人脂肪间充质干细胞分离培养及其生物学特性的实验研究

目的探索人脂肪间充质干细胞（adipose tissue—derived mesenchymal stem cells，ADMSCs）分离培养的方法及体外扩增的条件，观察ADMSCs的生物学特性。方法以腹部手术患者皮下脂肪组织为材料，采用I型胶原酶消化法及贴壁法分离培养ADMSCs，在含10％胎牛血清的低糖DMEM培养基中贴壁培养，倒置显微镜观察，流式细胞仪检测细胞表面标记CD29、CD44、CD105、CD31、CD34、CD106的表达，透射电镜及扫描电镜下观察ADMSCs超微结构，流式细胞仪测定细胞周期。结果原代和传代细胞呈梭形外观，生长增殖能力良好。CD29、CD44、CD105均呈阳性表达，阳性率分别为95.3％、98．6％和86．5％；而CD31、CD34、CD106阳性率分别为3．5％、2．6％、1．3％。透射电镜观察显示ADMSCs表现出早期幼稚细胞形态的特点，流式细胞仪检测显示84．8％的细胞处于G0/G1期。结论酶消化法能有效地从人脂肪组织分离培养人ADSCs，细胞生长稳定，增殖能力活跃，为今后ADMSCs的分离培养提供了更简单有效的方法。

A study on isolation,cultivation and biological characterisitics of human adipose derived mesenchymal stem cells in vitro

Objective To explore a practical method for effective isolation and culture of human adipose derived mesenchymal stem cells (ADMSCs) and observe its biological characteristics.Methods Subcutaneous adipose tissue were collected. Primary ADMSCs were isolated subsequently by the method of stirring type Ⅰ collagenase digestion and adhering to culture flask. The morphological changes of ADMSCs were observed under an inserted microscope. The expressions of CD29, CD44, CD105,CD31, CD34, and CD106 were detected by flow cytometry (FCM). Ultrastructure of ADMSCs were observed via electron microscopy. Cell cycle was analyzed by flow cytometry. Results ADSCs in both primary and passage culture were fusiform shapes, and stable in growth with active proliferation. The cells were in latency for 2 days, converted into growth period on day 3 and entered the stable phase on day 5. FCM results indicated that the positive rates of CD29, CD44, CD105, CD31, CD34, and CDl06were respectively 95.3%, 98.6%, 86.5%, 3.5%, 2.6%, and 1.3%. Cell cycle analysis showed that 84.8% of the cells were in G0/G1 phase and they revealed the structural characteristics of stem cells under an election microscope.Conclusions Stirring type Ⅰ collagenase digestion and adhering to culture flask can be effectively used to isolate and culture human ADSCs from the subcutaneous adipose tissue. The cultured ADSCs are stable in growth with active proliferation, which provides a much simple and effective collagenase digestion method.