伊氏锥虫和布氏锥虫动基体DNA酶切电泳比较

限制性内切酶ＭｂｏＩ，ＤｄｅＩ，Ｈｉｎｆｉ和ＴａｑＩ对布氏锥虫ＫＤＮＡ进行酶切后，电泳中均显示出多条ＤＮＡ区带，其总Ｋｂ数约等于２０ｋｂ，而各限制酶对伊氏锥虫ＫＮＤＡ消化后均显示出１至２条区带，总和约１ｋｂ明显区别于布氏锥虫。     

伊氏锥虫、马媾疫锥虫、布氏锥虫、刚果锥虫的18S rDNA部分序列测定与系统发育关系

对伊氏锥虫（Trypanosoma evansi）：新疆株（XJCA）、湖北株（HBM）、云南株（YNB）、广东株（GDB2）；马媾疫锥虫（Trypanosoma equiperdum）、布氏锥虫（Trypanosoma brucei）、刚果锥虫（Trypanosoma congolense）提取基因组DNA，根据已报道的伊氏锥虫株18SrDNA基因序列设计合成引物，用PCR扩增了锥虫虫株基因组DNA,伊氏锥虫新疆株、湖北株、云南株、广东株、布氏锥虫、刚果锥虫均为373bp的片段；马媾疫锥虫为372bp的片段，PCR产物经电泳鉴定后用试剂盒回收纯化，纯化后PCR产物经连接、转化后测序，将测得的序列用DNAMAN软件分析并与国外已发表的相应序列进行了同源性比较，并绘制了系统发育进化树。结果与国外AJ009153、AJ223564、D89527株同源性达到99％～100％，与另外11株同源性75％。本研究为锥虫分子流行病学研究及分类研究打下基础。     

布氏锥虫马媾疫亚种体外培养的研究

用小白鼠腹腔巨噬细胞滋养层系统和无细胞培养系统成功地培养了布氏锥虫马媾疫亚种。开始建立无细胞培养系统培养时,从第6天起每日补种少量滋养层细胞系统培养的虫体,于3周内逐步建立了适应于无细胞培养系统的锥虫群体。长期连续培养两种系统培养物的虫密度均可稳定保持在7—10.O×10~5/ml左右,虫体有规律地呈岛屿状密集分布。培养液内2—ME和马血清的适宜浓度分别为0.3mM和20%,添加0.1mM次黄嘌呤可使培养物虫密度增加1倍左右,用烛罐可取代CO_2培养箱。体外连续培养120天。锥虫仍保持细长形态,具表面被膜,对小白鼠有感染性。     

布氏锥虫的一种核苷酸运输系统与药物抗性有关

病原体的药物抗性是日益严重的问题，其机制尚不完全了解。一般认为细胞摄入抗布氏锥虫的药大多是通过一种至今尚未被识别的不平常的腺苷运输系统发生的，使用酿酒酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）以功能筛选的方法克隆了布氏锥虫指名亚种（Ｔｒｙｐａｎｏｓｏｍａ ｂｒｕｃｅｉ ｂｒｕｃｅｉ）的一个编码核苷酸运输系统的基因－ＴｂＡＴ１。ＴｂＡＴ１在酵母中表达时，使酵母能摄取腺苷，并赋予对密胺基苯胂类药物（Ｍｅｌａｍｉｎｏｐｈｅｎｙｌ ａｒｓｅｎｉｃａｌｓ）的敏感性，抗药锥虫藏匿缺损ＴｂＡＴ１的变种，杀锥虫药进入细胞途径的分子识别开辟了诊断和治疗抗药锥虫病的途径。     

Analysis of neutral glycosphingolipids from Trypanosoma brucei

Neutral glycosphingolipids (GSLs) were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC), TLC/secondary ion mass spectrometry (TLC/SIMS), and liposome immune lysis assay (LILA). Three species of neutral GSLs, designated as N-1, -2, and -3 were separated on TLC. N-1 GSL migrated very close to glucosylceramide (GlcCer) and N-2 GSL showed the same mobility as lactosylceramide (LacCer). On the other hand, the mobility of N-3 GSL on the TLC plate was slower than globotetraosylceramide (Gb4). In order to characterize the molecular species of neutral GSLs from T. brucei, N-1, -2 and -3 GSLs were analyzed by TLC/SIMS. The TLC/SIMS analysis of N-1 of the parasites revealed a series of (M–H)⁻ ions from m/z 698 to 825 representing the molecular mass range of ceramide monohexoside (CMH) (GlcCer or galactosylceramide). On the other hand, the TLC/SIMS spectra of N-2 GSL revealed a series of (M–H)⁻ ions from m/z 944–987 indicating the molecular mass range of LacCer. In the TLC/SIMS analysis of N-3 GSL, however, the characteristic molecular ions that can elucidate the structure of N-3 GSL were not obtained. In order to confirm the results obtained from TLC/SIMS, N-1, -2, and -3, GSLs were tested by LILA with specific antibodies against GlcCer, LacCer, and Gb4, respectively. N-1 GSL had reactivity to anti-GlcCer antibody and N-2 GSL reacted with the antibody against LacCer. However, N-3 GSL was not recognized by anti-Gb4 antibody. Using anti-GlcCer and anti-LacCer antibodies, furthermore, we studied the expression of GlcCer and LacCer in T. brucei parasites. Both GlcCer and LacCer were detected on the cell surface of T. brucei.     

