脂肪组织来源干细胞定向分化脂肪组织的体内外实验研究

目的 分离和培养脂肪组织来源干细胞（ASCs），鉴定其是否具有干细胞表面标志，研究携带GFP基因的ASCs向脂肪组织的体外定向诱导分化能力，同时判断种子细胞ASCs与Ⅰ型胶原支架混合培养后在体内构建组织工程化脂肪组织的可能性。方法 取GFP小鼠腹股沟部脂肪组织，使用酶消化法进行原代培养，流式细胞仪鉴定其表面干细胞标志，细胞传至第3代后使用脂肪分化培养基诱导2周，观察细胞形态及功能变化。将诱导分化后的细胞与支架材料混合培养后12h，将支架材料移植到裸鼠背部皮下，观察新生组织情况，并对新生组织使用HE及油红O染色进行鉴定。结果 原代培养的ASC形态类似于成纤维细胞，具有很强的增殖能力，能持续稳定表达表面干细胞标志。在脂肪分化培养基的作用下，胞浆内脂滴不断聚集，逐渐演变为成熟的脂肪细胞，油红O染色阳性。体内实验中在裸鼠皮下发现了0．5ml的新生组织块，常规病理及油红O染色均证实其为成熟脂肪组织块。结论 脂肪组织来源的干细胞ASC能在体外定向诱导分化为成熟脂肪细胞，且ASC能作用种子细胞与Ⅰ型胶原支架在体内成功构建脂肪组织。

Experimental study of adipose tissue differentiation using adipose-derived stem cells harvested from GFP transgenic mice

OBJECTIVE: To identify the expression of protein which is characteristic of stem cell, induce the adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice, and to assess the possibility of constructing adipose tissue via the attachment of ASCs to type I collagen scaffold. METHODS: Inguinal fat pads from GFP transgenic mice were used for the isolation of ASCs. Expression of CD29, CD34, CD45, CD133 and HLA-DR were detected by flow cytometry. After expansion to three passages, the ASCs were incubated in an adipogenic medium for two weeks. Then they were attached to collagen I scaffold and co-cultured for 12 hours, followed by transplantation under the dorsal skin of athymic mice for 2 months. Adipogenic differentiation of ASCs in vitro was assessed by morphological observation, Oil red O staining and newly formed tissue was detected by HE staining. RESULTS: The cultured stem cells were fibroblast-like cells and showed highly homogeneous appearance with active proliferation capacity. Stem cells' characteristic CD expression was proved. After being incubated in an adipogenic medium, they could differentiate into mature adipocytes. Accumulation of lipid droplets in the cytoplasm was testified by Oil red O staining, morphological and biological observation. 0.5 ml new tissue was formed and was confirmed by fluorescent observation and HE staining to be mature adipose tissue. CONCLUSIONS: Adipose derived stem cells can successfully differentiate into mature adipocytes exhibiting an adipose-like morphology and expression of intracytoplasmic lipid droplet. It was an efficient model for adipose tissue engineering with ASCs and type I collagen scaffold.