分离与培养人脂肪组织间充质干细胞的研究

Isolation,culture mesenchymal stem cells from human adipose tissue

目的:探索人脂肪组织源性间充质干细胞(ASCs)的分离、体外培养,为其广泛应用提供实验依据。方法:无菌条件下获取腹部手术病人皮下脂肪组织,酶消化法分离、培养ASCs,观察细胞形态并绘制细胞生长曲线,计算细胞群体倍增时间;对第2代细胞进行免疫组织化学染色,鉴定其表面分子CD44表达;取2-4代细胞用含体积分数为10%胎牛血清、1%青链霉素原液、1μmmol/L地塞米松1、0μmmol/L胰岛素、0.5mmmol/LIBMX的高糖DMEM培养基中诱导培养一周,观察细胞形态变化,并用油红“O”染色定性。结果:人脂肪组织中含有大量间充质干细胞,呈成纤维细胞样贴壁生长,细胞群体倍增时间为55h左右;免疫化学染色鉴定CD44阳性;成脂诱导分化一周,可见细胞内有大量脂滴,油红“0”染色可见胞浆内有大量红染颗粒。结论:建立了一种自人体脂肪组织分离,培养ASCs经济简便的方法,为其能够作为组织工程理想的种子细胞及广泛应用于临床提供实验依据。

Objective： To find a method which can be used for isolation and cultivation of mesenchymal stem cells from human adipose tissue in vitro. Methods： The adipose tissue was obtained from subcutaneous adipose tissue of abdominal surgery patients under aseptic condition, cultured with collagenase digestion, The cells were observed under inverted microscope each day and cell growth was studied with cell counting. Calculate the population doubling time of the cells; Immunoeytoehemistry was used to determine the surface molecule CD44; the cells was in DMEM medium supplemented with 10% fetal boven serum, 1%ABAM, 1μmmol/L dexamethasone, 10μmmol/L insulin,0.5mmmol/LIBMX for 1 week as adipogenic - inducing culture. Results： There was a large amount of mesenchymal stem cells in human adipose tissue, The population doubling time of the cells was about 55 hours; Immunocytochemical staining showed that most of the cultured cells were CD44 positive; and they could differentiate into mature adipocytes. Conclusion： A simple and convenient method to isolate and culture the adipose tissue - derived mesenehymal stem cells was successfully established, providing the foundation for the extensive use of ASCs in the field of tissue engineering and clinic.