感染日本血吸虫前后东方田鼠血清补体的动态变化和杀虫作用
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摘要
东方田鼠是迄今为止发现的唯一一种具有天然抗日本血吸虫病特性的哺乳动物,但具体抗病机制尚不明确。本研究应用组织培养法比较了东方田鼠和BALB/c鼠感染日本血吸虫尾蚴后1、3、8、12、15 d的皮肤、肺脏和肝脏中童虫回收情况,结果表明东方田鼠对日本血吸虫的杀伤主要发生在感染后1～3 d和8～12 d。应用RT-PCR和ELISA检测东方田鼠和BALB/c鼠的补体相关分子C3、C9、CD59和DAF的转录水平,结果显示BALB/c小鼠各指标变化不明显,东方田鼠感染后8 d各因子转录水平显著升高。体外杀虫试验结果显示,东方田鼠正常血清和灭活补体血清孵育的日本血吸虫童虫死亡率分别为85.50%±7.15%和60.99%±8.61%,差异具有显著统计学意义(P <0.001)。结果表明东方田鼠补体对日本血吸虫童虫具有显著杀伤效果,东方田鼠早期对日本血吸虫的杀伤作用可能和补体相关。
Microtus fortis is the only mammal that has natural resistance to Schistosomasis japonicum. However, the mechanism involved in this nature is unclear. In this study,portal perfusion and tissue culture were used to collect schistosomula from M. fortis and BALB/c mice that were infected with Schistosoma japonicum cercariae at 1, 3, 8, 12 and 15 days and calculate the recovery rate of worms at skin,lung and liver, respectively. The results showed that schistosomula died out mainly in 1-3 days and 8-12 days post infection in M. fortis. RT-PCR and ELISA methods were applied to analyze the expression levels of complement-related molecules C3, C9, CD59 and DAF in M. fortis and BALB/C mice. The transcript levels of complement-related molecules of M. fortis signi?cantly increased at 8 days after infection whereas these of BALB/c mice had no obvious changes. Worms were cultured for 48 h in media added with normal serum and heat inactivated complement serum from M. fortis to assess the lethal effect on schistosomula. The mortality rates of worms incubated with normal serum and heat inactivated complement serum were 85.50% ± 7.15% and 11.08% ± 3.95%, respectively,indicating signi?cant difference(P<0.001). Therefore, the serum complement of M. fortis may be related to killing effect on Schistosomula in early stages of infection.
Microtus fortis is the only mammal that has natural resistance to Schistosomasis japonicum. However, the mechanism involved in this nature is unclear. In this study,portal perfusion and tissue culture were used to collect schistosomula from M. fortis and BALB/c mice that were infected with Schistosoma japonicum cercariae at 1, 3, 8, 12 and 15 days and calculate the recovery rate of worms at skin,lung and liver, respectively. The results showed that schistosomula died out mainly in 1-3 days and 8-12 days post infection in M. fortis. RT-PCR and ELISA methods were applied to analyze the expression levels of complement-related molecules C3, C9, CD59 and DAF in M. fortis and BALB/C mice. The transcript levels of complement-related molecules of M. fortis signi?cantly increased at 8 days after infection whereas these of BALB/c mice had no obvious changes. Worms were cultured for 48 h in media added with normal serum and heat inactivated complement serum from M. fortis to assess the lethal effect on schistosomula. The mortality rates of worms incubated with normal serum and heat inactivated complement serum were 85.50% ± 7.15% and 11.08% ± 3.95%, respectively,indicating signi?cant difference(P<0.001). Therefore, the serum complement of M. fortis may be related to killing effect on Schistosomula in early stages of infection.
引文
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[15]鲍沁露,黄春兰,陈丹丹,等.染色法判别日本血吸虫尾蚴生命状态的实验研究[J].中国血吸虫病防治杂志,2015,27(1):64-65,69.
[16]马帅,韩艳辉,洪炀,等.日本血吸虫四跨膜孤儿受体(TOR)克隆及其第一个膜外区基因的表达[J].中国动物传染病学报,2014,22(6):46-52.
[17]邬国军,秦志强,罗赛群,等.东方田鼠血清组分对日本血吸虫童虫的体外杀伤力[J].中国寄生虫学与寄生虫病杂志,2005,23(6):404-407.
[18]邵国艳,冯洁,柏熊,等.东方田鼠感染日本血吸虫后的病理与相关基因表达改变[J].中国实验动物学报,2013,21(2):56-60.
[19]朱传刚,杨艺,李小红,等.日本血吸虫虫卵在BALB/c鼠肝脏各叶中的分布调查[J].中国动物传染病学报,2011,19(5):66-70.
