淋病奈瑟球菌表面蛋白A基因疫苗的构建及其诱导小鼠的免疫应答

目的构建淋病奈瑟球菌表面蛋白A（NspA）基因疫苗，并接种小鼠，评价其诱导的体液和细胞免疫应答。方法将NspA基因插入到真核表达载体pcDNA3．1（＋）中，构建重组真核表达质粒pcDNA3．1（＋）／NspA，并经PCR、双酶切及测序鉴定。反转录一聚合酶链反应（RT-PCR）和免疫组织化学法分别检测NspA基因转染细胞后mRNA和蛋白的表达。以pcDNA3．1（＋）／NspA重组质粒免疫45只雄性BALB／c小鼠，试管凝集法检测免疫小鼠抗体滴度，ELISA检测IFN-γ水平，四甲基偶氮唑蓝（MTT）比色法检测脾淋巴细胞增殖。提取接种部位股四头肌总DNA，PCR检测BALB／c小鼠肌细胞内NspA基因。结果成功构建pcDNA3．1（＋）／NspA基因疫苗，能在真核细胞中转录和表达。pcDNA3．1（＋）／NspA免疫组的抗体滴度达1：640，pcDNA3．1（＋）和PBS免疫组均无特异性抗体检出。空载体pcDNA3．1（＋）组IFN-γ为（23．79±11．85）pg／mL，pcDNA3．1（＋）／NspA免疫组为（169．71±30．52）pg／mL（P〈0．01）；脾淋巴细胞增殖的刺激指数（SI）分别为1．05±0．30和1．94±0．74（P〈0．01）。并证实NspA基因可在小鼠肌细胞中稳定存在。结论构建淋病奈瑟球菌NspA基因疫苗，将其免疫BALB／c小鼠可诱导特异性体液和细胞免疫应答，为进一步用于淋病预防奠定了基础。     

The protective effect of a DNA vaccine encoding the Toxoplasma gondii cyclophilin gene in BALB/c mice

Toxoplasmosis is a world‐wide zoonosis that causes significant public health and veterinary problems. The study of vaccines remains the most promising method for the future prevention and control of toxoplasmosis. Recombinant Toxoplasma gondii cyclophilin has been shown to have potent PPIase and IL‐12‐inducing activities, thus promoting the stabilization of T. gondii's life cycle and maintaining the survival of its host during evolution. In this study, the T. gondii cyclophilin gene was used to construct a DNA vaccine (pVAX1‐TgCyP). The immune response and protective efficacy of the vaccine against T. gondii infection in BALB/c mice were evaluated. All BALB/c mice that were vaccinated with pVAX1‐TgCyP developed a high response with TgCyP‐specific antibodies, and significant splenocyte proliferation (P < 0·05) compared with pVAX1 vector and PBS groups. pVAX1‐TgCyP also induced a significant Th1 type immune response, indicated by the higher production of IL‐2 and IFN‐γ (P < 0·05). The survival rate of BALB/c mice increased significantly after vaccination with pVAX1‐TgCyP (37·5%) (P < 0·05). These results indicate that TgCyP is a highly efficacious vaccine candidate that can generate protective immunity against T. gondii infection in BALB/c mice.     

苦参合剂对隐孢子虫感染小鼠空肠黏膜肥大细胞的影响

目的 阐明空肠黏膜肥大细胞激活在隐孢子虫致病机制中的作用, 探讨苦参合剂抗隐孢子虫感染的作用机理。方法 30只雄性BALB/c小鼠随机分为正常对照组、 隐孢子虫感染模型对照组和苦参合剂实验组。建立隐孢子虫肠道感染小鼠模型, 经苦参合剂治疗3周后, 光镜下观察小鼠空肠病理变化; 甲苯胺蓝染色, 计数肥大细胞数量并观察其脱颗粒变化。结果 隐孢子虫感染模型对照组小鼠小肠上皮出现明显炎性病理改变。苦参合剂实验组小鼠治疗3周后小肠上皮较完整, 接近正常。甲苯胺蓝染色显示隐孢子虫感染模型对照组小鼠肥大细胞数量 （12.80±0.84） 较正常对照组 （1.60±0.89）明显增多 （P < 0.01）, 且脱颗粒现象明显; 苦参合剂实验组小鼠肥大细胞数量 （2.00±0.71） 较隐孢子虫感染模型组明显减少（P < 0.01）, 肥大细胞数量和形态接近正常对照组。结论 肥大细胞活化参与了隐孢子虫感染引起的肠黏膜炎症反应。苦参合剂可减少隐孢子虫感染小鼠空肠黏膜肥大细胞数量及抑制肥大细胞活化脱颗粒, 从而减轻炎症、 修复受损肠黏膜, 发挥治疗隐孢子虫病的作用。     

