慢病毒介导PEDF基因修饰人脐带间充质干细胞的定量蛋白质组学研究

Quantitative proteomic analysis of human umbilical cord mesenchymal stem cells with pigment epithelium-derived factor gene modification mediated by lentivirus

目的探讨慢病毒介导色素上皮衍生因子(PEDF)基因修饰后人脐带间充质干细胞(hUCMSCs)的蛋白质组学变化。方法将hUCMSCs分为基因修饰组和对照组,基因修饰组采用慢病毒介导PEDF基因修饰hUCMSCs,对照组为正常hUCMSCs,提取各组细胞蛋白;采用FASP酶切法制备蛋白质样本,进行液相-质谱联用仪检测,采用SWATH模式采集数据。根据蛋白检测结果,进行差异蛋白分析及相应蛋白基因本体论(GO)分析和Reactome信号通路显著性富集分析。结果共鉴定出5361个可定量蛋白,与对照组相比,实验组慢病毒介导的PEDF基因修饰后差异表达倍数>1.5且P<0.05的蛋白共432个,其中上调蛋白219个,包括丝氨酸蛋白酶抑制剂家族F成员1(SERPINF1)(PEDF)、DEAD-box家族螺旋酶59(DDX59)、血小板反应蛋白1(THBS1)等;下调蛋白213个,包括Ⅰ型胶原蛋白α1链(COL1A1)、COL18A1等。GO分析结果显示,差异蛋白主要参与纤维蛋白溶解、细胞外结构构建、转运蛋白活性调节、仲醇及胆固醇生物合成、辅酶代谢、肽酶活性调节等多种生物学进程;Reactome信号通路显著性富集分析表明,差异蛋白主要涉及胰岛素样生长因子结合蛋白调节的胰岛素样生长因子的运输和摄取、蛋白质翻译后修饰磷酸化、细胞外基质构成、糖皮质激素代谢等信号通路。结论慢病毒介导PEDF基因修饰的hUCMSCs可通过调节多种蛋白的表达水平,改变细胞外基质结构,调节与细胞增生、自我更新和多能性相关的蛋白表达水平。

Objective To investigate the protein expression changes of human umbilical cord mesenchymal stem cells(hUCMSCs)modified with pigment epithelium-derived factor(PEDF)gene mediated by lentivirus.Methods The hUCMSCs were divided into the control group and experimental group.Cells in the control group were normal hUCMSCs and the cells in experimental group were hUCMSCs with PEDF modification.The proteins from the two groups were collected and processed by FASP method.Samples were fractionated by liquid chromatography and analyzed by tandem mass spectrometry^,and SWATH mode was applied.Differential protein analysis,Gene Ontology(GO)analysis and Reactome pathway enrichment analysis were performed.Results A total of 5361 quantified proteins were detected in this experiment,of which 432 proteins were differentially expressed(fold change>l.5,P<0.05).There were 219 of the 432 proteins up-regulated,including serpin family F member 1(SERPINF1)(PEDF),DEAD-box helicase 59(DDX59)and thrombospondin 1(THBS1),etc.,whereas 213 proteins were down-regulated,including collagen type I alpha 1(COL1A1),COL18A1,etc.GO analysis indicated that the differential proteins were mainly involved in fibrinolysis,extracellular structure organization,regulation of transporter activity,biosynthetic process of secondary alcohol and cholesterol,coenzyme metabolic process and regulation of peptidase activity,etc.Reactome pathway analysis showed that the differential proteins were mostly involved in regulation of insulin like growth factor(IGF)transport and uptake by IGF binding protein,post-translational protein phosphorylation,extracellular matrix organization,metabolism of steroids.Conclusions After gene modification with PEDF mediated by lentivirus,the expression of many proteins in hUCMSCs were changed.PEDF gene modification can alter the structure of extracellular matrix and regulate the expression of proteins associated with cell proliferation,selfrenewal and multipotency.