新孢子虫急性感染对小鼠器官中TLR3表达的影响
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摘要
为了研究新孢子虫急性感染对小鼠体内不同器官中Toll样受体3(TLR3)表达的影响,试验将培养、纯化的新孢子虫Nc-1株速殖子通过腹腔注射途径感染5周龄C57BL/6小鼠(8×106个速殖子/只),感染后第2天和第8天分别采集小鼠的心脏、肝脏、脾脏、肺脏和脑,通过荧光定量PCR和免疫组织化学方法,分别从mRNA和蛋白质水平检测各器官中TLR3的表达量。结果表明:感染组小鼠脾脏和肺脏中TLR3 mRNA的相对表达量较对照组显著或极显著升高(P<0. 05或P<0. 01),其中脾脏中第2天和第8天TLR3 mRNA的相对表达量为对照组的6倍或9倍,肺脏中TLR3 mRNA的相对表达量约为对照组的4倍;肝脏和心脏中TLR3 mRNA的相对表达量约为对照组的2倍,但差异均不显著(P>0. 05),脑中TLR3 mRNA的相对表达量未见明显变化。免疫组织化学结果与荧光定量PCR结果基本一致。说明新孢子虫急性感染可上调小鼠多个器官中TLR3的表达。
In order to study the effect of Neospora caninum acute infection on the expression of Toll-like receptor 3(TLR3) in different organ of mice,the cultured and purified tachyzoites of N. caninum Nc-1 strain(8×106 tachyzoites per mouse) were adopted in this experiment for the infection of 5-week-old C57 BL/6 mice by intraperitoneal injection. The organ heart,liver,spleen,lung and brain of mice were collected on 2 and8 days post infection(dpi). Fluorescence quantitative PCR and immunohistochemistry methods were used for the detection of the expression of TLR3 in organ at mRNA and protein levels,respectively. The results showed that the relative mRNA expression of TLR3 in mouse spleen and lung in the infection group was significantly or extremely higher than those in the control group(P < 0. 05 or P < 0. 01,respectively). Among them,the relative mRNA expression of TLR3 in spleen was 6 or 9 times as much as that of the control group. The relative mRNA expression of TLR3 in lung was about 4 times as much as that of the control group. The relative mRNA expression of TLR3 in liver and heart were about twice as much as that of the control group,but with no statistically significant difference(P>0. 05). The relative mRNA expression of TLR3 in brain had no obvious change. The result of immunohistochemistry was consistent with that of fluorescence quantitative PCR. The results suggested that Neospora caninum acute infection could upregulate TLR3 expression in multiple tissues of mice.
In order to study the effect of Neospora caninum acute infection on the expression of Toll-like receptor 3(TLR3) in different organ of mice,the cultured and purified tachyzoites of N. caninum Nc-1 strain(8×106 tachyzoites per mouse) were adopted in this experiment for the infection of 5-week-old C57 BL/6 mice by intraperitoneal injection. The organ heart,liver,spleen,lung and brain of mice were collected on 2 and8 days post infection(dpi). Fluorescence quantitative PCR and immunohistochemistry methods were used for the detection of the expression of TLR3 in organ at mRNA and protein levels,respectively. The results showed that the relative mRNA expression of TLR3 in mouse spleen and lung in the infection group was significantly or extremely higher than those in the control group(P < 0. 05 or P < 0. 01,respectively). Among them,the relative mRNA expression of TLR3 in spleen was 6 or 9 times as much as that of the control group. The relative mRNA expression of TLR3 in lung was about 4 times as much as that of the control group. The relative mRNA expression of TLR3 in liver and heart were about twice as much as that of the control group,but with no statistically significant difference(P>0. 05). The relative mRNA expression of TLR3 in brain had no obvious change. The result of immunohistochemistry was consistent with that of fluorescence quantitative PCR. The results suggested that Neospora caninum acute infection could upregulate TLR3 expression in multiple tissues of mice.
引文
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[13] ZHU W W,ZHAO S T,LIU Z H,et al. Pattern recognition receptor-initiated innate antiviral responses in mouse epididymal epithelial cells[J]. J Immunol,2015,194(10):4825-4835.
[14] PFAFFL M W. A new mathematical model for relative quantification in real-time RT-PCR[J]. Nucleic Acids Res,2001,29(9):e45.
