评价脐带间充质干细胞移植前细胞活性的指标  

Evaluation indexes for the viability of umbilical cord-derived mesenchymal stem cells before transplantation

作　　者：雷鑫[1] 陈彦[2] 张建林[1] 崔蕾[2] 牛玉虎[1] 牛勃[1] 

机构地区：[1]山西医科大学生物化学与分子生物学教研室,山西省太原市030001 [2]山西医科大学第二附属医院呼吸科,山西省太原市030001

出　　处：《中国组织工程研究》2013年第32期5847-5854,共8页Chinese Journal of Tissue Engineering Research

基　　金：山西医科大学生物化学与分子生物学教研究给予的支持

摘　　要：背景:脐带间充质干细胞已在临床多种疾病中展开研究,作为一种动态的活细胞,移植前的细胞活性将直接影响治疗效果,锥虫蓝染色虽然是目前评价细胞活性的常用指标,但由于其易受主观因素的影响,并不能准确反映细胞功能状态。目的:探寻可准确反映脐带间充质干细胞功能状态的活性指标。方法:体外分离培养人脐带间充质干细胞,生理盐水4℃条件下保存0,2,4,6h,采用多种具有代表性的细胞活性测定方法(锥虫蓝染色法、Annexin V-PI、TUNEL、CCK-8、Live-Dead Assay、贴壁实验)检测保存后细胞存活率,进一步观察它们与反映细胞功能状态指标克隆形成率之间的关联程度。结果与结论:人脐带来源的间充质干细胞体外培养呈梭形贴壁生长,第3代细胞表面标记CD29、CD44与CD105表达呈现阳性,CD34与CD45表达呈现阴性,成脂及成骨诱导均表达阳性。锥虫蓝染色法得到的细胞活力与相应的克隆形成率相关性不大,而其他检测方法较锥虫蓝染色法而言相关性较好,其中以贴壁实验最为明显。证明了贴壁实验是目前反映细胞活性状态相对最为精确的指标。BACKGROUND： Umbilical cord-derived mesenchymal stem cells are gaining more attention in clinical treatments. Cell viability prior to transplantation has a direct impact on clinical prognosis. Despite trypan blue staining is a widely performed procedure to assess the viability of umbilical cord-derived mesenchymal stem cells, it cannot reflect the functional capacity of those cells accurately because of some subjective factors. OBJECTIVE： To explore sensitive and accurate assay for the functions of umbilical cord-derived mesenchymal stem cells. METHODS： Human umbilical cord-derived mesenchymal stem cells were isolated and cultured in vitro. Cultured umbilical cord-derived mesenchymal stem cells were preserved in 0.9% saline for 0, 2, 4 and 6 hours at 4 ℃. Various methods （trypan blue staining, AnnexinV-PI, terminal deoxynucleotidyl transferase dutp nick end labeling, cell counting kit-8, live-dead assay, cell adherent assay） were used to determine the viability of post-storage umbilical cord-derived mesenchymal stem cells, and the results were compared with colony-forming efficiency, a measure of cell function. RESULTS AND CONCLUSION： Human umbilical cord-derived mesenchymal stem cells cultured in vitro showed a spindle shape and attached growth, the third-generation umbilical cord-derived mesenchymal stem cells were positive for CD29, CD44, CD105, and negative for CD 34 and CD 45. Umbilical cord-derived mesenchymal stem cells incubated in the adipogenic and osteogenic medium were both positive. Cell viability measured with trypan blue correlated moderately with colony-forming efficiency, while the percentage of viable cells measured with other methods correlated better with colony-forming efficiency,among which adherent assay was the most obvious. It is proved that cell adherent assay-measured viability is the most accurate indicator.

关 键 词：干细胞 脐带脐血干细胞 脐带间充质干细胞 细胞移植 体外 细胞保存 保存效果 细胞活性 细胞功能 克隆形成 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]