人脂肪干细胞体外培养特性及成脂成软骨分化  

In vitro culture and differentiation of human adipose-derived mesenchymal stem cells into adipocytes and chondrocytes

作　　者：郭宝锋[1] 尹维田[1] 刘浩宇[1] 魏壮[1] 

机构地区：[1]吉林大学中日联谊医院手外科,吉林省长春市130033

出　　处：《中国组织工程研究与临床康复》2007年第33期6625-6628,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：吉林省科技发展计划项目资助(200505164)~~

摘　　要：目的:观察人脂肪组织间充质干细胞体外分离培养及成脂、成软骨诱导分化的生物学性状,探讨其作为软骨组织工程种子细胞的可行性。方法:实验于2006-08/2007-03在吉林大学中日联谊医院前列腺疾病防治研究中心完成主要工作。实验方法:①人脂肪组织来源于健康成年女性腹部吸脂术,酶消化法分离出脂肪间充质干细胞,接种于含体积分数为0.10胎牛血清的高糖DMEM培养基进行原代培养。②取第3代脂肪间充质干细胞,采用细胞计数法测定细胞生长曲线,流式细胞仪测定CD13、CD34抗原的表达,免疫荧光法测定CD34抗原的表达。③取第3代脂肪间充质干细胞,用含体积分数为0.10胎牛血清、0.5mmol/LIBMX、1μmol/L地塞米松、10μmol/L胰岛素的成脂诱导培养基和含体积分数为0.01胎牛血清、10μg/L转化生长因子β1、50nmol/L抗坏血酸、6.25mg/L胰岛素的成软骨诱导培养基分别诱导分化。④每天用倒置显微镜观察细胞形态及增殖变化,油红"O"、AB-PSA染色和Ⅱ型胶原免疫细胞化学染色检测成脂、成软骨分化情况。结果:①体外培养的脂肪间充质干细胞呈扁平的长梭形,细胞形态均一,传代稳定。间质细胞相关标志CD13和干细胞相关标志CD34表达阳性。②定向诱导后表现出脂肪细胞和软骨细胞特性。经成脂诱导,细胞内出现空泡,油红"O"染色呈红色;经成软骨染色,AB-PSA染色呈紫红色,Ⅱ型胶原免疫细胞化学染色胞浆呈棕黄色。结论:脂肪干细胞能向软骨细胞方向诱导分化,可作为软骨组织工程种子细胞。AIM： To investigate the biological features of human adipose-derived mesenchymal stem cells （ADMSCs） to isolate and culture, then differentiate into adipocyte and chondrocyte in vitro, and discuss the feasibility of ADMSCs as seed cells in cartilage tissue engineering. METHODS： The expenment was conducted in the Laboratory of Prostate Diseases Prevention and Treatment Research Center, China-Japan Union Hospital of Jilin University from August 2006 to March 2007. ①Human adipose was obtained from the healthy womanhood dudng the abdominal liposuction procedure and digested with collagenase I to isolate ADMSCs, which ware incubated in high-glucose DMEM with 0.10 volume fraction fetal bovine serum （FBS） for primary culture. ②Growth curve of the third-passage ADMSCs was drawn by cytometry; the expressions of CD13, and CD34 ware assayed by flow cytometry and immunofluorescence technique. （3）The third-passage ADMSCs were cultured in high-glucose DMEM supplemented with 0.10 volume fraction FBS, 0.5 mmol/L IBMX, 1 μmol/L dexamethasone and 10 μmol/L insulin for adipogenic induction, and with 0.01 volume fraction FBS, 10 μg/L transforming growth factor-β1 （TGF-β1）, 50 nmol/L ascorbate-2-phosphate and 6.25 mg/L insulin for chondrogenic induction. ④The appearance and generation of ADMSCs were observed everyday under inverted microscope, and the differentiations to adipocyte and chondrocyte were verified by oil red O staining, AB-PSA staining and immunocytochemical staining. RESULTS： ①ADMSCs cultured in vitro were in flat fusiform shape and stably passaged, during which ADMSCs kept the same shape. The expression of stromal cell-associated marker CD13 and stem cell-associated marker CD34 was positive. ②The characteristics of adipocyte and chondrocyte were shown after the ADMSCs had exposed to the defined medium. ADMSCs cultured in adipogenic differentiation medium developed intracellular vacuoles that accumulates the lipid dye oil red O, and in chondrogenic differentiation medium, AB-PSA stai

关 键 词：组织工程 干细胞 软骨 

分 类 号：R394.2[医药卫生—医学遗传学]