氯化钴模拟低氧环境下人脐带间充质干细胞增殖及基因蛋白的表达  

Effect of cobalt chloride-induced hypoxia on proliferation of human umbilical cord-derived mesenchymal stem cells and related gene and protein expressions

作　　者：韩潇[1,2] 白海[1] 尹娇娇[1] 杨柯[1] 韩燕霞[1] 欧剑锋[1] 王存邦[1] 

机构地区：[1]解放军兰州军区兰州总医院血液科/全军血液病中心,甘肃省兰州市730050 [2]兰州大学第二医院血液内科,甘肃省兰州市730030

出　　处：《中国组织工程研究》2015年第45期7268-7273,共6页Chinese Journal of Tissue Engineering Research

基　　金：甘肃省科技重大专项(1102FKDA005)~~

摘　　要：背景:氯化钴可促进人脐带源性间充质干细胞的增殖,具有时间和浓度依赖性,并对其基因蛋白表达具有调控作用。目的:研究氯化钴模拟的低氧环境对人脐带源性间充质干细胞的增殖及基因蛋白表达的影响,旨在建立高效的间充质干细胞体外培养扩增体系。方法:采用组织块法提取人脐带间充质干细胞,氯化钴模拟化学低氧环境,在不同浓度(0,100,150,200,250μmol/L)和不同时间(0,1,2,3,4 d)条件下,用流式细胞仪进行细胞表面相关抗原的鉴定及细胞周期检测;CCK-8法测定细胞增殖情况;RT-PCR测定缺氧诱导因子1α、诱导型一氧化氮合酶、基质细胞衍生因子1、白细胞介素6、转化生长因子βmR NA的表达;Western blot测定缺氧诱导因子1α蛋白的表达。结果与结论:人脐带间充质干细胞表面相关抗原CD29、CD73、CD90、CD105表达阳性,CD31、CD14、CD34、CD45、CD11b、HLA-DR呈阴性表达,且不受氯化钴模拟的低氧环境影响。氯化钴模拟的化学性低氧可促进人脐带间充质干细胞增殖,且具有一定时间及浓度依赖性。RT-PCR检测到低氧组缺氧诱导因子1α、诱导型一氧化氮合酶、基质细胞衍生因子1 m RNA表达上调,而白细胞介素6及转化生长因子βmR NA表达下调,差异有显著性意义(P<0.05)。低氧组中缺氧诱导因子1α蛋白表达增高。结果表明氯化钴模拟的体外低氧环境能促进人脐带间充质干细胞增殖,最佳浓度为200μmol/L,但过高浓度(250μmol/L)时反而抑制其增殖,机制可能与缺氧诱导因子1α基因与蛋白表达增加相关。BACKGROUND： Cobalt chloride（Co Cl2） may promote the proliferation of human umbilical cord-derived mesenchymal stem cells（h UC-MSCs） in a time- and concentration-dependent manner, and meanwhile, Co Cl2 can regulate the expression of genes and proteins in h UC-MSCs.OBJECTIVE： To explore the effects of Co Cl2 induced-hypoxia on the proliferation of h UC-MSCs and gene and protein expressions in h UC-MSCs, thereby establishing an effective method for MSCs culture and amplification in vitro. METHODS： h UC-MSCs were extracted using tissue explant method. Under hypoxia conditions induced by Co Cl2（0, 100, 150, 200, 250 μmol/L） for different periods（0, 1, 2, 3, 4 days）, flow cytometry was used to identify cell surface-associated antigens; cell counting kit-8 was used to detect cell proliferation; RT-PCR was used todetermine levels of hypoxia inducible factor-1α, inducible nitric oxide synthase, stromal cell-derived factor-1, interleukin-6, transforming growth factor-β m RNA; western blot assay was used to detect protein expression of hypoxia inducible factor-1α. RESULTS AND CONCLUSION： The cells were positive for CD29, CD73, CD90, CD105, while negative for CD31, CD14, CD34, CD45, CD11 b, HLA-DR. Moreover, the antigen expression was not affected by Co Cl2 induced-hypoxia. Co Cl2 induced-chemical hypoxia could promote the proliferation of h UC-MSCs in a time- and concentration-dependent manner. RT-PCR results showed that under hypoxia, hypoxia inducible factor 1α, inducible nitric oxide synthase and stromal cell-derived factor-1 m RNA expressions were significantly up-regulated, but interleukin-6 and transforming growth factor-β m RNA expressions were down-regulated significantly（P〈0.05）. Additionally, the protein expression of hypoxia inducible factor 1α was increased under hypoxia conditions. These findings indicate that Co Cl2 induced-hypoxia environment may promote the proliferation of h UC-MSCs and the optimal concentration of Co Cl2 is 200 μmol/L. However, a higher concentration o

关 键 词：脐带 间质干细胞 细胞低氧 细胞增殖 缺氧诱导因子1 组织工程 干细胞 脐带脐血干细胞 人脐带间充质干细胞 低氧 氯化钴 增殖 基因表达 缺氧诱导因子1α 

分 类 号：R394.2[医药卫生—医学遗传学]