弓形虫ME49虫株α-微管蛋白对神经瘤母细胞自噬的影响
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摘要
为研究刚地弓形虫α-微管蛋白(TgTUBA1)与神经瘤母细胞(N2a)自噬的关系,利用TgTUBA1基因的CDS序列设计引物并引入HA标签序列,提取弓形虫ME49虫株总RNA并反转录获得cDNA,以cDNA为模板进行PCR扩增,获得目的片段,并克隆至pcDNA3.1(+)载体上。将构建的真核表达质粒pcDNA3.1(+)-HA-TUBA1转染至N2a细胞内,通过透射电子显微镜和荧光显微镜观察自噬小体,并通过Western-blot检测LC3Ⅰ/Ⅱ的表达变化。结果显示,成功构建了pcDNA3.1(+)-HA-TUBA1真核表达载体,Western-blot试验检测到50 ku大小的特异目的条带。与空载体组相比,pcDNA3.1(+)-HA-TUBA1转染N2a细胞48 h后,N2a细胞皱缩、突触减少;观察到特征性的自噬小体;自噬相关蛋白LC3Ⅰ/Ⅱ表达上调。本研究证明了TgTUBA1蛋白可以促进N2a细胞自噬现象的发生,这为宿主细胞抗弓形虫感染提供了新的研究思路。
In order to study the relationship between Tg TUBA1 of Toxoplasma gondii and the autophagy of neuroblastoma(N2 a) cells,the CDS sequence of Tg TUBA1 gene was used to design primers with HA tag sequence,and the total RNA was extracted from T.gondii ME49 strain and used to synthesize c DNA by using reverse transcription.Then target fragment was amplified by PCR,and cloned into the pc DNA3.1(+) vector.An eukaryotic expression plasmid pcDNA3.1(+)-HA-TUBA1 was constructed and was transfected into N2 a cells.Afterwards,autophagosomes were observed by transmission electron microscopy and immunofluorescence microscopy.The expression of LC3 I/II was detected by Western-blot.The results showed that the eukaryotic expression plasmid pc DNA3.1(+)-HA-TUBA1 was successfully constructed and a specific band about 50 ku was detected by Western-blot.And after 48 hours of pcDNA3.1(+)-HA-TUBA1 transfection,compared with the control group,transfected N2 a cells were shrinkage and their synapses were decreased.Moreover,characteristic autophagosomes were found and the expression of LC3Ⅰ/Ⅱ was up-regulated.Therefore,Tg TUBA1 protein promoted the autophagy of N2 a cells,which provides a new idea for the research of host cells against T.gondii infection.
In order to study the relationship between Tg TUBA1 of Toxoplasma gondii and the autophagy of neuroblastoma(N2 a) cells,the CDS sequence of Tg TUBA1 gene was used to design primers with HA tag sequence,and the total RNA was extracted from T.gondii ME49 strain and used to synthesize c DNA by using reverse transcription.Then target fragment was amplified by PCR,and cloned into the pc DNA3.1(+) vector.An eukaryotic expression plasmid pcDNA3.1(+)-HA-TUBA1 was constructed and was transfected into N2 a cells.Afterwards,autophagosomes were observed by transmission electron microscopy and immunofluorescence microscopy.The expression of LC3 I/II was detected by Western-blot.The results showed that the eukaryotic expression plasmid pc DNA3.1(+)-HA-TUBA1 was successfully constructed and a specific band about 50 ku was detected by Western-blot.And after 48 hours of pcDNA3.1(+)-HA-TUBA1 transfection,compared with the control group,transfected N2 a cells were shrinkage and their synapses were decreased.Moreover,characteristic autophagosomes were found and the expression of LC3Ⅰ/Ⅱ was up-regulated.Therefore,Tg TUBA1 protein promoted the autophagy of N2 a cells,which provides a new idea for the research of host cells against T.gondii infection.