[15] MADSON D M,MAGSTADT D R,ARRUDA P H E,et al. Pathogenesis of porcine epidemic diarrhea virus isolate(US/Iowa/18984/2013)in 3-week-old weaned pigs[J]. Vet Microbiol,2014,174(1/2):60-68.
[16]何小兵,贾怀杰,景志忠.Toll样受体对病原寄生虫的天然免疫识别[J].生物工程学报,2012,28(12):1401-1413.
[17]张馨月,刘晓雷,吴秀萍,等.旋毛虫感染对肌肉卫星细胞的TLR3及B7-H1表达的影响[J].中国预防兽医学报,2014,36(4):323-326.
[18]刘瀛,颜超,华慧,等. TLR3和IRF3在华支睾吸虫感染C3H/He N小鼠中表达的初步分析[J].徐州医学院学报,2013,33(9):602-605.
[2]刘群.新孢子虫病[M].北京:中国农业大学出版社,2013.
[3] REGIDOR-CERRILLO J,ARRANZ-SOLIS D,BENAVIDES J,et al. Neospora caninum infection during early pregnancy in cattle:how the isolate influences infection dynamics,clinical outcome and peripheral and local immune responses[J]. Vet Res,2014,45:10.
[4] MACALDOWIE C,MALEY S W,WRIGHT S,et al. Placental pathology associated with fetal death in cattle inoculated with Neospora caninum by two different routes in early pregnancy[J]. J Comp Pathol,2004,131(2):142-156.
[5] MALEY S W,BUXTON D,RAE A G,et al. The pathogenesis of neosporosis in pregnant cattle:inoculation at mid-gestation[J]. J Comp Pathol,2003,129(2):186-195.
[6] BEITING D P,PEIXOTO L,AKOPYANTS N S,et al. Differential induction of TLR3-dependent innate immune signaling by closely related parasite species[J]. PLoS One,2014,9:e88398.
[7] GURURAJAN M,JACOB J,PULENDRAN B. Toll-like receptor expression and responsiveness of distinct murine splenic and mucosal B-cell subsets[J]. PLoS One,2007,2(9):e863.
[8]陆天明,许庆梅,马乐.TLR3信号通路的研究进展[J].世界最新医学信息文摘,2017,17(52):94,96.
[9] MARTIN M U,WESCHE H. Summary and comparison of the signaling mechanisms of the toll/interleukin-1 receptor family[J]. Biochim Biophys Acta,2002,1592(3):265-280.
[10] MARIN M S,HECKER Y P,QUINTANA S,et al. Toll-like receptors 3,7 and 8 are upregulated in the placental caruncle and fetal spleen of Neospora caninum experimentally infected cattle[J]. Vet Parasitol,2017,236:58-61.
[11] QIAN W F,WANG H,SHAN D,et al. Activity of several kinds of drugs against Neospora caninum[J]. Parasitol Int,2015,64(6):597-602.
[12]邓冲,张维,刘群,等.Balb/c小鼠新孢子虫病动物模型的建立及在其体内发育过程的研究[J].中国农业科学,2009,42(3):1123-1128.
[13] ZHU W W,ZHAO S T,LIU Z H,et al. Pattern recognition receptor-initiated innate antiviral responses in mouse epididymal epithelial cells[J]. J Immunol,2015,194(10):4825-4835.
[14] PFAFFL M W. A new mathematical model for relative quantification in real-time RT-PCR[J]. Nucleic Acids Res,2001,29(9):e45.
[15] MADSON D M,MAGSTADT D R,ARRUDA P H E,et al. Pathogenesis of porcine epidemic diarrhea virus isolate(US/Iowa/18984/2013)in 3-week-old weaned pigs[J]. Vet Microbiol,2014,174(1/2):60-68.
[16]何小兵,贾怀杰,景志忠.Toll样受体对病原寄生虫的天然免疫识别[J].生物工程学报,2012,28(12):1401-1413.
[17]张馨月,刘晓雷,吴秀萍,等.旋毛虫感染对肌肉卫星细胞的TLR3及B7-H1表达的影响[J].中国预防兽医学报,2014,36(4):323-326.
[18]刘瀛,颜超,华慧,等. TLR3和IRF3在华支睾吸虫感染C3H/He N小鼠中表达的初步分析[J].徐州医学院学报,2013,33(9):602-605.