转移性人小细胞肺癌裸鼠模型的建立

目的建立转移性人小细胞肺癌(SCLC)的裸鼠模型,探讨其用于人SCLC的治疗研究的可行性。方法 RPMI 1640培养基培养的人SCLC细胞株NCI-H446用无血清培养液重悬成1×107个/mL,取0.1 mL细胞悬液接种于BALB/c-nu裸鼠胫骨结节骨髓腔内,构建裸鼠模型并观察腿部肿瘤生长情况及肺等器官的转移情况。免疫组化染色检测小细胞肺癌标志物CD56及神经上皮干细胞标志物netsin的表达情况。结果经胫骨结节骨髓腔注射的方法注射细胞后裸鼠腿部均形成原位瘤,肺部转移6/6只,肝、脾等脏器未见转移灶。肿瘤组织细胞CD56阳性率>75%,nestin阳性率为4%～12%。结论经胫骨结节骨髓腔注射的方法可以形成弥散性肺部肿瘤,其他各器官则未见肿瘤块,可以为人SCLC实验性治疗的研究提供一种高效的实验模型。     

低硒小鼠模型的建立及低硒对心肌的影响

目的建立低硒实验动物模型,观察低硒对心肌的影响。方法利用黑龙江地产酵母配制低硒小鼠饲料,使用配制的小鼠饲料喂养BALB/c幼鼠,经过4个月的喂养,测定血清、肝脏、心肌细胞的硒含量,观察心肌超微结构的变化,测定血清心肌酶的变化。结果利用黑龙江地产酵母配制的低硒饲料,硒含量为0.016 mg/kg,符合低硒标准。BALB/c鼠用该饲料喂养4个月,心肌、肝脏、血清硒含量分别为0.187 mg/kg、0.219 mg/kg、0.241mg/kg,符合低硒诊断标准。观察低硒鼠心肌超微结构,可见心肌细胞线粒体肿胀,细胞核出现了异型性,血清心肌酶较常硒鼠升高。结论利用黑龙江地产酵母成功配制了低硒饲料。经过低硒饲料饲养可建立低硒鼠模型。低硒可以引起BALB/c鼠心肌细胞损伤。     

肉苁蓉苯乙醇苷对环磷酰胺致小鼠生精障碍的治疗作用及其机制

目的： 探讨肉苁蓉苯乙醇苷对环磷酰胺致生精障碍小鼠的治疗作用,并初步阐明作用机制。方法：乙醇浸提法提取肉苁蓉苯乙醇苷。40只BALB/C小鼠随机分为对照组、
模型组、肉苁蓉苯乙醇苷低剂量组（50 mg/kg^-1）和高剂量组（100 mg/kg^-1）,每组10只。除对照组外,其余各组小鼠每天注射环磷酰胺（80 mg?kg-1）连续5 d,制备生精障碍小鼠模型。末次给药后,肉苁蓉苯乙醇苷低剂量组和高剂量组小鼠分别给予相应剂量灌胃,对照组和模型组小鼠给予等体积生理盐水灌胃,每天1次,连续30 d。末次灌胃后24 h,取小鼠睾丸组织。酶联免疫法测定小鼠睾丸组织匀浆中睾酮水平,比较各组小鼠精子密度、精子活率及精子畸形率,HE染色观察睾丸组织形态学变化。结果：与对照组比较,模型组小鼠精子密度和精子活率降低（P<0.01）,精子畸形率升高（P<0.01）,睾丸组织匀浆中睾酮水平降低（P<0.01）;与
模型组比较,肉苁蓉苯乙醇苷各剂量组小鼠精子密度和精子活率明显升高（P<0.01）,精子畸形率降低（P<0.01）,睾丸组织匀浆中睾酮水平升高（P<0.05或P<0.01）。组织学观察,模型组小鼠睾丸生精小管萎缩、退化,生精上皮明显变薄、生精细胞排列紊乱,腔内精子数目减少;肉苁蓉苯乙醇苷组小鼠睾丸生精上皮层数明显增多,层次分明,生精细胞排列整齐、紧密、规则,管腔内可见精子。结论：肉苁蓉苯乙醇苷对环磷酰胺所致小鼠生精障碍具有明显的治疗作用,其机制可能与改善睾丸组织中睾酮水平有关。     

