夏氏、伯氏鼠疟原虫实验感染状况比较

用夏氏、伯氏疟原虫血传感染小鼠，采尾血制薄血片，姬氏染色，计１万个红细胞内疟原虫感染数。结果不同虫种同等数量或同虫种不同数量原虫其红细胞感染率不同，均有高度显著性差异。昆明鼠对夏氏疟原虫不敏感；对伯氏疟原虫敏感度高，易接种成功。血传伯氏疟原虫以１０５红细胞感染数为宜，其红细胞感染率高，原虫密度上升速度较快，动物不易死亡。     

伯氏疟原虫类巨噬细胞移动抑制因子的表达和纯化

目的 表达和纯化伯氏疟原虫类巨噬细胞移动抑制因子（PbMIF）。方法 根据GenBank中PbMIF mRNA的预测序列，设计特异引物，采用RT-PER方法从伯氏疟原虫ANKA株红内期RNA扩增获得PbMIF基因。将PbMIF基因与T载体连接并测序，利用NCBI中Blast程序分析测序结果。阳性T／A克隆质粒经BamH Ⅰ和XhoⅠ双酶切后，将目的片段克隆至原核表达载体pET28a，并转化大肠埃希菌BL21（DE3），收集经IPTG诱导表达的细菌进行SDS-PAGE分析，并对表达产物进行亲和纯化。结果 从伯氏疟原虫RNA中扩增到PbMIF cDNA，长度为351bp，编码116个氨基酸。测序结果显示，其核苷酸序列与GenBank中PbMIF cDNA的预测序列完全一致，编码的氨基酸在一级结构上与恶性疟原虫类巨噬细胞移动抑制因子（PfbbMIF）的同源性为76％．与人类和小鼠MIF的同源性为31％。表达产物为可溶性的PbMIF蛋白，亲和纯化后的PbMIF蛋白纯度为71％。结论 表达并纯化出可溶性PbMIF蛋白，为进一步研究疟原虫的生物学特性奠定了物质基础。     

人群TLR基因多态性与间日疟原虫感染的相关性

目的　比较间日疟原虫感染人群的Toll样受体（Toll-like receptor, TLR）5、TLR9基因3个单核苷酸多态性（single nucleotide polymorphism, SNP）位点差异,探讨其与间日疟原虫不同感染类型的相关性。方法　收集中缅边境地区（云南省盈江县和缅甸拉咱市）2017—2019年间日疟原虫感染者202例及健康对照者104例,将上述研究对象分为无症状感染组、有症状感染组及健康对照组,其中无症状感染组与有症状感染组统称为感染组;采用飞行时间质谱分型技术检测以上研究对象TLR5、TLR9基因3个SNP位点,分析不同组之间TLR5、TLR9基因SNP位点等位基因和基因型的分布差异。结果　3组研究对象之间性别与年龄分布差异均无统计学意义（P均＞0.05）;3组之间TLR9 1486C/T位点、TLR9 G1174A位点和TLR5 R392Stop位点的等位基因分布差异均无统计学意义（P均＞0.05）;健康对照组与感染组间及健康对照组与无症状感染组间TLR9 1486C/T位点的AG基因型分布差异均有显著性统计学意义（P均＜0.05）;3组之间TLR9 G1174A位点、TLR5 R392Stop位点的基因型分布差异均无统计学意义（P均＞0.05）。结论　TLR9 1486C/T位点的基因型多态性与间日疟原虫感染相关,但尚未发现TLR9 G1174A位点、TLR5 R392Stop位点与间日疟原虫感染的相关性,本研究结果可为揭示宿主基因多态性对间日疟原虫感染的影响提供重要线索。     

