大鼠脂肪干细胞分离培养及细胞表型的研究

摘要

Objective To search a method for isolation, cultivation, identification and cryopreservation of mesenchymal stem cells from rat adipose tissue in vitro. Method Three rats was anaesthetized and disinfected con-ventionally, their inguinal adipose tissue were taken and sheared to paste. The fragments were digested by collagenase typeⅠ, suspended by DMEM, and planted in culture dish. Then the cells were cultured in incubator culture. Their first medium change happen after 24~48 hours,then the change occur 2~3 days once again,after 5~7 days and 80%cell fu-sion, cells were digested by Trypsin and passaged by EDTA. Cell microscopy was used to morphological observation and the third generation was used to identify by flow cytometric cell cycle. Results Rats fat had a large amount of adipose-derived mesenchymal stem cells (ADMSCs). The shape of primary cultured ASCs was a star or much horn;af-ter passage of the ASCs relatively uniform shape,fusiform shape,spiral growth. The ASCs surface markers are identi-fied by flow cytometry and showed positive expression of CD44, CD106. Cryopreserved ASCs remains a good activity, high survival rate. Conclusion A simple and convenient method was successfully established to isolate, culture and store the adipose tissue derived mesenchymal stem cells, which provides the foundation for the future use of ADMSCs in the further research.%目的：分离、培养和冻存大鼠脂肪干细胞，并对其相关的细胞表型进行研究，为利用脂肪干细胞治疗梗阻性肾病肾纤维化等实验提供依据。方法取大鼠3只，经消毒麻醉，采集其腹股沟处脂肪组织分离培养其中的脂肪干细胞，用相差倒置显微镜观察细胞生长方式及形态变化。取第三代细胞用流式细胞仪作细胞免疫表型的鉴定。冻存复苏后再次检测干细胞生长情况及表型。结果大鼠脂肪组织中含有大量间充质干细胞，原代培养的ASCs呈小圆形或星形，经传代后的脂肪干细胞形态比较均一，呈长梭形，个别略呈多角形、漩涡样生长。其细胞表型为CD29、CD44高表达，CD73、CD105中等程度表达，CD34极低表达。脂肪干细胞经低温冻存3个月，复苏后，生长活力和存活率与冻存前未见明显区别，CD44和CD29检测与冻存前表达率也一致。结论建立了一种脂肪干细胞的有效分离、培养及保存方法，并对其表面标志物进行鉴定，为利用脂肪干细胞进行进一步的实验研究提供了依据。