[20]王涛,洪炀,傅志强,等.大、小鼠来源32d日本血吸虫凋亡相关基因表达分析[J].中国动物传染病学报,2016,24(3):55-59.
[21]Santoro F,Vandemeulebroucke B,Liebart M C,et al.Schistosoma mansoni:role in vivo of complement in primary infection of mice[J].Exp Parasitol.1982,54(1):40-46.
[22]刘金明,傅志强,李浩,等.东方田鼠ADCC体外杀伤日本血吸虫童虫效果的初步观察[J].寄生虫与医学昆虫学报,2001,8(4):212-219.
[2]李浩,何艳燕,林矫矫,等.东方田鼠抗日本血吸虫病现象的观察[J].中国兽医寄生虫病,2000,8(2):12-15.
[3]孙军,李浩,王喜乐,等.东方田鼠和小鼠感染日本血吸虫后血清NO的变化以及肝、肺病变的比较[J].中国人兽共患病学报,2006,22(5):433-436,439.
[4]孙军,林矫矫,程国锋,等.利用基因表达谱芯片研究东方田鼠和小鼠感染日本血吸虫前后基因的差异性表达[J].北京大学学报(自然科学版),2004,40(4):532-537.
[5]王庆林,易新元,罗新松,等.日本血吸虫肠道蛋白酶对小鼠和东方田鼠血红蛋白降解作用的比较研究[J].中国人兽共患病杂志,2000,16(4):35-36.
[6]朱政斌,喻鑫玲.日本血吸虫童虫在东方田鼠和昆明小鼠中的蛋白差异初探[J].湖南理工学院学报(自然科学版),2014,27(4):71-73.
[7]张新跃,何永康,刘宗传,等.实验室繁殖东方田鼠生理指标的检测[J].中国实验动物学杂志,2001,11(2):128.
[8]Jin J,He S.The complement system is also important in immunogenic cell death[J].Nat Rev Immunol,2017,17(2):143.
[9]Boyle M J,Reiling L,Feng G,et al.Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with Protection against malaria[J].Immunity,2015,42(3):580-590.
[10]Garza J J,Greiner S P,Bowdridge S A.Serum-mediated Haemonchus contortus larval aggregation differs by larval stage and is enhanced by complement[J].Parasite Immunol,2017,39(3).doi:10.1111/pim.12409.
[11]Vignali D A,Bickle Q D,Taylor M G,et al.Comparison of the role of complement in immunity to Schistosoma mansoni in rats and mice[J].Immunology,1988,63(1):55-61.
[12]成钢,王京仁,王兴平,等.东方田鼠和小鼠感染日本血吸虫后组织与血清蛋白组分差异比较研究[J].中国畜牧兽医,2012,39(7):99-102.
[13]蒋守富,邱倩雯,何艳燕,等.东方田鼠血清蛋白组分体外杀伤日本血吸虫童虫作用的研究[J].中国血吸虫病防治杂志,2008,20(4):260-264.
[14]欧阳理,易新元,曾宪芳,等.东方田鼠血清及其不同部分对日本血吸虫童虫的体外杀伤作用[J].中国血吸虫病防治杂志,2003,15(2):98-101.
[15]鲍沁露,黄春兰,陈丹丹,等.染色法判别日本血吸虫尾蚴生命状态的实验研究[J].中国血吸虫病防治杂志,2015,27(1):64-65,69.
[16]马帅,韩艳辉,洪炀,等.日本血吸虫四跨膜孤儿受体(TOR)克隆及其第一个膜外区基因的表达[J].中国动物传染病学报,2014,22(6):46-52.
[17]邬国军,秦志强,罗赛群,等.东方田鼠血清组分对日本血吸虫童虫的体外杀伤力[J].中国寄生虫学与寄生虫病杂志,2005,23(6):404-407.
[18]邵国艳,冯洁,柏熊,等.东方田鼠感染日本血吸虫后的病理与相关基因表达改变[J].中国实验动物学报,2013,21(2):56-60.
[19]朱传刚,杨艺,李小红,等.日本血吸虫虫卵在BALB/c鼠肝脏各叶中的分布调查[J].中国动物传染病学报,2011,19(5):66-70.
[20]王涛,洪炀,傅志强,等.大、小鼠来源32d日本血吸虫凋亡相关基因表达分析[J].中国动物传染病学报,2016,24(3):55-59.
[21]Santoro F,Vandemeulebroucke B,Liebart M C,et al.Schistosoma mansoni:role in vivo of complement in primary infection of mice[J].Exp Parasitol.1982,54(1):40-46.
[22]刘金明,傅志强,李浩,等.东方田鼠ADCC体外杀伤日本血吸虫童虫效果的初步观察[J].寄生虫与医学昆虫学报,2001,8(4):212-219.