脂肪细胞去分化及构建组织工程化脂肪的实验研究

Experimental study of human adipocyte dedifferentiation for adipose tissue engineering

目的探讨成熟脂肪细胞去分化及去分化脂肪细胞作为种子细胞构建组织工程化脂肪组织的可行性。方法取健康女性脂肪抽吸术的抽吸物,使用酶消化法获取成熟脂肪细胞,采用天花板贴壁法培养,取第3代细胞进行实验。体外实验:成脂、成软骨和成骨诱导培养,并分别采用油红"O"染色、阿尔辛蓝染色和茜素红染色法鉴定。体内实验:实验组(n=6):取第3代细胞使用荧光染料DiI标记,与纤维蛋白胶混合后注射于裸鼠背部一侧皮下,8周后,从大体观察、组织学、油红"O"染色及荧光显微镜观察新生物形成情况;对照组(n=6):以空白的纤维蛋白胶注射于同一裸鼠背部另一侧皮下,于同一时间点进行检测。结果成熟脂肪细胞呈单房圆形,经天花板贴壁法培养后变为长梭形成纤维细胞状,即去分化脂肪细胞。去分化脂肪细胞在定向诱导培养下能够分别成脂、成软骨及成骨分化。体内实验中实验组8周后在裸鼠背部皮下有新生组织块形成,经检测后证实其为成熟脂肪块并来源于植入的去分化脂肪细胞,对照组无新生组织形成,纤维蛋白胶被降解吸收。结论成熟脂肪细胞可通过体外培养实现去分化,去分化脂肪细胞具有成脂、成软骨和成骨等多向分化能力,以去分化脂肪细胞为种子细胞可在裸鼠皮下构建出脂肪组织。

Objective To investigate the dedifferentiation of mature adipocytes and the possibility of adipose tissue engineering using dedifferentiated adipocytes. Methods Human adipose tissue was harvested from healthy women undergoing abdominal liposuction procedures, and mature adipocytes were isolated with enzymatic digestion and cultured by ceiling adherent culture method, using the third-passage cells for subsequent experiment. The cells were cultured in adipogenic, chondrogenic or osteogenic media, and Oilred-O staining, Alcian blue staining and Alizarin red staining were used for dedifferentiation identification. The third-passage cells labeled with DiI were mixed with fibrin glue and injected subcutaneously on one side of the nude mouse back （n=6）, and fibrin glue solution without cells as control was injected on the other side （n=6）. After 8 weeks of cell implantation, the specimens were harvested for general observation and histological, Oil red-O staining and fluorescent microscope analyses. Results Mature adipocytes were round, unilocular cells. After ceiling adherent culture, the adipocytes underwent morphological changes into fibroblast-like cells indicating their dedifferentiation. The dedifferentiated adipocytes were induced for adipogenic, chondrogenic and osteogenic differentiation in specified media. Eight weeks after the cell injection in nude mice, newly formed tissue occurred which was identified as mature adipose tissue. The implants without cells were completely absorbed in the control group. Conclusions Mature adipocytes can dedifferentiate in vitro culture, and the dedifferentiated adipocytes are capable of differentiating into adipogenic, chondrogenic and osteogenic lineages. Adipose tissue engineering can be achieved in vivo using the dedifferentiated adipocytes as the seed cells.