过表达Akt1对小鼠骨髓间充质干细胞生物学功能的影响

发布时间：2018-01-15 17:36

本文关键词：过表达Akt1对小鼠骨髓间充质干细胞生物学功能的影响　出处：《生物医学工程与临床》2014年04期 　论文类型：期刊论文

【摘要】：目的探讨Akt1过表达对小鼠骨髓间充质干细胞(BMSCs)生物学功能的影响。方法雄性健康C57小鼠,鼠龄7~8周,体质量18~25 g。取该小鼠胫骨和股骨,利用BMSCs的贴壁生长能力进行分离,并对获取的细胞进行流式鉴定。构建Akt1-IRES-GFP逆转录病毒载体,感染BMSCs,并筛选、扩增Akt1过表达的BMSCs。逆转录-聚合酶链反应(RT-PCR)、Western blot测定转染Akt1表达情况。用不同浓度(0、10、50、100 ng/mL)的γ干扰素(IFN-γ)处理Akt1-BMSCs及BMSCs,采用四唑氮化合物(MTS)法绘制生长曲线,通过Ca2+依赖性磷脂结合蛋白/碘化丙啶(AnnexinV/PI)复染,观察不同浓度IFN-γ对Akt1-BMSCs及GFP-BMSCs细胞凋亡的影响。结果成功培养出小鼠BMSCs,高表达CD29、CD44、Sca-1,而不表达CD45、CD34,CD11b、CD31、FLK-1、MHC-Ⅱ。成功筛选并扩增出Akt1-BMSCs;RT-PCR显示,Akt1-BMSCs的Akt1基因表达量明显高于BMSCs(P0.01);Western blot显示,Akt1-BMSCs的总Akt1蛋白及磷酸化Akt蛋白均高于BMSCs。流式细胞仪检测Akt1-BMSCs及GFP-BMSCs的绿色荧光蛋白(GFP)的表达量均高于95%。细胞增殖及凋亡实验显示,在正常培养及高浓度IFN-γ(100 ng/mL)的刺激下,Akt1-BMSCs显示出了增殖及抗凋亡优势。结论 Akt1-BMSCs比相同代数的BMSCs在正常培养及高浓度IFN-γ(100 ng/mL)的刺激下增殖及抗凋亡能力增强,可用于进行下一步的研究。
[Abstract]:Objective to investigate the expression of Akt1 on mouse bone marrow mesenchymal stem cells (BMSCs) on biological function. Methods healthy male C57 mice, mice 7~8 weeks old, weighing 18~25 g. from the tibia and femur of mice, using BMSCs adherent growth ability were isolated, and the cells were analyzed by flow cytometry to construct. Akt1-IRES-GFP retroviral vector, BMSCs infection, and screening, amplification of Akt1 over expression of BMSCs. reverse transcriptase polymerase chain reaction (RT-PCR), Western blot was determined by the expression of Akt1. (0,10,50100 ng/mL) with different concentrations of interferon gamma (IFN- gamma) Akt1-BMSCs and BMSCs, with four of nitrogen (MTS) growth curve drawing method and through the Ca2+ dependent phospholipid binding protein / propidium iodide (AnnexinV/PI) staining, observe the effects of different concentrations of IFN- on Akt1-BMSCs and GFP-BMSCs cell apoptosis. Results the successful cultivation of a high expression of CD29 in BMSCs mice. CD44, Sca-1, and CD34, the expression of CD45, CD11b, CD31, FLK-1, MHC- II. Successful screened and amplified Akt1-BMSCs Akt1 gene; RT-PCR showed that the expression of Akt1-BMSCs was significantly higher than that of BMSCs (P0.01); Western blot, green fluorescent protein Akt1-BMSCs Akt1 protein and phosphorylated Akt protein were higher than that of BMSCs. detection of Akt1-BMSCs and GFP-BMSCs by flow cytometry (GFP) expression were higher than that cell proliferation and apoptosis in experimental 95%., in normal culture and high concentration of IFN- (100 ng/mL) under the stimulation of Akt1-BMSCs showed proliferation and anti apoptosis. Conclusion Akt1-BMSCs is the same as the algebra of BMSCs in normal and high culture the concentration of IFN- gamma (100 ng/mL) enhanced the stimulation of proliferation and anti apoptosis ability, can be used for the next research.

【作者单位】：中国医学科学院血液学研究所·血液病医院;
【基金】：天津市应用基础与前沿技术研究计划(重点项目)(14JCZDJC33000)
【分类号】：R329.2
【正文快照】： 骨髓间充质干细胞(bonemorrowmesenchymalste-m cells,BMSCs)是一种存在于骨髓、脂肪、脐带血等组织的成体干细胞。BMSCs具有成脂、成骨、成软骨[1~3]、免疫调节[4]和组织修复等多种功能。Akt1基因居PI3K/Akt1信号传导通路核心部位,Akt1参与介导细胞生长、增殖等,其既是细胞存

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