不同类型血清对大鼠脂肪干细胞分离培养的影响  被引量:6

The Effect of Different Types of Serum on the Isolation and Culture of Rat Adipose Mesenchymal Stem Cells

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

机构地区:[1]昆明医学院第一附属医院头颈外科,云南昆明650031 [2]昆明医学院第一附属医院实验中心,云南昆明650031

出  处:《昆明医学院学报》2010年第3期4-10,共7页Journal of Kunming Medical College

基  金:国家自然科学基金资助项目(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资助项目(KM2007D03)

摘  要:目的研究不同血清培养基对脂肪干细胞分离培养的影响.方法用4种不同类型的血清培养基分离培养大鼠脂肪干细胞,免疫荧光法检测培养细胞CD34、CD90、CD133、Flk-1表面标志;成骨诱导液诱导分化,于14 d进行碱性磷酸酶和钙染色检验各细胞成骨分化特性.结果(1)新生牛血清组可见贴壁单个核细胞形态单一,呈梭形生长,无重叠现象;胎牛血清组均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)新生牛血清组梭形贴壁细胞免疫荧光检测可见CD90为阳性,CD34、CD133、Flk-1染色均为阴性;胎牛血清组多角形细胞CD34、Flk-1均为阳性,CD133组部分为阳性,CD90为阴性;(3)新生牛血清组贴壁细胞多数呈碱性磷酸酶阳性;胎牛血清组细胞多角状细胞死亡,形成空白区,梭形细胞呈碱性磷酸酶阳性;(4)新生牛血清组均可见大量红色钙结节影;胎牛血清组细胞多角状细胞死亡,形成空白区,梭形细胞红色钙结节影.结论(1)新生牛血清培养脂肪干细胞可以得到较纯的细胞;(2)胎牛血清组培养含有大量的血管内皮细胞.Objective Toanalyze the influence ofdifferent types ofserumon the isolation and culture of rat adipose mesenchymal stem cells.Methods Rat adipose mesenchymal stem cells(AMSCs) were isolated and cultured by four types of blood serum culture-medium,and the immunofluorescence method was used to observe expressions and distributions of some special surface markers,such as CD34,CD90,CD133 and Flk-1 on adherent cells.AMSCs were induced by osteoinductive culture medium,and alkaline phosphatase(ALP) and Von Kossa staining were done on the 14 th day to test the characteristic of ossification.Results(1) It was clear that the cell shape in new-born calf serum(NBCS) group was single,presenting fusiform and no overlapping phenomenon;but that in fetal bovine serum(FBS) was complex,we found that numerous polyhedral cells showed nest-like growth and a cobblestone-like morphology,the fusiform cells was existing near here.(2) The results in the immunofluorescent staining in the NBSCS group were negative in CD34,CD133 and Flk-1 and positive in CD90;but the results of polyhedral cells in the FBS group were positive in CD34 and Flk-1,the part of polyhedral cells were positive in CD133,it is negatively in CD90,the positive cells were not found in the control group.(3) The ALP staining of major cells in the NBSCS group was positive;but,in the FBS group,numerous polyhedral cells died,and frmed blank sites,that of some fusiform cells was positive.(4) There were many red calcium nodus in the NBSCS group;but the polyhedral cells died in the FBS group,presenting blanked sites,a part of fusiform cells formed red calcium nodus.Conclusions(1) The comparatively pure ADSCs can be obtained with nutrient medium contains NBCS.(2) The cells cultured with the medium contains FBS are abound with vascular endothelial cells.

关 键 词:脂肪干细胞 免疫荧光 血清 血管内皮细胞 

分 类 号:Q813.1[生物学—生物工程]

 

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