引文
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[20]廖依婧,高天毅,胡振良,等.Tau蛋白在神经元细胞质和细胞核内的不同功能及其磷酸化对阿兹海默病的影响[J].中国组织化学与细胞化学杂志,2016,25(6):543-549.LIAO Yi-jing,GAO Tian-yi,HU Zhen-liang,et al.The different functions of Tau protein in the cytoplasm and nucleus of neuron and the effect of its phosphorylation on Alzheimer’s Disease[J].Chinese Journal of Histochemistry and Cytochemistry,2016,25(6):543-549.(in Chinese)
[21]武强,吴乐,徐志鹏,等.人脂氧素A4对β淀粉样蛋白所致N2a细胞损伤的保护作用及机制研究[J].中国药师,2017,20(8):1340-1344.WU Qiang,WU Le,XU Zhi-peng,et al.Protective effect and mechanism of human lipoxin A4 on N2a cell injury induced byβ-amyloid protein 25-35[J].China Pharmacist,2017,20(8):1340-1344.(in Chinese)
[22]周凡琳,熊晓敏,梁洁,等.姜黄素通过提高Rab家族蛋白的表达增强N2a/APP695swe细胞的自噬作用[J/OL].重庆医科大学学报,2018:1-7.DOI:10.13406/j.cnki.cyxb.001885ZHOU Fan-lin,XIONG Xiao-min,LIANG Jie,et al.Curcumin enhances the autophagy of N2a/APP695swe cells by up-regulating expression of the Rab family proteins[J/OL].Journal of Chongqing Medical University,2018:1-7.DOI:10.13406/j.cnki.cyxb.001885(in Chinese)
[23]NODA T,KIRISAKO T,MIZUSHIMA N,et al.LC3,a mammalian homologue of yeast Apg8p,is localized in autophagosome membranes after processing[J].The EMBO Journal,2000,19(21):5720-5728.
[24]张庆伟,包军,贾志龙,等.柴胡皂苷A诱导Huh7细胞凋亡并上调细胞自噬水平的研究[J/OL].海南医学院学报,2018:1-15.DOI:10.13210/j.cnki.jhmu.20181119.005ZHANG Qing-wei,BAO Jun,JIA Zhi-long,et al.Saikos aponin A induces apoptosis and upregulates autophagy in Huh7 cells[J/OL].Journal of Hainan Medical College,2018:1-15.DOI:10.13210/j.cnki.jhmu.20181119.005(in Chinese)
[25]THYBERG J,MOSKALEWSKI S.Role of microtubules in the organization of the Golgi complex[J].Experimental Cell Research,1999,246(2):263-279.
[26]胡雅彬.N-乙酰半胱氨酸萝卜硫素通过微管断裂抑制自噬导致非小细胞肺癌细胞凋亡[D].北京:首都医科大学,2018.HU Ya-bin.Sulforaphane N-acetyl-cysteine inhibited autophagy leading to apoptosis via microtubule disruption in non-small cell lung cancer cells[D].Beijing:Capital Medical University,2018.(in Chinese)
[27]LUN Z,LAI D,WEN Y,et al.Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii[J].Proceedings of the National Academy of Sciences,2015,112(29):8835-8842.
[28]郭晓强,张晶晶.微管抑制类抗癌药物---大自然抗癌药物的瑰宝[J].生命世界,2016(12):78-83.GUO Xiao-qiang,ZHANG Jing-jing.Microtubule inhibitory anticancer drugs:A treasure of natural anticancer drugs[J].Life World,2016(12):78-83.(in Chinese)
[29]MOREJOHN L C,FOSKET D E.The biochemistry of compounds with anti-microtubule activity in plant cells[J].Pharmacology&Therapeutics,1991,51(2):217-230.
[30]LYONS-ABBOTT S,SACKETT D L,WLOGA D,et al.α-Tubulin mutations alter oryzalin affinity and microtubule assembly properties to confer-siserenilinaortinid cnate[J].oyraku Eclle Cit,1020,9(12):1825-1834.
[31]MARQUEZ-NAVARRO A,PEREZ-REYES A,ZEPEDA-RODRIGUEZA,et al.RCB20,an experimental benzimidazole derivative,affects tubulin expression and induces gross anatomical changes in Taenia crassiceps cysticerci[J].Parasitology Research,2013,112(6):2215-2226.
[32]COTTINGHAM K.A new PTM for tubulin is found in a parasite[J].Journal of Proteome Research,2010,9(1):4-5.
[2]WITOLA W H,LIU S R,MONTPETIT A,et al.ALOX12 in human toxoplasmosis[J].Infection and Immunity,2014,82(7):2670-2679.
