硫氧还蛋白还原酶对弓形虫速殖子增殖的影响
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摘要
目的探讨siRNA抑制弓形虫硫氧还蛋白还原酶对弓形虫增殖的影响。方法将针对弓形虫硫氧还蛋白还原酶合成3组siRNA通过一定的电压和脉冲时间电转弓形虫,将弓形虫在电转30min后接种到MDA-MB-231细胞,24h后Giemsa染色,并拍照,光镜下计数细胞内平均的弓形虫速殖子数目;36h后采用荧光定量PCR检测硫氧还蛋白还原酶基因的相对表达量,试剂盒法测定酶的活性。结果 Giemsa染色后计数细胞内平均弓形虫速殖子数分别为(9±7)个、(13±10)个、(6±5)个、(37±17)个,RNA干扰后弓形虫硫氧还蛋白还原酶基因相对表达量siRNA001、002、003组分别为0.54±0.09、0.33±0.04、0.21±0.03,与阴性对照组0.93±0.08比较差异有统计学意义(t1=1.865,t2=1.898,t3=1.843,P<0.05),酶活性也显著下降。结论通过siRNA干扰弓形虫弓形虫硫氧还蛋白还原酶基因的表达,能抑制弓形虫在细胞内的增殖。
Objective To investigate the effect that inhibiting thioredoxin reductase with siRNA has on the proliferation of Toxoplasma gondii. Methods Three groups of siRNAs were synthesized with T.gondii thioredoxin reductase,and T.gondii was electrotransformed with a certain voltage and pulse time.After 30 minutes of electroporation,T.gondii was inoculated into MDA-MB-231 cells.After 24 hours,it was stained with Giemsa stain and photographed.The number of T.gondii tachyzoites in the cells was counted under a light microscope.After 36 hours,the relative expression of the thioredoxin reductase gene was detected using quantitative PCR,and the activity of the enzyme was determined using a kit. Results After Giemsa staining,the average number of T.gondii tachyzoites in the cells was 9±7,13±10,6±5,and 37±17.After RNA interference,the relative levels of expression of the thioredoxin reductase genes of T.gondii in groups treated with siRNA001,002,003 were 0.54±0.09,0.33±0.04,and 0.21±0.03 respectively.Relative levels of expression differed significantly(t1=1.865,t2=1.898,t3=1.843,P<0.05)from the level in the negative control group(0.93±0.08),and enzyme activity also decreased significantly.Enzyme activity in the three experimental groups and the negative control group was 0.000 441±0.000 098,0.0 002 205±0.000 084,0.000 147±0.000 049,and0.001 764±0.000 147 respectively. Conclusion siRNA inhibited the proliferation of T.gondii in cells by blocking the expression of the thioredoxin reductase gene of T.gondii.
Objective To investigate the effect that inhibiting thioredoxin reductase with siRNA has on the proliferation of Toxoplasma gondii. Methods Three groups of siRNAs were synthesized with T.gondii thioredoxin reductase,and T.gondii was electrotransformed with a certain voltage and pulse time.After 30 minutes of electroporation,T.gondii was inoculated into MDA-MB-231 cells.After 24 hours,it was stained with Giemsa stain and photographed.The number of T.gondii tachyzoites in the cells was counted under a light microscope.After 36 hours,the relative expression of the thioredoxin reductase gene was detected using quantitative PCR,and the activity of the enzyme was determined using a kit. Results After Giemsa staining,the average number of T.gondii tachyzoites in the cells was 9±7,13±10,6±5,and 37±17.After RNA interference,the relative levels of expression of the thioredoxin reductase genes of T.gondii in groups treated with siRNA001,002,003 were 0.54±0.09,0.33±0.04,and 0.21±0.03 respectively.Relative levels of expression differed significantly(t1=1.865,t2=1.898,t3=1.843,P<0.05)from the level in the negative control group(0.93±0.08),and enzyme activity also decreased significantly.Enzyme activity in the three experimental groups and the negative control group was 0.000 441±0.000 098,0.0 002 205±0.000 084,0.000 147±0.000 049,and0.001 764±0.000 147 respectively. Conclusion siRNA inhibited the proliferation of T.gondii in cells by blocking the expression of the thioredoxin reductase gene of T.gondii.
引文
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[16] Conrad M,Jakupoglu C,Moreno SG,et al.Essential role for mitochondrial thioredoxin reductase in hematopoiesis,heart development,and heart function[J].Mol Cell Biol,2004,24(21):9414-23.
[17] Zhang J,Qin X,Zhu YU,et al.Mechanism of dexamethasone in the context of Toxoplasma gondii infection[J].Parasitology,2017,144(11):1551-9.
[18] Zheng Y,Tao M,Li Z,et al.Cloning and characterization of selenoprotein thioredoxin reductase gene in Haematococcus pluvialis[J].Algal Res,2016(17):97-104.
[19] Zhao S,Gong H,Zhou Y,et al.Identification of a thioredoxin reductase from Babesia microti during mammalian infection[J].Parasitol Res,2016,115(8):3219-27.
[20] Arias DG,Carranza PG,Lujan HD,et al.Immunolocalization and enzymatic functional characterization of the thioredoxin system in Entamoeba histolytica[J].Free Radic Biol Med,2008,45(1):32-9.