猪胸腺免疫抑制提取物超滤组分2对细胞免疫的影响

目的　探讨猪胸腺免疫抑制提取物超滤组分 2 (简称U2 组分 )对细胞免疫的影响。方法　用3H TdR掺入DNA法检测了不同剂量的U2 组分注射BALB c小鼠后的淋巴细胞增殖情况和U2 组分对PHA诱导的正常人外周血淋巴细胞增殖的影响。结果　注射 0 .2mgU2 组分的试验组小鼠淋巴细胞增殖能力比对照组降低 (P<0 .0 5 ) ;注射 1mg和 3mg两个试验组小鼠淋巴细胞增殖能力均比对照组显著降低 (P <0 .0 1) ;5 0 μg和 10 0 μg的U2 组分对PHA诱导的正常人外周血淋巴细胞增殖也均具有显著抑制作用 (P <0 .0 1)。结论　用一定量的U2 组分对体内和体外淋巴细胞增殖均具有显著抑制作用     

Distribution of vesicular stomatitis virus proteins in the brain of BALB/c mice following intranasal inoculation: an immunohistochemical analysis

Earlier studies have shown that intranasal instillation of vesicular stomatitis virus (VSV), a negative-sense RNA virus, in mice and rats can result in infection of the brain, hind-limb paralysis and death. Using an antiserum directed against VSV proteins, we sought to determine the potential neuronal and non-neuronal pathways VSV utilize, for central nervous system dissemination in BALB/c mice. Within 12 h following intranasal inoculation of VSV, VSV antigen could be detected in the olfactory nerve layer of the ipsilateral olfactory bulb. Within 3–4 days post-inoculation (p.i.), VSV had disseminated into the glomeruli of the olfactory bulb as well as the anterior olfactory nuclei that were ipsilateral to the VSV instillation. Within the glomeruli, VSV antigen was more prevalent in the granule cells than in the mitral cells. Correspondingly, the lateral olfactory tract, where axons of mitral cells course, remained VSV negative throughout 7 days p.i. By 7 days p.i., viral proteins were detected in several additional regions extending to the brainstem. These included regions involved in -rhythm generation during exploration and REM sleep, i.e. the septal nuclei, the suprammamilary body, and the hippocampal formation, as well as the amygdaloid complex and brainstem neuromodulatory centers, such as the dorsal raphe´and locus coerullus. Structures abutting the ventricular surfaces, such as the dorsal cochlear nucleus, were also labeled. Tracts immunoreactive to VSV included the dorsal tegmental tract, fascia retroflexus, Probst tract, and mesencephalic tract of the trigeminal motor nerve. Besides the lateral olfactory tract, tracts that remained VSV negative included the anterior commissure, the corpus callosum and the mammilary peduncle. The pattern of VSV immunoreactivity supports the idea that following infection of the olfactory bulb glomeruli, VSV spreads via both ventricular surfaces and retrograde transport within axons of neuromodulatory transmitter systems innervating the olfactory bulb. Conversely, regions exhibiting low levels of VSV antigen are not likely to be involved in VSV dissemination. In particular, the paucity of VSV antigen in some of the terminal fields of neuromodulatory systems indicate that anterograde transport is more selective than retrograde transport. Surprisingly, the principal neurons of the olfactory glomeruli, thalamus, cerebral cortex and the hippocampus, all of which usel-glutamate as the excitatory neurotransmitter, are much less involved in viral dissemination.     

BALB/c mice resistant to Toxoplasma gondii infection proved to be highly susceptible when previously infected with Myocoptes musculinus fur mites

The immune response induced by Toxoplasma gondii is characterized by Th1 immune mechanisms. We previously demonstrated that C57BL/6 mice infested with Myocoptes musculinus and infected with T. gondii by intraperitoneal route undergo accelerated mortality according to Th2 immune mechanisms induced by the acarian. To evaluate whether infection with M. musculinus influences T. gondii-induced Th1 response in a resistant mouse lineage, BALB/c, which develops latent chronic toxoplasmosis in a way similar to that observed in immunocompetent humans, this study was done. The animals were infected with T. gondii ME-49 strain 1 month after M. musculinus infestation, being the survival and the immune response monitored. The double-infected displayed higher mortality rate if compared with the mono-infected mice. In addition, infection with M. musculinus changed the T. gondii-specific immune response, converting BALB/c host to a susceptible phenotype. Spleen cells had increased the levels of IL-4 in double-infected mice. This alteration was associated with severe pneumonia, encephalitis and wasting condition. In addition, a higher tissue parasitism was observed in double-infected animals. It can be concluded that infection with these two contrasting parasites, M. musculinus and T. gondii, may convert an immunocompetent host into a susceptible one, and such a host will develop severe toxoplasmosis.     

Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model

Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.