人脂肪来源干细胞复合Ⅰ型胶原支架构建工程化脂肪组织的实验研究

Study of adipose tissue engineering with human adipose-derived stem cells and collagen type Ⅰ scaffold

目的探讨构建组织工程化脂肪组织的可行性，为临床修复软组织缺损寻找一种新方法。方法以酶消化法从人脂肪抽吸术抽吸物脂质部分获取人脂肪来源干细胞作为种子细胞，并行DiI体外荧光标记，以Ⅰ型胶原支架为载体材料，将细胞成脂诱导后，以1×10^7／ml细胞密度与支架复合后接种于裸鼠左侧背部皮下，未诱导组不对细胞进行任何诱导，以相同方式接种于裸鼠右侧背部皮下，空白对照组将Ⅰ型胶原空白支架接种于裸鼠颈部正中皮下，每组各6只实验鼠；于第12周取材，通过大体和荧光显微镜观察、湿重测定、组织学检测和油红O染色定性判断体内成脂能力。结果原代培养的脂肪来源干细胞，经成脂诱导能演变为成熟脂肪细胞，油红O染色阳性。诱导组裸鼠皮下均发现新生组织块，新生物平均湿重为0．020g，常规病理切片及油红O染色均证实其为成熟脂肪组织，DiI荧光显色阳性证实其为外源性；未诱导组4只裸鼠皮下发现新生组织块，新生物平均湿重为0．014g，常规病理切片及油红O染色证实其含有部分成熟脂肪组织，DiI荧光显色阳性证实其为外源性，两组新生物湿重比较差异有统计学意义（P〈0．01）；空白对照组未见新生组织形成。结论用酶消化法从人脂肪抽吸术抽吸物脂质部分提取的细胞为脂肪组织来源干细胞，该细胞能作为种子细胞经成脂诱导后，与Ⅰ型胶原支架在体内成功构建脂肪组织。

Objective To explore the possibility of building tissue-engineered adipose tissue and looking for a new approach for the repair of soft tissue defects. Methods The cells using enzymatic digestion from human liposuction part of the lipid extract were used as adipose tissue-derived cells and labeled with DiI fluorescent marker, the induced group using Ⅰ collagen scaffold material as a carrier, the induced cell were planted into left back subcutaneously in nude mice at 1 ×10^7/ml cell density, in the unindueed group cells were not induced by any, in the same cell density and type Ⅰ collagen scaffold composite inoculated in nude right mouse back skin, the blank control group Ⅰ collagen scaffold gaps in nude mice inoculated subcutaneously center of the neck, each of the six miee; Remove implants after 12 weeks and judge the adipogenic capacity through general and fluorescence microscopy, wet - determination, histological detection and oil red O staining qualitative. Results The primary source of fat euhured stem cells, similar to the fibroblast morphology, and has a strong proliferative capacity. In the role of adipose differentiation medium, it can be the mature fat cells in which cytoplasmic lipid droplets gather, oil red O staining was positive. In the induced group, newborn tissue were found in the experimental groups of nude mice and its average weight is about 0.020 g. Conventional pathological slices and oil red O staining confirmed it is mature adipose tissue, the fluorescence staining positive confirm them are exogenous. Uninduced group newborn tissue are found in the experimental groups of nude mice and its average weight is about 0.014 g. Conventional pathological slices and oil red O staining confirmed it include some mature adipose tissue, the fluorescence staining positive confirm them are exogenous. Two groups of the new wet weight with have statistical significance （ P 〈 0.01 ） ; gaps in the control group no new organization formed. Conclusions The cells using enzymatic digestion from human liposuction part of the lipid extract are adipose tissue-derived cells. The cells can be as seed cells and with solid scaffold of collagen type Ⅰ it can become fat tissue in vivo successfully.