华支睾吸虫感染小鼠肝脏中TLR2 mRNA动态表达的初步研究

目的观察华支睾吸虫感染小鼠肝脏中TLR2 mRNA的动态表达。方法建立华支睾吸虫感染小鼠模型,在感染不同时间点(1、2、4、7、14、28、568、4 d)取肝脏,采用逆转录聚合酶链反应检测肝脏TLR2 mRNA的表达,同时行HE染色观察感染小鼠各时间点肝脏病理学变化。结果与未感染对照组相比,感染组自感染后第7 d起TLR2 mRNA表达升高(P<0.05),至第28 d达到高峰,之后开始下降,至第56 d仍高于未感染对照组(P<0.05),第84 d下降至未感染对照组相近水平。病理学观察发现,感染小鼠肝脏于第4 d始,汇管区轻度炎症反应;第7 d,炎症反应加重,或见嗜酸性脓肿;第14 d,嗜酸性脓肿增多,纤维组织增生,周围小胆管增生;第28 d,胆管壁增厚,周围炎性渗出明显;第56 d,胆管上皮不同程度增生,增生的上皮呈腺样结构;第84 d,胆管周围炎性细胞减少,纤维增多,管壁增厚。结论 TLR2 mRNA在华支睾吸虫感染过程中表达升高,推测TLR2可能参与调控华支睾吸虫感染引发的Th1/Th2免疫应答和病理改变。     

TLR2、TLR4和TLR9在慢性重型肝炎患者及肝衰竭大鼠中的表达

目的:观察重型肝炎患者及肝衰竭大鼠中TLR2、TLR4和TLR9的表达,为重型肝炎肝衰竭发病机制的研究提供思路.方法:免疫组织化学法检测慢性重型肝炎患者肝组织及外周血单个核细胞(PBMC)中TLR2、TLR4和TLR9表达,同时平行设立慢性乙型肝炎、肝硬化患者和正常人作为对照.免疫组织化学法检测急性肝衰竭、慢加急性肝衰竭及正常大鼠肝组织中TLR2、TLR4和TLR9表达.阳性对照为大鼠结肠组织.结果:正常人、慢性乙型肝炎和肝硬化患者肝组织中TLR2和TLR9未见表达;慢性重型肝炎患者肝组织中TLR2和TLR9有表达;正常人及慢性乙型肝炎患者PBMC中TLR2、TLR9未见表达,慢性重型肝炎患者PBMC中TLR2和TLR9有表达;正常大鼠肝组织TLR2、TLR9未见表达,急性肝衰竭和慢加急性肝衰竭大鼠肝组织中TLR2、TLR9有表达;TLR4在所有标本中均未见表达,但在阳性对照中有表达;TLR2、TLR9的表达多见于炎性细胞中,在肝实质细胞上表达很少.结论:TLR2、TLR9的高表达与机体的免疫状态密切相关,可能参与了重型肝炎和肝衰竭的发病过程.     

蒿甲醚与瑞香素伍用对感染伯氏疟原虫小鼠的治疗作用

目的　研究蒿甲醚与瑞香素伍用(A+D)对感染伯氏疟原虫ANKA株小鼠的疗效及其联合作用方式。　方法　按“四天抑制法”d0 感染,d0 ～d3 给药,每天1次,d4涂薄血膜检查,并计算d4减虫率及各药丰数有效置(ED50),用等效应图解法分析A+D的合并作用。　结果　蒿甲醚0.4mg/(kg·d)× 4d的d4减虫率与对照组相比差异无显著性 ;[A0 .4mg/(kg·d) +D7.7mg/(kg·d) ]× 4d的抗疟效果提高 ,与对照组相比差异有显著性(P<0.05)。A+D各组的ED50均低于单用药组;不同配伍比例的R值皆大于0.4,小于2.7(0.4     

伯氏疟原虫氯喹抗性株感染小鼠的氯喹代谢动力学研究

[目的 ]比较氯喹在正常小鼠、感染伯氏疟原虫药物敏感 (N)株和氯喹抗药性 (RC)株小鼠体内的药物动力学差异。 [方法 ]应用反相高效液相色谱法分别测定正常小鼠、感染伯氏疟原虫N株和RC株小鼠血浆中的氯喹浓度 ,采用 3P87药物动力学分析软件对数据进行分析 ,从而获得有关药物动力学参数。 [结果 ]感染RC鼠的t1/2 β与其他两组间有显著的统计学差异 (P <0 .0 5 ) ,而感染N株鼠与正常鼠间无显著差异。 [结论 ]氯喹在感染了伯氏疟原虫RC株鼠体内的消除速度显著快于正常鼠及感染N株鼠。     

