大鼠脂肪干细胞培养中一种特异细胞的表型与成骨诱导  

Ossification induction and phenotype of a special cell in culture process of rat adipose-derived stem cells

作　　者：王福科[1] 刘流[1] 何晓光[1] 赵德萍[2] 代晓明[1] 李逸松[1] 

机构地区：[1]昆明医学院第一附属医院头颈外科,云南省昆明市650032 [2]昆明医学院第一附属医院实验中心,云南省昆明市650032

出　　处：《中国组织工程研究与临床康复》2010年第40期7509-7512,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30960388);云南科技厅联合专项基金资助项目(2007C0007R);昆明医学院博士创新基金项目(KM2007D03)~~

摘　　要：背景:前期实验在大鼠脂肪干细胞培养中发现了梭形细胞中混杂一种特异细胞,呈巢状生长,形成铺路石样形态。这种细胞和脂肪干细胞关系密切,有必要明确其性质。目的:分析这种特异细胞的表型及诱导成骨特性以判断这种细胞的性质。方法:采用Ⅰ型胶原酶分离大鼠脂肪干细胞,置于含有胎牛血清的培养基中培养,免疫荧光法检测培养梭形细胞、多角细胞的CD34、CD133、CD90,Flk-1表面标志;取第2代细胞,以成骨诱导液诱导分化,于培养14d进行碱性磷酸酶和钙染色检验特异细胞的成骨分化特性。结果与结论:①胎牛血清培养的脂肪干细胞可见大量多角细胞呈巢状生长,形成铺路石样形态,间或有梭形细胞存在。②梭形细胞CD90为阳性,CD34、Flk-1、CD133为阴性;多角形细胞CD34、Flk-1均为阳性,CD133部分为阳性,CD90为阴性。③碱性磷酸酶染色多角状细胞多数死亡,形成空白区,梭形细胞呈碱性磷酸酶阳性。④茜素红染色多角状细胞死亡,形成空白区,梭形细胞集聚形成红色钙结节影。结果证实脂肪干细胞培养中的特异细胞为血管内皮细胞。BACKGROUND：In previous experiment,a type of special cell that mixed fusiform adipose-derived stem cells（ADSCs） was found.A great quantity of polyhedral cells offer nest shape growth and take on cobblestone shape morphology.This is closely related cells to ADSCs,and it is necessary to clarify its nature.OBJECTIVE：To determine the nature of the special cell by analysis of this specific phenotype and the induction of bone formation of these cells.METHODS：Rat ADSCs were isolate and cultured by collagenaseⅠ,cultured by the medium containing fetal bovine serum.The immunofluorescence method was used to observe expressions and distributions of some special surface marker of the polygonal and fibroblastic cells,such as CD34,CD90,CD133 and Flk-1.Cells at passage 2 were obtained and induced by osteoinductive culture medium.Alkaline phosphatase（ALP） and Von Kossa staining were done on day 14 to test the characteristic of ossification.RESULTS AND CONCLUSION：①Shape of cells cultured in fetal bovine serum（FBS） showed that a great quantity of polyhedral cells offered nest growth and took on cobblestone morphology,and the fusiform cells were visible.②The fusiform cells were positive for CD90 and negative for CD34,CD133 and Flk-1;the polyhedral cells were positive for CD34 and Flk-1,and a part of polyhedral cells were positive for CD133,and negative for CD90.③ In the process of ALP staining,a great quantity of polyhedral cells died,forming blank region;that of some fusiform cells was positive.④The polyhedral cells died in the process of Von Kossa staining,presenting blank region;a part of fusiform cells formed red calcium nodus.The results have shown that the special cell is vascular endothelial cells in the culture process of rat ADSCs.

关 键 词：脂肪干细胞 免疫荧光 血清 血管内皮细胞 干细胞培养与分化 

分 类 号：R394.2[医药卫生—医学遗传学]