布氏锥虫C型凝集素类似蛋白的鉴定

C型凝集素在机体免疫系统中具有重要的作用.为了解布氏锥虫(T.brucei)基因组编码的C型凝集素类似蛋白(CLLPs),本研究在GeneDB数据库中通过软件鉴定了13种T.brucei基因组编码的CLLPs家族基因序列,并分析它们之间的遗传进化关系.同时,采用RT-PCR方法检测了其中3种CLLPs基因在血液阶段(BSF)T.brucei虫体内的转录.利用PCR扩增并在大肠杆菌中对所选3种基因进行表达,以表达的重组蛋白免疫小鼠制备多克隆抗体.实验结果表明,本研究选择的3种CLLPs基因在BSF T.brucei虫体内均能够进行转录;而通过间接免疫荧光检测表明所选的3种CLLPs基因中,只有一种CLLP (Tb5290)在T.brucei的BSF阶段和昆虫阶段(PCF)表达.本研究首次在T.brucei中鉴定了C型凝集素家族的新成员,为进一步了解CLLPs在T.brucei中的生物学功能的研究奠定了基础.     

体外培养对布氏锥虫伊氏亚种药敏性的影响

用小鼠治疗试验和体外药敏试验观察了4个布氏锥虫伊氏亚种虫株在长期体外培养条件下药敏性的稳定性.各虫株的原始群体、连续培养30d和90d的群体对贝尼尔、苏拉灭、安锥赛和硫胂聚氰胺的敏感性基本相同,说明连续培养90d各虫株对上述4种抗锥虫药的敏感性无明显改变.     

Effect of anti-ganglioside antibody in experimental Trypanosoma brucei infection in mice

The effect of antibody against ganglioside antigen on Trypanosoma brucei parasites was examined in vitro and in vivo using anti-ganglioside GM1 (AGM-1) monoclonal antibody. The antibody showed complement-dependent cytotoxicity against T. brucei with mouse complement. Furthermore, mice given AGM-1 were challenged intraperitoneally with T. brucei. Although all non-treated control mice died within six days after infection, all of AGM-1-injected mice had survived by six days post-infection. These data suggest that antibody against ganglioside antigen on T. brucei has potential in protection against T. brucei infection.     

沟叶结缕草蛋白质二硫键异构酶(ZmPDIs)基因家族耐盐功能分析

蛋白质二硫键异构酶（protein disulfide isomerase,PDI）及其类蛋白,是硫氧还蛋白超家族的重要成员,具有分子伴侣活性及钙离子结合位点,负责催化蛋白质二硫键的氧化、还原和异构。本研究从沟叶结缕草基因组中鉴定获得了5个蛋白质二硫键异构酶（ZmPDIs）基因,对其进行了系统进化与基因结构的生物信息学分析,利用同源基因缺失酵母突变体对其耐盐功能进行了初步分析。荧光定量RT-PCR结果表明5个ZmPDIs基因的表达均受到盐胁迫的诱导。这些结果为进一步研究ZmPDIs的耐盐生理功能及分子机制提供参重要参考。     

Flavonoid mixture ameliorates increase in erythrocyte osmotic fragility and malondialdehyde concentration induced by Trypanosoma brucei brucei-infection in Wistar rats

The experiment was performed with the aim of investigating the effect of a flavonoid mixture, Daflon® 500 mg (DF) on the erythrocyte fragility and lipoperoxidative changes, induced by Trypanosoma brucei brucei infection in Wistar rats. Fifty adult male rats randomly divided into five groups of 10 animals each were used. Rats in the control group were administered (1 mL/kg) distilled water only, while the other groups were infected with T. brucei brucei and treated with Daflon® 500 mg and/or Diminazene aceturate. At the end of 5 weeks, EDTA-blood samples and serum samples were collected from the rats, and were used to determine erythrocyte osmotic fragility (EOF) and serum malondialdehyde (MDA) concentration respectively. The results showed that EOF and MDA concentration significantly (P < 0.05) increased in the infected untreated group when compared to the treatment groups. Treatment with Daflon® 500 mg and Diminazene aceturate significantly (P < 0.05) reduced trypanosome-induced increases in EOF and lipoperoxidative changes, suggesting possible antioxidant properties of Daflon® 500 mg and its therapeutic value in trypanosomosis.     

