TRIM22与HIV衣壳蛋白p24在脑胶质瘤细胞中的表达特点及共定位研究
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摘要
目的观察外源性三基序蛋白22(TRIM22)与HIV衣壳蛋白p24在U-251脑胶质瘤细胞中的表达特点及共定位情况。方法分别将pEGFP-N3-TRIM22或pDsRed1p24载体转染U-251细胞,通过激光共聚焦显微镜观察TRIM22-EGFP和p24-DsRed1的表达和分布情况。通过激光共聚焦xyz轴扫描,经ImageJ 1.50i软件进行3D结构重建,观察p24-Dsred1在U-251细胞中的分布特点。将pEGFP-N3-TRIM22和pDsRed1p24载体在U-251细胞中共表达,观察TRIM22-EGFP和p24-DsRed1是否存在共定位关系。结果单独转染pEGFP-N3-TRIM22或pDsRed1p24载体时,TRIM22-EGFP和p24-DsRed1在U-251细胞的胞体或突起中均有较高水平的表达;且3D结构重建显示外源性p24-DsRed1可迁移至U-251细胞的足突。在U-251细胞共转染pEGFP-N3-TRIM22和pDsRed1p24载体时,TRIM22-EGFP与p24-DsRed1存在共定位关系,并能以出胞形式脱离细胞。结论 TRIM22与HIV衣壳蛋白p24在U-251脑胶质瘤细胞中存在共定位关系。
Objective To observe the expression characteristics and co-localization of exogenous TRIM22 and HIV capsid protein p24 in glioma cells. Methods The vectors of pEGFP-N3-TRIM22 or pDsRed1-p24 were transfected into U-251 glioma cells respectively to examine the expression of TRIM22-EGFP or p24-DsRed1 by confocal microscopy. Moreover, we used a confocal z-stacking program to achieve series of optical sections and to rebuild 3-D images by ImageJ 1.50 i software to detect the expression characteristics of p24-DsRed1 in U-251 cells. In the end, the vectors of pEGFP-N3-TRIM22 and pDsRed1-p24 were co-transfected into U-251 cells to detect the co-localization between TRIM22 and p24 by confocal microscopy. Results Confocal microscopy results showed that TRIM22-EGFP or p24-Dsred1 was localized to the cell body as well as to protuberance in U-251 cells, and 3 D structural reconstruction showed that p24-Dsred1 could be transferred to foot processes of U-251 cells. Simultaneously, confocal microscopy results also showed that TRIM22 and p24 could be co-localized and their combination could be released by budding in U-251 cells co-transfected with pEGFP-N3-TRIM22 and pDsRed1-p24. Conclusion TRIM22 co-localized to HIV capsid protein p24 and their combination can be released by budding in glioma cells.
Objective To observe the expression characteristics and co-localization of exogenous TRIM22 and HIV capsid protein p24 in glioma cells. Methods The vectors of pEGFP-N3-TRIM22 or pDsRed1-p24 were transfected into U-251 glioma cells respectively to examine the expression of TRIM22-EGFP or p24-DsRed1 by confocal microscopy. Moreover, we used a confocal z-stacking program to achieve series of optical sections and to rebuild 3-D images by ImageJ 1.50 i software to detect the expression characteristics of p24-DsRed1 in U-251 cells. In the end, the vectors of pEGFP-N3-TRIM22 and pDsRed1-p24 were co-transfected into U-251 cells to detect the co-localization between TRIM22 and p24 by confocal microscopy. Results Confocal microscopy results showed that TRIM22-EGFP or p24-Dsred1 was localized to the cell body as well as to protuberance in U-251 cells, and 3 D structural reconstruction showed that p24-Dsred1 could be transferred to foot processes of U-251 cells. Simultaneously, confocal microscopy results also showed that TRIM22 and p24 could be co-localized and their combination could be released by budding in U-251 cells co-transfected with pEGFP-N3-TRIM22 and pDsRed1-p24. Conclusion TRIM22 co-localized to HIV capsid protein p24 and their combination can be released by budding in glioma cells.
