C57BL/6小鼠对PbK173青蒿素敏感性不同虫株的耐受性对比探讨

Resistance of C57BL/6 Mice to Different Artemisinin-sensitive Strains of PbK173

目的:比较C57BL/6小鼠对伯氏疟原虫K173(Pb K173)青蒿素敏感株与抗性株的耐受性及感染过程中的血液参数、脾脏系数、脾脏结构的差异,探究疟原虫对青蒿素产生耐药性是否会加重疟疾感染。方法:Pb K173青蒿素敏感株与抗性株进行平行实验,使用C57BL/6小鼠,根据体质量随机分为1个空白组、4个敏感株感染组与4个抗性株感染组,各感染组小鼠分别同时腹腔接种1×10^(7)个敏感/抗性株的感染红细胞(iRBCs),测生存期组感染后每天记录小鼠的体质量及尾静脉采血涂片计算染虫率,其他感染组分别在感染后第2、5、9天采集小鼠外周血液、脾。检测各组小鼠外周血液参数、脾脏系数、脾脏病理切片及脾细胞。结果:感染第1~3天,抗性株小鼠的染虫率分别为(0.4±0.0、0.8±0.1、1.9±0.4)%,一直较敏感株小鼠的染虫率(0.2±0.1、0.4±0.1、1.1±0.3)%高(P<0.01)。在感染第4天及后期,两组的染虫率逐渐接近。敏感株小鼠生存期(20.5±1.2)d较抗性株小鼠的生存期(23.3±1.4)d短(P<0.01)。感染第9天,敏感株小鼠血液中的白细胞计数(16.2±1.1×10^(9))个/L较抗性株小鼠(10.6×10^(9)±1.8×10^(9))个/L高(P<0.01);流式细胞仪分析染虫小鼠脾细胞发现,敏感株染虫鼠在感染第9天的CD4^(+)/CD8^(+)值(3.6±0.4)较抗性株(2.3±0.2)小鼠高(P<0.01)。C57BL/6染虫鼠的脾脏在感染期间会逐渐肿大,感染第9天,抗性株小鼠的脾脏系数(3.1±0.1)%较敏感株小鼠(2.7±0.2)%大(P<0.01)。C57BL/6染虫鼠感染初期,脾脏红髓发生充血肿胀;感染第9天,脾脏边缘区消失,红髓与白髓的结构被破坏。结论:不经药物治疗时,C57BL/6小鼠外周血液及脾脏的保护免疫反应对Pb K173青蒿素敏感株更敏感;采用鼠-鼠间血传、青蒿素剂量递增法培育的Pb K173青蒿素抗性株生长周期缩短,毒性降低。

Objective:The tolerance of C57BL/6 mice to artemisinin-sensitive and-resistant strains of Plasmodium berghei(Pb)K173 and the differences in blood parameters,spleen coefficient and spleen structure during infection were compared to explore whether the artemisinin resistance of Pb would aggravate malaria infection.Method:Pb K173 artemisinin-sensitive and-resistant strains were tested in parallel.C57BL/6 mice were randomly divided into 1 control group,4 artemisinin-sensitive strain groups and 4 artemisinin-resistant strain groups by body weight.Each infection group was simultaneously inoculated(ip)with 1×10^(7) infected red blood cells(iRBCs)of sensitive/resistant strain.For the mice in the survival test group,the body weight was recorded every day post infection,and the tail vein blood smear was collected to calculate the Pb infection rate.In the other infection groups,peripheral blood and spleen were collected on 2,5 and 9 d after infection.Peripheral blood parameters,spleen coefficient,pathological section of spleen and spleen cells were detected in each group.Result:On 1-3 d after infection,the infection rate of the resistant strain(0.4±0.0,0.8±0.1,1.9±0.4)%was always higher than that of the sensitive strain(0.2±0.1,0.4±0.1,1.1±0.3)%(P<0.01).From the 4^(th) d of infection,the infection rate of the two groups gradually approached.The survival period of the sensitive strain group(20.5±1.2)d was shorter than that of the resistant strain group(23.3±1.4)d(P<0.01).On the 9^(th) d,the white blood cell count of the sensitive strain group(16.2±1.1)×10^(9) cells/L was higher than that of the resistant strain group(10.6±1.8)×10^(9) cells/L(P<0.01).Flow cytometry analysis of spleen cells showed that the sensitive strain group(3.6±0.4)demonstrated a higher CD4^(+)/CD8^(+) value than the resistant strain group(2.3±0.2)on the 9thd(P<0.01).The spleen of C57BL/6 infected mice was gradually enlarged during infection,and on the9thd,the resistant strain group(3.1±0.1)%showed a higher spleen coefficient than the sensitive strain group(2.7±0.2)%(P<0.01).In the early stage of C57BL/6 infected mice,the red pulp of spleen was hyperemic and swollen.On the 9thd,the marginal area of the spleen disappeared and the structure of the red and white pulp was destroyed.Conclusion:Without drug treatment,the protective immune responses of peripheral blood and spleen of C57BL/6 mice were more sensitive to Pb K173 artemisinin-sensitive strain.The artemisinin-resistant strain of Pb K173 bred with mouse-to-mouse blood transmission and increased artemisinin dose exhibited shortened growth period and reduced toxicity.