过表达Periostin蛋白对内皮前体细胞功能影响的实验研究
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摘要
目的:构建Periostin蛋白过表达慢病毒载体,并检测其对内皮前体细胞(EPCs)功能的影响。方法:从小鼠骨髓中获取EPCs,并进行鉴定。采用Oligos设计获得目的基因,构建慢病毒载体LV5/Periostin。慢病毒感染EPCs,荧光显微镜下观察被感染细胞绿色荧光蛋白(GFP)的表达情况,Western blotting检测Periostin蛋白表达情况。CCK-8法和流式细胞计数检测被感染细胞的增殖和凋亡情况,划痕法和Transwell小室法检测被感染细胞迁移和侵袭能力的改变情况。结果:被重组慢病毒感染的EPCs明显表达GFP;过表达慢病毒组细胞增殖活性较阴性对照组低,而凋亡细胞较阴性对照组多(P<0.05);过表达慢病毒组细胞的迁移率较空白对照组细胞高,侵袭细胞数目较多(P<0.05)。结论:可以从小鼠骨髓中成功分离和培养EPCs,过表达Periostin蛋白会降低EPCs体外增殖的活性,促进其凋亡;但会增加EPCs的体外迁移和侵袭能力。
Objective:To construct the over-expression of Periostin protein lentiviral vector,and investigate its effects on the biological function of endothelial progenitor cells(EPCs).Methods:The mouse EPCs were isolated from bone marrow,cultured and identified.The LV5/Periostin lentiviral vector was constructed,and transferred into the EPCs.The expression of green fluorescent protein(GFP) in transfected cells was observed under fluorescence microscope.The expression level of Periostin in EPCs was verified by Western blotting,the proliferation and apoptosis of infected cells were detected using the cell counting kit-8(CCK-8) assay and flow cytometry,and the migration ability and invasion ability of cells were detected using the scratch and Transwell assgy.Results:The expression of GFP in EPCs was obvious.Compared with the control group,the proliferation activity of cells was lower,and the number of apoptosis cells were more in lentiviral vector group(P<0.05).Compared with the control group,the migration rate of cells was higher,the number of invasion cells were more in lentiviral vector group(P<0.05).Conclusions:EPCs can be isolated from bone marrow,and cultured and identified.The over-expression lentiviral vector of Periostin protein can reduce the growth and proliferation,promote the apotosis,and enhance the migration and invasion ability of EPCs.
Objective:To construct the over-expression of Periostin protein lentiviral vector,and investigate its effects on the biological function of endothelial progenitor cells(EPCs).Methods:The mouse EPCs were isolated from bone marrow,cultured and identified.The LV5/Periostin lentiviral vector was constructed,and transferred into the EPCs.The expression of green fluorescent protein(GFP) in transfected cells was observed under fluorescence microscope.The expression level of Periostin in EPCs was verified by Western blotting,the proliferation and apoptosis of infected cells were detected using the cell counting kit-8(CCK-8) assay and flow cytometry,and the migration ability and invasion ability of cells were detected using the scratch and Transwell assgy.Results:The expression of GFP in EPCs was obvious.Compared with the control group,the proliferation activity of cells was lower,and the number of apoptosis cells were more in lentiviral vector group(P<0.05).Compared with the control group,the migration rate of cells was higher,the number of invasion cells were more in lentiviral vector group(P<0.05).Conclusions:EPCs can be isolated from bone marrow,and cultured and identified.The over-expression lentiviral vector of Periostin protein can reduce the growth and proliferation,promote the apotosis,and enhance the migration and invasion ability of EPCs.
引文
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[14] 汪丽燕,李滨,刘艳华.上调Periostin基因表达对人胃癌细胞增殖的影响[J].胃肠病学,2013,18(9):526.
[15] KANEMITSU Y,MATSUMOTO H,IZUHARA K,et al.Increased periostin associates with greater airflow limitation in patients receiving inhaled corticosteroids[J].J Allergy Clin Immunol Pract,2013,132(2):305.
[16] 周武.内皮祖细胞的生物学性状及其治疗作用的研究进展[J].东南大学学报(医学版),2014,33(6):783.
[2] UNIZONY S,MENENDEZ ME,RASTALSKY N.Inpatient complications in patients with giantcell arteritis:decreased mortality and increased risk of thromboembolism,delirium and adrenal insufficiency[J].Rheumatology (Oxford),2015,54(8):1360.
[3] LIAO YF,FENG Y,CHEN LL,et al.Coronary heart disease risk equivalence in diabetes and arterial diseases characterized by endothelial function and endothelial progenitor cell[J].J Diabetes Complications,2014,28(2):214.
[4] WILLIAMS PA.The role of synthetic extracellular matrices in endothelial progenitor cell homing for treatment of vascular disease[J].Ann Biomed Eng,2015,43(10):2301.
[5] 王惠洁,石金凤,谢远杰,等.Periostin过表达对鼻咽癌6-10B细胞侵袭和迁移的影响及机制[J].解剖学报,2014,45(4):500.
[6] DUCHAMP DE LAGENESTE O,JULIEN A,ABOU-KHALIL R,et al.Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin[J].Nat Commun,2018,9(1):773.
[7] KIM GE,LEE JS,PARK MH.Epithelial periostin expression is correlated with poor survival in patients with invasive breast carcinoma[J].PLoS One,2017,12(11):e0187635.
[8] EKIGUCHI H,LI M,JUJO K,et al.Improved culture-based isolation of differentiating endothelial progenitor cells from mouse bone marrow mononuclear cells[J].PLoS One,2011,6(12):e28639.
[9] LI M,NISHIMURA H,IWAKURA A,et al.Endothelial progenitor cells are rapidly recruited to myocardium and mediate protective effect of ischemic preconditioning via “imported” nitric oxide synthase activity[J].Circulation,2005,111(9):1114.
[10] MAJESKY MW.Vascular development[J].Arterioscler Thromb Vasc Biol,2018,38(3):e17.
[11] HU WW,CHEN PC,CHEN JM,et al.Periostin promotes epithelial-mesenchymal transition via the MAPK/miR-381 axis in lung cancer[J].Oncotarget,2017,8(37):62248.
[12] LIU C,FENG X,WANG B,et al.Bone marrow mesenchymal stem cells promote head and neck cancer progression through Periostin-mediated phosphoinositide 3-kinase/Akt/mammalian target of rapamycin[J].Cancer Sci,2018,109(3):688.
[13] NAKAMA T,YOSHIDA S,ISHIKAWA K,et al.Inhibition of choroidal fibrovascular membrane formation by new class of RNA interference therapeutic agent targeting periostin[J].Gene Ther,2015,22(2):127.
[14] 汪丽燕,李滨,刘艳华.上调Periostin基因表达对人胃癌细胞增殖的影响[J].胃肠病学,2013,18(9):526.
[15] KANEMITSU Y,MATSUMOTO H,IZUHARA K,et al.Increased periostin associates with greater airflow limitation in patients receiving inhaled corticosteroids[J].J Allergy Clin Immunol Pract,2013,132(2):305.
[16] 周武.内皮祖细胞的生物学性状及其治疗作用的研究进展[J].东南大学学报(医学版),2014,33(6):783.