶消化法提取人网膜脂肪干细胞,复苏冻存的3T3-L1,进行培养,显微镜观察细胞在培养瓶中的生长过程,拍照记录细胞密度、形态;将两种细胞接种入96孔板,定时加入CCK-8试剂,测定吸光值,绘制生长曲线;将两种细胞诱导为成熟脂肪细胞,油红O染色后,观察脂滴分布。结果人网膜脂肪干细胞1~3代细胞倍增时间6 d,随着传代次数增多,细胞贴壁后停止增殖,第4天出现老化凋亡;复苏后3T3-L1倍增时间48 h,每72 h传代1次。根据人网膜脂肪干细胞和3T3-L1的吸光值,二者的指数增长期分别为第2~5天、第2天,平台期分别为第6~8天、第3~10天,人网膜脂肪干细胞第9天进入衰亡期。人网膜脂肪干细胞和3T3-L1经过10天成脂诱导分化,油红染色后均可见红染的脂滴,两种细胞均可分化为成熟脂肪细胞,并且在诱导过程中出现漂浮裂解,贴壁细胞减少。结论与3T3-L1相比,人网膜脂肪干细胞也具有成脂分化的能力,但增殖能力有限。

Objective To compare the in vitro proliferation and differentiation between the human omental adipose stem cells and mouse embryonic fibroblasts(3 T3-L1).Methods Human adipose stem cells were extracted by collagenase digestion from human omental adipose tissues.We resuscitated the frozen mouse 3 T3-L1 cells and cultured them.The growth process of the two kinds of cells in the culture bottle was observed by microscope,and the cell density and cell morphology of the two kinds of cells were photographed and recorded.The two kinds of cells were inoculated into 96-well plate,and CCK-8 reagent was added regularly;the absorbance value was measured and the growth curve was drawn;the two kinds of cells were induced into mature adipocytes.After oil red O staining,the distribution of lipid droplets was observed.Results The doubling time of human omental adipose stem cells from the first to the third generations was 6 days.With the increased generations of stem cells,the proliferation stopped and aging apoptosis appeared during the 4 th generation.The doubling time of resuscitated 3 T3-L1 cells was 48 h and the passaged time was 72 h.According to the light absorption values of human omental adipose stem cells and 3 T3-L1 cells,their exponential growth periods were day 2-5 and day 2,respectively;the plateau periods were day 6-8 and day 3-10,respectively;human omental adipose stem cells entered the decline period on the 9 th day.The human omental adipose stem cells and 3 T3-L1 cells were differentiated into mature adipocytes after 10 days of induction.Red stained lipid droplets were seen after oil red staining in both cells.During the induction process,floating lysis appeared and adherent cells decreased in both cells.Conclusion Compared with 3 T3-L1,human omental adipose stem cells also have the ability to differentiate into mature adipocytes,but the proliferation ability is limited.