鸟苷酸结合蛋白2对人脐带来源间充质干细胞成骨分化的调控作用  

Guanylate binding protein 2 regulates osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells

作　　者：白海涛 刘伟江 袁福临 李雪 王洋 刘元林 王振山[1] 张毅[1,2] BAI Hai-tao;LIU Wei-jiang;YUAN Fu-lin;LI Xue;WANG Yang;LIU Yuan-lin;WANG Zhen-shan;ZHANG Yi(Institute of Life Science,Hebei University,Baoding 071002,Hebei Province,China;不详)

机构地区：[1]河北大学生命科学院,河北保定071002 [2]军事科学院军事医学研究院辐射医学研究所,北京100850

出　　处：《中国生物制品学杂志》2022年第7期822-828,835,共8页Chinese Journal of Biologicals

基　　金：国家重点研发计划资助项目(2016YFC1000305)。

摘　　要：目的探讨鸟苷酸结合蛋白2(guanylate binding protein 2,GBP2)对人脐带来源间充质干细胞(human umbilical cord-derived mesenchymal stem cell,hUC-MSC)成骨分化的调控作用。方法培养hUC-MSC,采用吉姆萨染色、流式细胞术及体外诱导分化等方法对hUC-MSC生物学特性进行鉴定;构建过表达GBP2的慢病毒载体,转染hUCMSC,荧光显微镜及流式细胞术检测转染效率,实时荧光定量PCR(real time fluorescence quantitative PCR,q-PCR)法检测转染后hUC-MSC中GBP2的表达,获得稳定过表达GBP2的hUC-MSC(GBP2^(high)hUC-MSC);采用流式细胞术和体外诱导分化试验检测GBP2^(high)hUC-MSC的生物学特性,油红O染色评价体外成脂分化能力,碱性磷酸酶(alkaline phosphatase,ALP)染色评估成骨分化能力,q-PCR法检测成脂和成骨分化相关转录因子的表达。结果培养的hUCMSC的细胞形态、细胞表型和诱导分化均符合MSC国际金标准。经GBP2慢病毒载体转染的hUC-MSC中GBP2的表达显著增加,获得稳定过表达GBP2的hUC-MSC(GBP2^(high)hUC-MSC)。GBP2^(high)hUC-MSC的细胞形态和细胞表型以及体外成脂分化能力均未发生变化,但成骨分化能力发生改变,GBP2^(high)hUC-MSC组ALP染色明显高于hUC-MSC组,成骨分化关键转录因子OPN和ALP的表达也显著高于hUC-MSC组(P均<0.05)。结论GBP2可促进hUCMSC的成骨分化,并上调成骨标志基因OPN和ALP的表达。Objective To investigate the regulatory effect of guanylate binding protein 2(GBP2)on osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells(hUC-MSCs).Methods The hUC-MSCs were cultured,of which the biological characteristics was identified by Giemsa staining,flow cytometry and induced differentiation assay in vitro.Lentiviral vector for overexpression of GBP2 was constructed and transfected into hUC-MSCs.The transfection efficacy was determined by fluorescent microscopy and flow cytometry,while the expression of GBP2 in transfected hUCMSCs by real time fluorescence quantitative PCR(q-PCR)to obtain the hUC-MSCs for stable overexpression of GBP2(GBP2^(high)hUC-MSC).The biological characteristics of GBP2^(high)hUC-MSC was analyzed by flow cytometry and induced differentiation assay in vitro,while the adipogenic differentiation was evaluated by oil red O staining,the osteogenic differentiation by alkaline phosphatase(ALP)staining,and the expressions of adipogenic and osteogenic differentiationsrelated transcription factors were determined by q-PCR.Results The morphology,phenotype and induced differentiation ability of cultured hUC-MSCs met the international gold standard of MSCs.The expression level of GBP2 in hUC-MSCs transfected with lentiviral vector containing GBP2 gene increased significantly,indicating that the hUC-MSCs for stable overexpression of GBP2(GBP2^(high)hUC-MSC)were obtained.The morphology,phenotype as well as adipogenic differentiation ability in vitro showed no change,while the osteogenic differentiation ability changed.The ALP activity and the expressions of osteogenic differentiation-related transcription factors OPN and ALP in GBP2^(high)hUC-MSC group were significantly higher than those in hUC-MSC group(each P<0.05).Conclusion GBP2 promoted the osteogenic diffe-rentiation of hUC-MSCs and up-regulated the expression of osteogenic marker genes OPN and ALP.

关 键 词：间充质干细胞 成骨诱导分化 鸟苷酸结合蛋白2 

分 类 号：Q28[生物学—细胞生物学]