[3]KEMPF M C,CESBRON-DELAUW M F,DESLEE D,et al.Different manifestations of Toxoplasma gondii infection in F344 and LEW rats[J].Medical Microbiology and Immunology,1999,187(3):137-142.
[4]WELLS T N,BURROWS J N,BAIRD J K.Targeting the hypnozoite reservoir of Plasmodium vivax:the hidden obstacle to malaria elimination[J].Trends in Parasitology,2010,26(3):145-151.
[5]姚嘉青,刘丛珊,薛剑,等.细粒棘球蚴原头节β4微管蛋白的可溶性表达、纯化及生物信息学分析[J].中国寄生虫学与寄生虫病杂志,2018,36(3):231-237.YAO Jia-qing,LIU Cong-shan,XUE Jian,et al.Soluble expression,purification and bioinformatics analysis ofβ4-tubulin of Echinococcus granulosus protoscoleces[J].Chinese Journal of Parasitology and Parasitic Diseases,2018,36(3):231-237.(in Chinese)
[6]岳东梅,王金磊,黄思扬,等.大片吸虫α-微管蛋白基因的克隆表达及免疫反应性的研究[J].中国兽医科学,2017,47(4):495-499.YUE Dong-mei,WANG Jin-lei,HUANG Si-yang,et al.Cloning,expression and immune-reactivity ofα-tubulin gene of Fasciola gigantica[J].Chinese Veterinary Science,2017,47(4):495-499.(in Chinese)
[7]LONG S,ANTHONY B,DREWRY L L,et al.A conserved ankyrin repeat-containing protein regulates conoid stability,motility and cell invasion in Toxoplasma gondii[J].Nature Communications,2017,8(1):2236.
[8]SOLEILHAC E,BRILLET-GUEGUEN L,ROUSSEL V,et al.Specific targeting of plant and Apicomplexa parasite tubulin through differential screening using in silico and assay-based approaches[J].International Journal of Molecular Sciences,2018,19(10):E3085.
[9]KOCHL R,HU X W,CHAN E Y W,et al.Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes[J].Traffic,2006,7(2):129-145.
[10]DUSZENKO M,GINGER M L,BRENNAND A,et al.Autophagy in protists[J].Autophagy,2011,7(2):127-158.
[11]FASS E,SHVETS E,DEGANI I,et al.Microtubules support production of starvation-induced autophagosomes but not their targeting and fusion with lysosomes[J].The Journal of Biological Chemistry,2006,281(47):36303-36316.
[12]OHSHIMA J,LEE Y,SASAI M,et al.Role of mouse and human autophagy proteins in IFN-gamma-induced cell-autonomous responses against Toxoplasma gondii[J].Journal of Immunology,2014,192(7):3328-3335.
[13]SELLECK E M,ORCHARD R C,LASSEN K G,et al.A noncanonical autophagy pathway restricts Toxoplasma gondii growth in a strain-specific manner in IFN-γ-activated human cells[J].mBio,2015,6(5):e01157-15.
[14]WANG Y,WEISS L M,ORLOFSKY A.Host cell autophagy is induced by Toxoplasma gondii and contributes to parasite growth[J].The Journal of Biological Chemistry,2009,284(3):1694-1701.
[15]SUBAUSTE C S.Autophagy in immunity against Toxoplasma gondii[J].Current Topics in Microbiology and Immunology,2009,335:251-265.
[16]SKOKOS D,BOTROS H G,DEMEURE C,et al.Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo[J].Journal of Immunology,2003,170(6):3037-3045.
[17]COAKLEY G,MAIZELS R M,BUCK A H.Exosomes and other extracellular vesicles:The new communicators in parasite infections[J].Trends in Parasitology,2015,31(10):477-489.
[18]WOWK P F,ZARDO M L,MIOT H T,et al.Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii[J].Proteomics,2017,17(15-16):201600477.
