兔脂肪干细胞的分离培养鉴定及诱导分化研究

Isolation,Characterization and Differentiation of Rabbits Adipose-derived Stem Cells

目的:分离培养家兔脂肪干细胞(Adipose-derived stem cells,ASCs),观察体外培养生长特性,并在特定条件下诱导分化,探讨其成脂成骨分化能力。方法:无菌条件下切取家兔腹股沟处脂肪组织,0.25%Ⅰ型胶原酶消化,分离培养原代细胞,取第3代ASCs做实验,用HE染色观察细胞形态,MTT比色法测定细胞生长曲线;用流式细胞仪检测其细胞表面标志物;用油红O染色和茜素红染色鉴定成脂和成骨诱导分化。结果:从家兔脂肪组织中分离获得的ASCs形态为长梭形,成纤维细胞样,生长活力旺盛,具有较强的增殖能力;流式细胞仪分析CD29呈阳性表达,CD31呈阴性表达;成脂诱导的细胞油红O染色阳性,成骨诱导的细胞茜素红染色阳性。结论:本实验分离培养的细胞为ASCs,具有成脂和成骨分化潜能。

Objective： To isolate and culture rabbits adipose--derived stem cells （ASCs） ,and to observe the biolog- ical characteristics of rabbits ASCs and to analyze the multipotent properties of differentiation with proper condition in vitro. Method： ASCs were prepared by collagenase Ⅰ digestion of the inguinal fat pads from rabbits, and cul- tured at 37℃ in a 5% CO2 humidified incubator. Cell passage was performed following about 800%-90% conflu- ence. The growth curve was drawn. And Hematoxylin-eosin（HE） staining was used to observe the morphology of ASCs. The surface antigens of ASCs were analyzed by flow cytometry. Passage 3 of ASCs were used to differentia- ted into adipocytes, osteoblasts, and identified by the Oil Red O staining and Alizarin red staining. Results： Adher- ent ASCs showed fibroblast-like and spindle shape or polygon shape, strong proliferative ability and active differ-entiation. The growth curve was like ‘S＇ shape. Flow cytometry showed that ASCs expressed CD29, but did not express CD31. The Oil Red O staining and Alizarin red staining showed positive reaction.