巴西日圆线虫诱导小鼠T辅助细胞的变化对伯氏疟原虫感染的影响

[目的 ]观察预感染巴西日圆线虫后 ,小鼠抵御伯氏疟原虫攻击感染的能力 ,并着重探讨T辅助细胞亚型在感染过程中的变化以及这些变化对宿主免疫力和预后的影响。 [方法 ]皮下注射巴西日圆线虫感染C5 7BL/ 6小鼠 ,建立线虫预感染模型 ,于 3wk后腹腔注射伯氏疟原虫ANKA株攻击感染小鼠。观察每天原虫血症变化情况 ,并于疟原虫感染后 0、3和 9d取脾 ,提取RNA ,用RT PCR扩增法定性观察细胞因子IFN γ和IL 4的变化。[结果 ]与对照组相比 ,实验组感染疟原虫后 ,原虫血症的峰值出现时间明显延长 ,小鼠对疟原虫感染的耐受程度以及小鼠生存时间显著提高。实验组Th2型细胞合成IL 4的量在疟原虫感染 0d时明显高于对照组 ;而在 3与 9d时两组均异常升高。Th1型细胞合成IFN γ的量在疟原虫感染后 3d时实验组高于对照组 ,但在 9d时实验组IFN γ有所下降。 [结论 ]预感染巴西日圆线虫的小鼠具有较高的抗感染能力。但在攻击感染疟原虫后Th2型细胞被提前激活而抑制了Th1型细胞的正常功能 ,最终仍导致小鼠死亡。     

伯氏疟原虫氯喹抗性株感染ICR小鼠脾脏树突状细胞成熟和B细胞活化

目的 研究伯氏疟原虫氯喹抗性株(RC株)和氯喹敏感株(N株)感染鼠脾脏B细胞活化与树突状细胞(DC)的关系。 方法 分别用感染N株或RC株疟原虫的红细胞(iRBC)腹腔接种感染ICR小鼠(1×10⁶个iRBC/只)。当小鼠原虫血症N株达50%～80%、 RC株达61.7%～68.4%时, 断颈处死取脾脏。 常规方法制作石蜡切片, HE染色或免疫组织化学染色, 进行组织学观察。 制作超薄切片, 透射电镜观察脾脏细胞的变化。制作冰冻切片进行免疫荧光观察。流式细胞仪分析比较B细胞和DC变化。 结果 RC株感染小鼠脾脏白髓增生明显, 抗B细胞的特异性表面分子CD45R/B220和CD19抗体同时表达阳性的B细胞在脾细胞中的百分比增加, 中、 小淋巴细胞数量增多, 在红髓内浆母细胞与成熟的浆细胞数量增多。而N株感染小鼠脾小体则以大、 小淋巴细胞为主, 生发中心不明显, 红髓可见大量的含疟原虫的红细胞、 小淋巴细胞, 而浆母细胞和其他发育期浆细胞则少见。 RC株感染小鼠脾脏内白细胞分化抗原11c(CD11c) 阳性的DC数量明显增多, 尤其动脉周围淋巴鞘T细胞区。并且这些DC表面主要组织相容性复合体Ⅱ (MHCⅡ) 类分子表达明显升高, 表明主要是成熟的DC增多。DC外形不规则, 胞质丰富, 电子密度高, 含发达的高尔基复合体和吞噬泡样结构。 结论 RC株感染小鼠脾脏成熟的DC 明显增加, 从而诱导B细胞的活化增殖。     

TLR2在溃疡性结肠炎患者结肠黏膜中的表达

目的 检测溃疡性结肠炎(UC)患者结肠肠黏膜TLR2蛋白及TLR2 mRNA的表达及其与UC临床活动度、内镜分级的关系.方法 取47例UC患者结肠黏膜标本根据内镜及临床活动度进行分级,同时收集正常对照者结肠黏膜标本13例.用Western Blot技术和半定量反转录聚合酶链反应方法(RT-PCR)检测结肠黏膜中TLR2蛋白及TLR2 mRNA的表达.结果 UC患者结肠黏膜TLR2蛋白及TLR2mRNA的表达量高于正常对照者,且随着内镜下及临床病变分级的加重逐渐增加.结论 UC患者肠黏膜中TLR2及TLR2 mRNA的表达增加,且其表达均随临床及内镜下病变程度加重,表达逐渐增加,在一定程度上可以反映疾病的活动度.     