引文
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[4] GARCIA-MESA Y,JAY TR,CHECKLEY MA,et al.Immortalization of primary microglia:A new platform to study HIV regulation in the central nervous system[J].J Neurovirol,2017,23(1):47-66.
[5] GRAY LR,ROCHE M,FLYNN JK,et al.Is the central nervous system a reservoir of HIV-1 [J].Curr O Opin HIV AIDS,2014,9(6):552-558.
[6] NATH A.Eradication of human immunodeficiency virus from brain reservoirs[J].J Neurovirol,2015,21(3):227-234.
[7] 孙大康,安新业,周玉明,等.HIV衣壳蛋白P24与TRIM22在HEK293T细胞中的表达及共定位[J].细胞与分子免疫学杂志,2015,8(31):1081-1084.
[8] COCKBILL LM,MURK K,LOVE S,et al.Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells[J].Mol Bio Cell,2015,26(25):4552-4561.
[9] XING D,WANG J,OU S,et al.Expression of neonatal Nav1.5 in human brain astrocytoma and its effect on proliferation,invasion and apoptosis of astrocytoma cells[J].Oncol Rep,2014,31(6):2692-2700.
[10] LIANG Q,MA C,ZHAO Y,et al.Inhibition of STAT3 reduces astrocytoma cell invasion and constitutive activation of STAT3 predicts poor prognosis in human astrocytoma[J].PloS One,2013,8(12):e84723.
[11] AMBROSINI E,SLEPKO N,KOHLEISEN B,et al.HIV-1 Nef alters the expression of betaII and epsilon isoforms of protein kinase C and the activation of the long terminal repeat promoter in human astrocytoma cells[J].Glia,1999,27(2):143-151.
[12] 安新业,程艳丽,孙大康,等.pEGFP-N3-TRIM22真核表达载体的构建及在HEK293T细胞中的表达[J].免疫学杂志,2015,7(31):557-561.
[13] CHURCHILL M,NATH A.Where does HIV hide?A focus on the central nervous system[J].Curr Opin HIV AIDS,2013,8(3):165-169.
[14] STURDEVANT CB,JOSEPH SB,SCHNELL G,et al.Compartmentalized replication of R5 T cell-tropic HIV-1 in the central nervous system early in the course of infection[J].PLoS Pathog,2015,11(3):e1004720.
[15] CHURCHILL MJ,WESSELINGH SL,COWLEY D,et al.Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia[J].Ann Neurol,2009,66(2):253-258.
[16] LI GH,ANDERSON C,JAEGER L,et al.Cell-to-cell contact facilitates HIV transmission from lymphocytes to astrocytes via CXCR4[J].AIDS (London,England),2015,29(7):755-766.
[17] LUO X,HE JJ.Cell-cell contact viral transfer contributes to HIV infection and persistence in astrocytes[J].J Neurovirol,2015,21(1):66-80.
[18] LIU X,KUMAR A.Differential signaling mechanism for HIV-1 Nef-mediated production of IL-6 and IL-8 in human astrocytes[J].Sci Rep,2015,5:9867.
[19] GRAY LR,TURVILLE SG,HITCHEN TL,et al.HIV-1 entry and trans-infection of astrocytes involves CD81 vesicles [J].PloS One,2014,9(2):e90620.
[20] SUNDQUIST WI,KRAUSSLICH HG.HIV-1 assembly,budding,and maturation [J].CSH Perspect Med,2012,2(7):a006924.
[21] JARVIS LM.Conquering HIV’s capsid [J].Chem Eng News,2017,95(31):23-25.
[22] LIAN Q,SUN B.Interferons command Trim22 to fight against viruses [J].Cell Mol Immunol,2017,14(9):794-796.
[23] OHMINE S,SAKUMA R,SAKUMA T,et al.The antiviral spectra of TRIM5alpha orthologues and human TRIM family proteins against lentiviral production[J].PLoS One,2011,6(1):e16121.