人细胞外基质支架联合脂肪干细胞构建脂肪组织

Experimental study on adipose tissue engineering with human adipose-derived stem cells and adipose tissue extracellular matrix scaffold

目的探讨人脂肪组织细胞外基质（ECM）支架联合人脂肪来源干细胞（ADSCs）构建工程化脂肪组织的可行性。方法以酶消化法从人抽脂术抽吸物脂质部分获取人ADSCs，体外进行多向分化诱导鉴定，并行DiI荧光标记。从抽脂术的脂质部分分离提取人脂肪组织细胞外基质，经过低温冻干、粉碎、灭菌等处理，制备成粉末状，电镜扫描观察表面特征并将其与ADSCs进行黏附实验，探讨其作为支架材料的可行性。收集人ADSCs，以2×10^9／L的细胞密度与提取的细胞外基质支架复合后移植于裸鼠背部皮下，同鼠对侧背部皮下移植ECM支架和细胞培养液作为对照，每侧移植0．5ml，共6只实验鼠。8周后取材，称量标本湿重。取出的标本行苏木素-伊红（HE）染色和油红O染色进行定性判断，分析人脂肪组织ECM支架联合人ADSCs构建工程化脂肪组织的能力。结果从脂肪组织中分离得到人ADSCs和ECM支架。ADSCs在相应的诱导环境下能够分化成为脂肪细胞、骨细胞和软骨细胞。ECM支架电镜扫描和大体观察具有疏松、多孔的结构特征，适合ADSCs的黏附生长。ADSCs与支架相容性良好，黏附率达（89．87±2．59）％，细胞在支架表面可充分伸展生长。体内移植8周后，实验组和对照组都能够形成新生物，湿重比较实验组较对照组重（P〈0．05）。经HE切片及油红O染色均证实实验组形成成熟的脂肪组织，对照组不能形成脂肪组织。结论人脂肪组织ECM支架联合人ADSCs在体内能够成功构建成熟的脂肪组织，8周后支架并无明显吸收。

Objective To explore the possibility of building tissue-engineered adipose tissue with human adipose-derived stem cells （ADSCs） and adipose tissue extracellular matrix （ECM） scaffold, and provide experimental basis for clinical application of tissue-engineered adipose tissue for the repair of soft tissue defects. Methods ADSCs were isolated from adipose tissue by liposuction with the method of enzymatic digestion. The cells were induced for three-line differentiation and marked with DiI. Human ECM was extracted from adipose tissue obtained by liposuction. The ECM was freeze-dried, sterilized and crushed to powders. The surface characteristics of ECM were observed under the electron microscopy in vitro. The ADSCs were adhered to ECM powers, and the feasibility of ECM powers as scaffold was explort- ed. Human ADSCs in a density of 2 x 109/L were collected, and mixed them with ECM powers. They were transplanted subcutaneously into nude mice, and the opposite side of the same mice transplanted with scaffold only served as control group. There were 6 mice in total, and each side was transplanted by 0. 5 ml. At 8th week post-transplantation, the mice were sacrificed and the implanted tissue were taken to record the wet weight, The hematoxylin and eosin （HE） sections and red oil O staining were detected. Two-sample test of SPSS 13.0 was used for statistical comparison between wet-determination. Results The ADSCs and human ECM powder were obtained successfully from adipose tissue. The ADSCs could differentiate into adipose cells, bone cells and chondrocytes. Scanning election microscopy （SEM） images showed that the powers had porous structure to which ADSCs could adhere easily, with the adhesion rate being （89. 87 ± 2. 59）%. Eight weeks after transplantation, both experimental and control groups formed new tissues. There were significant differences between two groups （ P 〈 0.05 ） in the wet weight. HE and red oil O staining confirmed that the experimental group could form mature adipose tissue. Conclusion ADSCs and human ECM scaffold could form tissue-engineered adipose tissue in vivo. It can be used as an ideal method to construct tissue-engineered adipose tissue.