[21] Arias DG,Regner EL,Iglesias AA,et al.Entamoeba histolytica thioredoxin reductase:molecular and functional characterization of its atypical properties[J].Biochim Biophys Acta,2012,1820(12):1859-66.
[22] Li T,Ziniel PD,He PQ,et al.High-throughput screening against thioredoxin glutathione reductase identifies novel inhibitors with potential therapeutic value for schistosomiasis[J].Infect Dis Poverty,2015,4(1):40.
[23] Zhao S,Gong H,Zhou Y,et al.Identification of a thioredoxin reductase from Babesia microti during mammalianinfection[J].Parasitol Res.2016(8):3219-27.
[2] Tedford E,Mcconkey G.Neurophysiological changes induced by chronic Toxoplasma gondii infection[J].Pathogens,2017,6(2):19.
[3] Wang H,Li M,Liu J,et al.Toxoplasma gondii infection induces high mobility group box 1released from mouse macrophages[J].Front Microbiol,2017(8):658.
[4]谢曙英,殷旭仁,华万全,等.日本血吸虫硫氧还蛋白谷胱甘肽还原酶基因克隆和序列分析[J].中国血吸虫病防治杂志,2008,20(6):427-30.
[5] Takahashi K,Cohen HJ.Selenium-dependent glutathione peroxidase protein and activity:immunological investigations on cellular and plasma enzymes[J].Blood,1986,68(3):640-5.
[6] Takahashi K,Yamamoto Y,Kobayashi C,et al.Primary structure of human plasma glutathione peroxidase deduced from cDNA sequences[J].Phosphorus&Sulfur&the Related Elements,2010,67(1-4):455-60.
[7]张笑天,熊咏民.硫氧还蛋白还原酶研究进展[J].国外医学医学地理分册,2005,26(4):148-51.
[8] Budachetri K,Karim S.An insight into the functional role of thioredoxin reductase,a selenoprotein,in maintaining normal native microbiota in the Gulf Coast tick(Amblyomma maculatum)[J].Insect Mol Biol,2015,24(5):570-81.
[9] Rundlof AK,Arner ES.Regulation of the mammalian selenoprotein thioredoxin reductase 1in relation to cellular phenotype,growth,and signaling events[J].Antioxid Redox Signal,2004,6(1):41-52.
[10] Park YS,Misonou Y,Fujiwara N,et al.Induction of thioredoxin reductase as an adaptive response to acrolein in human umbilical vein endothelial cells[J].Biochem Biophys Res Commun,2005,327(4):1058-65.
[11] Lindner DJ,Ma X,Hu J,et al.Thioredoxin reductase plays a critical role in IFN retinoid-mediated tumor-growth control in vivo[J].Clin Cancer Res,2002,8(10):3210-8.
[12] Ma X,Hu J,Lindner DJ,et al.Mutational analysis of human thioredoxin reductase 1.Effects on p53-mediated gene expression and interferon and retinoic acid-induced cell death[J].J Biol Chem,2002,277(25):22460-8.
[13] Kim JR,Kwon KS,Yoon HW,et al.Oxidation of proteinaceous cysteine residues by dopamine-derived H2O2in PC12cells[J].Arch Biochem Biophys,2002,397(2):414-23.
[14]赵少若,周金林.原虫硫氧还蛋白还原酶研究进展[J].动物医学进展,2016,37(3):86-90.
[15]刘楠,张晓磊,郭玲玲,等.刚地弓形虫ROP16-ROP18复合基因真核表达载体的构建[J].中国病原生物学杂志,2017,12(1):57-61.
[16] Conrad M,Jakupoglu C,Moreno SG,et al.Essential role for mitochondrial thioredoxin reductase in hematopoiesis,heart development,and heart function[J].Mol Cell Biol,2004,24(21):9414-23.
[17] Zhang J,Qin X,Zhu YU,et al.Mechanism of dexamethasone in the context of Toxoplasma gondii infection[J].Parasitology,2017,144(11):1551-9.
[18] Zheng Y,Tao M,Li Z,et al.Cloning and characterization of selenoprotein thioredoxin reductase gene in Haematococcus pluvialis[J].Algal Res,2016(17):97-104.
[19] Zhao S,Gong H,Zhou Y,et al.Identification of a thioredoxin reductase from Babesia microti during mammalian infection[J].Parasitol Res,2016,115(8):3219-27.
[20] Arias DG,Carranza PG,Lujan HD,et al.Immunolocalization and enzymatic functional characterization of the thioredoxin system in Entamoeba histolytica[J].Free Radic Biol Med,2008,45(1):32-9.
[21] Arias DG,Regner EL,Iglesias AA,et al.Entamoeba histolytica thioredoxin reductase:molecular and functional characterization of its atypical properties[J].Biochim Biophys Acta,2012,1820(12):1859-66.
[22] Li T,Ziniel PD,He PQ,et al.High-throughput screening against thioredoxin glutathione reductase identifies novel inhibitors with potential therapeutic value for schistosomiasis[J].Infect Dis Poverty,2015,4(1):40.
[23] Zhao S,Gong H,Zhou Y,et al.Identification of a thioredoxin reductase from Babesia microti during mammalianinfection[J].Parasitol Res.2016(8):3219-27.