[19]张蕊,苑玉和,赵明,等.细胞骨架与神经退行性疾病[J].中国药理学通报,2011,27(8):1041-1044.ZHANG Rui,YUAN Yu-he,ZHAO Ming,et al.Cytoskeleton and neurodegenerative diseases[J].Chinese Pharmacological Bulletin,2011,27(8):1041-1044.(in Chinese)
[20]廖依婧,高天毅,胡振良,等.Tau蛋白在神经元细胞质和细胞核内的不同功能及其磷酸化对阿兹海默病的影响[J].中国组织化学与细胞化学杂志,2016,25(6):543-549.LIAO Yi-jing,GAO Tian-yi,HU Zhen-liang,et al.The different functions of Tau protein in the cytoplasm and nucleus of neuron and the effect of its phosphorylation on Alzheimer’s Disease[J].Chinese Journal of Histochemistry and Cytochemistry,2016,25(6):543-549.(in Chinese)
[21]武强,吴乐,徐志鹏,等.人脂氧素A4对β淀粉样蛋白所致N2a细胞损伤的保护作用及机制研究[J].中国药师,2017,20(8):1340-1344.WU Qiang,WU Le,XU Zhi-peng,et al.Protective effect and mechanism of human lipoxin A4 on N2a cell injury induced byβ-amyloid protein 25-35[J].China Pharmacist,2017,20(8):1340-1344.(in Chinese)
[22]周凡琳,熊晓敏,梁洁,等.姜黄素通过提高Rab家族蛋白的表达增强N2a/APP695swe细胞的自噬作用[J/OL].重庆医科大学学报,2018:1-7.DOI:10.13406/j.cnki.cyxb.001885ZHOU Fan-lin,XIONG Xiao-min,LIANG Jie,et al.Curcumin enhances the autophagy of N2a/APP695swe cells by up-regulating expression of the Rab family proteins[J/OL].Journal of Chongqing Medical University,2018:1-7.DOI:10.13406/j.cnki.cyxb.001885(in Chinese)
[23]NODA T,KIRISAKO T,MIZUSHIMA N,et al.LC3,a mammalian homologue of yeast Apg8p,is localized in autophagosome membranes after processing[J].The EMBO Journal,2000,19(21):5720-5728.
[24]张庆伟,包军,贾志龙,等.柴胡皂苷A诱导Huh7细胞凋亡并上调细胞自噬水平的研究[J/OL].海南医学院学报,2018:1-15.DOI:10.13210/j.cnki.jhmu.20181119.005ZHANG Qing-wei,BAO Jun,JIA Zhi-long,et al.Saikos aponin A induces apoptosis and upregulates autophagy in Huh7 cells[J/OL].Journal of Hainan Medical College,2018:1-15.DOI:10.13210/j.cnki.jhmu.20181119.005(in Chinese)
[25]THYBERG J,MOSKALEWSKI S.Role of microtubules in the organization of the Golgi complex[J].Experimental Cell Research,1999,246(2):263-279.
[26]胡雅彬.N-乙酰半胱氨酸萝卜硫素通过微管断裂抑制自噬导致非小细胞肺癌细胞凋亡[D].北京:首都医科大学,2018.HU Ya-bin.Sulforaphane N-acetyl-cysteine inhibited autophagy leading to apoptosis via microtubule disruption in non-small cell lung cancer cells[D].Beijing:Capital Medical University,2018.(in Chinese)
[27]LUN Z,LAI D,WEN Y,et al.Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii[J].Proceedings of the National Academy of Sciences,2015,112(29):8835-8842.
[28]郭晓强,张晶晶.微管抑制类抗癌药物---大自然抗癌药物的瑰宝[J].生命世界,2016(12):78-83.GUO Xiao-qiang,ZHANG Jing-jing.Microtubule inhibitory anticancer drugs:A treasure of natural anticancer drugs[J].Life World,2016(12):78-83.(in Chinese)
[29]MOREJOHN L C,FOSKET D E.The biochemistry of compounds with anti-microtubule activity in plant cells[J].Pharmacology&Therapeutics,1991,51(2):217-230.
[30]LYONS-ABBOTT S,SACKETT D L,WLOGA D,et al.α-Tubulin mutations alter oryzalin affinity and microtubule assembly properties to confer-siserenilinaortinid cnate[J].oyraku Eclle Cit,1020,9(12):1825-1834.
[31]MARQUEZ-NAVARRO A,PEREZ-REYES A,ZEPEDA-RODRIGUEZA,et al.RCB20,an experimental benzimidazole derivative,affects tubulin expression and induces gross anatomical changes in Taenia crassiceps cysticerci[J].Parasitology Research,2013,112(6):2215-2226.
[32]COTTINGHAM K.A new PTM for tubulin is found in a parasite[J].Journal of Proteome Research,2010,9(1):4-5.