Cognitive dysfunction in mice infected with Plasmodium berghei strain ANKA

Cerebral malaria complicated by cognitive sequelae is a major cause of morbidity in humans infected with Plasmodium falciparum. To model cognitive function after malaria, we created a rodent model of cerebral malaria by infecting C57BL/6 mice with Plasmodium berghei strain ANKA. After 7 days, an object-recognition test of working memory revealed a significant impairment in the visual memory of infected mice. This impairment was observed in the absence of confounding effects of infection. The cognitive dysfunction correlated with hemorrhage and inflammation. Furthermore, microglial activity and morphological changes detected throughout the brains of infected mice were absent from the brains of control mice, and this correlated with the measured cognitive defects. Similar testing methods in human studies could help identify subjects at risk for an adverse cognitive outcome. This murine model should facilitate the study of adjunctive methods to ameliorate adverse neurological outcomes in cerebral malaria.     

Serum-soluble malarial antigens and immune complex nephritis in Plasmodium berghei berghei infected mice.

Swiss albino mice infected with Plasmodium berghei berghi showed the serum-soluble malarial antigen and antibody on day 10 of infection onward. Immune complex nephritis in these mice developed on the seventh day after inoculation. The infected kidneys revealed the deposition of mouse gamma globulin, mouse beta1C globulin and malaria antigen along the capillary wall of the glomeruli. Proteinuria was detected on seventh day of infection. Serum-soluble malaria antigen in probably responsible for forming the soluble immune complex which causes glomerulonephritis in infected mice.     

Time course of serum nuclease activity in mice infected with Plasmodium berghei

The present paper shows that murine serum nuclease activity increases following P.berghei infection. DNA activity begins increasing just 48 hours after infection. Following 78 hours, it achieves the maximum, by exceeding the baseline level by 6 times. Then DNA activity starts decreasing and following 94 hours after infection it is just thrice higher than the baseline. Serum RNA activity shows only 30% increases 72 hours after infection and returns to the baseline following 94 hours. Microscopic monitoring indicates that only single malaria causative agents appear in the red blood cells in this period. The peak increases in nuclease activities after P.berghei infection are assumed to be associated with the induction of serum infection.     

Topographical distribution of the cerebral lesions in mice infected with Plasmodium berghei

In the mouse P. berghei malaria model systematic studies were carried out on the relationship between the type and the topographical distribution of the brain lesion in cerebral malaria. As previously stated for pernicious P. falciparum malaria in man, petechial haemorrhage was not the sole morphologic lesion. In addition to severe brain oedema, microthrombosis, sludging of mononuclear cells, arteriolar spasms, scattered disturbances of the microcirculation, and the occasional proliferation of gliocytes were the prevailing morphologic changes. Pronounced perivascular oedema with compression of capillaries and ischaemic demyelinisation were particular frequent in the nucleus caudatus putamen, while the adjacent regions (radiatio corporis callosi, claustrum, hippocampus, and fimbria hippocampi) were the sites of predilection of petechial haemorrhage. Arteriolar spasms were particularly frequent in branches of the posterior choroidal artery. The proliferation of gliocytes was practically restricted to the tubercula olfactoria and to the subependymal zone of the lateral wall of the lateral ventricle. The present results indicate a neurovascular component in the pathogenesis of cerebral malaria. The preponderance of a special histopathological lesion in a certain cerebral region may be the result of a particular sensitivity of cells of these areas to noxious events (pathoclisis), for instance hypoxia, and/or exaggeration of autoregulatory phenomena that exist between the cerebral parenchyma and the supplying vasculature.     

Catalase activity in red cell and liver of mice infected with Plasmodium berghei

Hydrogen peroxide (H2O2) has been incriminated to have an oxidative killing malaria parasite. As P. berghei-infected mouse red cells generated H2O2 in vivo, this would result in the alteration of catalase status of the host. The present study was undertaken to determine catalase activity in red cells and liver of mice infected with P. berghei. The studies were performed in 17 samples of infected red cells as well as 20 samples of the normal red cells. Results showed that the catalase activity in red cells of the infected group was significantly lower (p less than 0.01) than that of the normal group. There was a reverse relationship between catalase activity and parasitemia. Crude parasite lysates possessed no catalase activity. Liver catalase content in the infected group was also found to be significantly lower (p less than 0.05) than that of the control group. All these findings indicated that P. berghei-infected mice caused a depressed catalase activity in red cells and liver which was possibly due to the catalatic function in detoxifying the increased H2O2 to water and free oxygen.