人与大鼠脂肪干细胞提取方法及生物学特性的比较研究

Comparison of isolation and biological characteristics of adipose-derived stem cells from human and rat

目的:研究人脂肪干细胞与大鼠脂肪干细胞的提取方法并对比差异,对培养出的细胞做形态学观察及表面标记鉴定.为今后ADSCs分化能力相关研究提供方法及依据.方法:获取人脂肪及大鼠脂肪,利用胶原酶消化法进行ADSCs提取,消化传代以纯化细胞.对比观察细胞形态,并取第3代的细胞进行细胞爬片HE染色、生长曲线测定、成脂和成骨诱导,流式鉴定表面标记物.结果:在相同的培养环境下,两种细胞均呈成纤维细胞样贴壁生长,h ADSCs原代第3天,r ADSCs原代第1天即可见较多细胞伸展为长梭形或短梭形,HE染色可见二者大小形态差异;成脂及成骨诱导组分别用油红"O"、茜素红染色为阳性,对照组为阴性.传代培养后,h ADSCs约1周,r ADSCs约6 d长满.流式鉴定示:二者均CD29(+),CD45(-),CD90(+),CD106(-),符合间充质干细胞特性.结论:本实验建立了高效提取ADSCs的方法,经过比较发现r ADSCs成骨能力较弱,在进行成骨向分化研究时不推荐首选;成脂能力与h ADSCs相似.r ADSCs是较好的动物来源脂肪干细胞,可广泛应用于再生医学研究.

Aim: Compare the isolation and biological characteristics differences between adipose-derived stem cells from human and rat,provide a method and basis for future related experiment about differentiation potency of ADSCs. Methods: Obtain human and rats fats,ADSCs were isolated by the collagenase digestion method,purified cells by digestion and extended. Observe the cells every day and take photos,use the third generation of cells to make HE staining,cell growth curve determination,induction into fat and osteogenic,flow cytometric analysis,etc. Results: Two kinds of cells were growed like fibroblasts; HE staining expresses the size difference; After fat induction and osteogenic induction,the experimental group ADSCs were positive and the control group were negative for staining. After extend culture,the h ADSCs about 1 week,the r ADSCs about 6 days grow full of the culture dish. The flow cytometric analysis show: the two kinds of cells both express the specific positive surface markers,CD29,CD90,and did not express the negative surface markers,CD106,CD45,consistent with the characteristics of mesenchymal stem cells. Conclusion: This experiment has established a efficient extraction method of ADSCs. Through comparing we found that r ADSCs' s osteogenesis ability is weaker than h ADSCs,so not recommended for the osteogenesis study. It's adipogenesis ability is similar to h ADSCs. Rat is a good animal source for ADSCs,can be widely used in regenerative medicine.