小鼠脂肪间充质干细胞的分离培养及肠道归巢  

Isolation and culture of mouse adipose-derived stem cells and their homing to the intestinal tract

作　　者：侯晓琳[1] 郁卫东[2] 崔梅花[1] 何湘君[2] 梁君[1] 

机构地区：[1]北京大学航天临床医学院消化科,北京市100049 [2]北京大学人民医院临床分子生物学研究所,北京市100044

出　　处：《中国组织工程研究》2015年第6期854-860,共7页Chinese Journal of Tissue Engineering Research

基　　金：航天中心医院特色临床医学发展科研项目(YN2013TS03)<间充质干细胞联合内皮祖细胞治疗小鼠溃疡性结肠炎的疗效及机制研究>~~

摘　　要：背景:脂肪来源间充质干细胞因其取材安全、创伤小、易纯化、增殖快等优点而备受关注。目的:建立一种有效、快速分离培养较高纯度小鼠脂肪间充质干细胞的方法,荧光标记后探索其是否可在体内向肠道归巢。方法:取小鼠腹股沟及附睾脂肪,用0.1%Ⅰ型胶原酶消化得到单细胞,并与剩余未消化脂肪组织块一起接种,贴壁培养获得脂肪间充质干细胞,通过细胞形态、细胞表型、生长动力学、成骨成脂分化潜能4个方面进行鉴定,并将脂肪间充质干细胞用PKH67标记后经尾静脉注射移植到溃疡性结肠炎模型小鼠体内,观察其向肠道的归巢情况。结果与结论:脂肪间充质干细胞镜下呈梭形,快速增殖呈漩涡状排列,细胞高表达CD29、CD44、CD90,不表达CD45。成骨诱导碱性磷酸酶染色显示有黑色颗粒生成,茜素红S染色呈红色结节,成脂诱导油红O染色显示有大量脂滴。细胞生长曲线显示第3-5天为对数增长期,细胞生长活力良好。结肠冰冻切片在荧光显微镜下可见绿色荧光光点,并随时间推移有增加趋势。以上结果表明采用胶原酶消化加组织块贴壁法体外分离培养的脂肪间充质干细胞增殖快、纯度高,可向成骨成脂细胞分化,在体内可向结肠归巢并增殖。BACKGROUND: Adipose-derived mesenchymal stem cells have gained more and more attention due to their high safety, less invasiveness, easy purification and rapid proliferation.OBJECTIVE: To isolate and culture adipose-derived mesenchymal stem cells efficiently and rapidly with high purity and then to explore the cell homing to the intestinal tract after fluorescence labeling.METHODS: Mouse adipose tissue obtained from the groin and epididymis was aseptically isolated and digested with 0.1% collagenase I. Then the digested cells and undigested adipose tissue were cultured together in the dish to harvest adipose-derived mesenchymal stem cells. The cells were identified by morphology, surface markers, growth kinetics, osteogenic and adipogenic differentiation potential, and then labeled by PKH67 before injected into ulcerative colitis mouse models through the tail vein to observe their homing to the intestinal tract.RESULTS AND CONCLUSION: In vitro, adipose-derived mesenchymal stem cells isolated by this method exhibited spindle-like appearance, grew intensively and arranged in a swirling shape. Adipose-derivedmese alkalmesenchymal stem cells expressed CD29, CD44 and CD90, but not expressed CD45. After osteogenic induction,alkaline phosphatase staining showed black particles and alizarin red S staining showed red mineralized nodules.After adipogenic induction, oil red O staining showed many lipid droplets were dyed red. Cell growth curve showed cells at 3-5 days were in logarithmic growth phase and they were active. Under fluorescence microscopy, frozen sections of the colon were found green fluorescence points that were increased with time. Results suggest that adipose-derived mesenchymal stem cells isolated and cultured in vitro can proliferate rapidly, purify easily and can be induced to osteoblasts and adipocytes; in vivo, PKH67-labeled cells can home to and proliferate in the colon.

关 键 词：脂肪组织 间质干细胞 结肠炎 溃疡性 干细胞 脂肪干细胞 细胞归巢 脂肪间充质干细胞 小鼠 生物学特性 诱导分化 分离培养 溃疡性结肠炎 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]