The Effect of Cordyceps Militaris Polysaccharides on the Antioxidant Function in Mice Injected With Cyclophosphamide

To investigate the antioxidant effects of the Cordyceps militaris polysaccharides(CMP),90 male BALB/c mice were randomly divided into six groups, three experimental groups (CMP groups), model control group (CY group), blank control group (BC group) and positive control group (PC group). The mice in CMP and CY groups were given cyclophosphamide at 80 mg/kg via intraperitoneal injection for 3 d. Then the CMP groups were administrated 17.5, 35.0 and 70.0 mg/(kg·BW) CMP via gavage, respectively, BC and CY groups were given physiological saline, and PC group was orally administered 70 mg/(kg·BW) CMP, lasting 18 d. At 24 h after the last administration, heart, liver and kidney were collected to measure the levels of MAD, CAT, SOD, GSH-Px and T-AOC in the homogenate.SOD and TAOC activity of three CMP groups increased significantly (P<0.01), and the level of MDA decreased significantly (P<0.01) compared with CY group. As for CAT activity of heart, the value in low-dose CMP group had no significant change (P>0.05), while in middle and high-dose groups had significant (P<0.05) and extremely significant(P<0.01) increase respectively. Except that the hepatic GSH-Px activity increased significantly (P<0.05) in low-dose CMP group, the rest values of GSH-Px activities in all CMP groups increased extremely significantly (P<0.01). The results indicated that CMP could effectively improve the antioxidant function of mice and had the potential to be a good resource of new antioxidant drugs.     

蛹虫草多糖对注射环磷酰胺小鼠抗氧化功能的影响

试验旨在阐明蛹虫草多糖（CMP）对小鼠抗氧化活性的影响。选取90只雄性BALB/c小鼠,体重18~20 g,随机分为6组,3个CMP组（低、中、高CMP组）,模型对照组（CY组）、阳性对照组（PC组）及空白对照组（BC组）各1个。CMP组和CY组分别腹腔注射80 mg/kg环磷酰胺,连续3 d,然后3个CMP组分别灌胃给予17.5、35.0、70.0 mg/kg体重的CMP,BC组和CY组给予生理盐水,PC组灌胃给予70 mg/kg体重的CMP,持续18 d。于最后一次给药24 h后,采集小鼠内脏,测定心脏、肝脏及肾脏匀浆液中T-AOC、MAD、CAT、SOD和GSH-Px水平。结果显示,与CY组相比,3个CMP处理组小鼠内脏匀浆液中的SOD和T-AOC活性均极显著增加（P<0.01）,MDA水平极显著降低（P<0.01）;低剂量CMP组心脏CAT无显著变化（P>0.05）,中、高剂量CMP组心脏CAT水平分别显著（P<0.05）、极显著（P<0.01）提高;除低剂量CMP组肝脏GSH-Px活性显著增加（P<0.05）外,其余CMP组内脏GSH-Px活性均极显著增加（P<0.01）。本试验中CMP可以有效提高小鼠心脏、肝脏及肾脏的抗氧化活性,这为抗氧化新药的研发提供了借鉴。     

伊氏锥虫安锥赛抗药性相关基因-TbTA1的分析

为探讨伊氏锥虫对安锥赛抗药性的分子机理，以布氏锥虫对砷剂药物抗药性相关的TbTA1基因设计引物，以55℃、57℃、60℃、63℃和65℃不同退火温度，分别从伊氏锥虫敏感株的cDNA和基因组DNA中扩增出TbTA1基因的全长序列，但是从安锥赛抗药株伊氏锥虫的cDNA和基因组DNA中都未扩增出TbTA1基因，这表明基因TbTA1可能与伊氏锥虫安锥赛抗药性的产生有关。伊氏锥虫与布氏锥虫的，TbTA1基因序列比较，它们有10个碱基不同，即第88位的G（T.e）-A（T.b）、第144位的T（T．e）-C（T．b）、第224位的C（T．e）-T（T．b）、第471位的C（T.e）-T（T.b）、第472位的A（T．e）-G（T.b）、第549位的G（T．e）-T（T．b）、第1008位的C（T．e）-T（T.b）、第1033位的A（T．e）-G（T．b）、第1293位的A（T．e）-G（T．b）和第1384位的T（T．e）-C（T．b），其中有5个碱基所编码的氨基酸不同，即Val^30（T.e）Ile（T．b）、Ala^75（T．e）-Val（T．b）、Ile^158（T．e）-Val（T．b）、Thr^345（T．e）-Ala（T．b）和Ser^462（T.e）-Pro（T．b），这表明伊氏锥虫与布氏锥虫的TbTA1差异可能是它们的种间差异。