人脂肪来源干细胞复合纤维蛋白胶构建可注射型工程化脂肪组织的实验研究

Adipose tissue engineering with human adipose-derived stem cells and fibrin glue injectable scaffold

目的探讨构建可注射型组织工程化脂肪组织的可行性，为临床修复软组织缺损寻求简便、微创的方法。方法采用酶消化法从人脂肪抽吸术的抽吸物中脂质部分获取人脂肪来源干细胞（ASC），以经成脂诱导和未经成脂诱导的ASC作为种子细胞，行DiI体外荧光标记后，分别以1×10^7个／ml细胞密度与纤维蛋白胶可注射支架复合。取6只裸鼠，将成脂诱导ASC-支架复合物注射于其背部左下方皮下（诱导组），未诱导ASC-支架复合物注射于背部右下方皮下（未诱导组），不加细胞的纤维蛋白胶注射于颈部正中皮下（空白支架组），每点注射0．2ml。第12周末处死裸鼠取出移植物，通过大体观察、湿重测定、常规组织病理学检测、油红O染色和DiI荧光标记检测判断体内成脂能力。结果诱导组和未诱导组均获得了外观类似脂肪组织的新生物，新生物湿重分别为（28±15）、（22±16）mg（t=23．238，P〈0．01）。诱导组新生物常规组织病理学检查及油红O染色均证实为成熟脂肪组织，DiI荧光标记呈阳性，证实为外源性ASC；未诱导组新生物中见少量成熟脂肪组织，大部分为纤维样结构；空白对照组未见新生组织形成。结论从人吸脂术抽吸物脂质部分提取的ASC能作为种子细胞，经成脂诱导后与纤维蛋白胶可注射支架复合，可在体内成功构建成熟脂肪组织。

Objective To explore the possibility of building tissue-engineered adipose tissue and find a new approach for repairing soft tissue defects. Methods Using enzymatic digestion, adipose tissue- derived stem cells （ASCs） were extracted from the lipid part of human liposuction aspirate, cultured, and underwent adipogenic induction or not. The adipogenic-induced and non-adipogenic-induced ASCs were labeled with 3, 3, 3＇, 3＇-tetramethylindo-carbocyanine perchlorate （DiI）, a fluorescent marker, in vitro to be used as seed cells. Then, they were combined with injectable fibrin glue scaffold at 1 × 10^7/ml cell density. Six athymic BALB/C mice underwent subcutaneous injection of adipogenic-induced ASCs with fibrin glue scaffold at the density of 1 × 10^7 cells/ml into the left side of the low back （induced group）, subcutaneous injection of non-adipogenic-induced ASCs into the right side of the low back （non-induced group）, and subcutaneous injection of injectable fibrin glue scaffold into the middle part of the neck （ blank control group） ,with 0.2 ml per injection. Twelve weeks later the mice were killed and the implants were taken out. The wet weight was measured. HE and oil red O staining and light and fluorescence microscopy were used for morphological observation. Results Adipose tissue-like new-born tissues were found in the injection sites of the induced and un-induced groups. The average wet-weight of the induced group new-born tissue was （28 ± 15 ） mg, significantly heavier than that of the un-induced group [ （22 ±16） mg, P 〈 0.01 ]. HE staining and oil red O staining confirmed that the new-born tissue of the induced group was mature adipose tissue and DiI fluorescent staining approved its exogenousness. Most part of the new-born tissues of the un-induced group was fibroid tissue with only a few mature adipose tissues. Conclusion ASCs extracted from the lipid part after liposuction can be used as seed cells, mixed, after adipose-induction, with injectable scaffold of fibrin glue, and injected into the body to build mature adipose tissue.