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余德立, 鲍朗. 利用基因芯片对赖型钩体毒力基因差异表达与致病相关性的探讨[J]. koko体育app 学报(医学版), 2015, 46(1): 11-15.
引用本文: 余德立, 鲍朗. 进行什么是遗传基因存储芯片对赖型钩体毒力什么是遗传基因差异性抒发与病发想关性的论述[J]. 成都社会学报(临床医学版), 2015, 46(1): 11-15.
YU De-li, BAO Lang. Research on the Relevance Between the Virulent Genes Differential Expression and Pathogenecity ofLeptospira with Microarray[J]. Journal of Sichuan University (Medical Sciences), 2015, 46(1): 11-15.
Citation: YU De-li, BAO Lang. Research on the Relevance Between the Virulent Genes Differential Expression and Pathogenecity ofLeptospira with Microarray[J]. Journal of Sichuan University (Medical Sciences), 2015, 46(1): 11-15.

利用基因芯片对赖型钩体毒力基因差异表达与致病相关性的探讨

Research on the Relevance Between the Virulent Genes Differential Expression and Pathogenecity ofLeptospira with Microarray

  • 摘要: 目的 通过对连续动物传代和试管培养的赖型钩端螺旋体部分致病基因表达差异性的检测,分析赖型钩端螺旋体特异性基因表达与毒力改变之间的联系,了解赖型钩端螺旋体致病过程和发病机制。方法 将27只豚鼠分为3组(n=9),分别经腹部皮下接种1 mL体内培养钩体(体内培养组)、体外培养钩体(体外培养组)或钩体培养基(培养基组),接种钩体浓度为108/mL并处于对数生长期。在接种后15 d内观察其发病和死亡状况,测量其体温体质量变化情况,并于接种后第15 d将存活豚鼠处死,测量脏器系数,了解经不同环境培养的钩体的毒力情况;以赖型钩端螺旋体标准株DNA为模板,采用PCR技术扩增出所选特定基因作为探针,使用点样仪进行芯片点样。分别提取经豚鼠体内连续数代培养的钩端螺旋体RNA与EMJH培养基中培养的钩端螺旋体RNA,逆转录后分别使用Cy5和Cy3进行荧光标记,并与基因芯片进行杂交。扫描后通过分析两种荧光强度的比值,了解赖型钩端螺旋体相关毒力基因的差异表达情况。结果 接种后,体内培养组豚鼠存活率为0%,体外培养组豚鼠存活率为88.9%,培养基组豚鼠全部存活;体内培养组豚鼠较其余两组豚鼠体温升高(P<0.05)、体质量下降(P<0.05);体内培养组豚鼠心、肺、肾的脏器系数较体外培养组及培养基组豚鼠更大(P<0.05);解剖后,体内培养组豚鼠肺部出血情况较其余两组更为严重;通过基因芯片,检测到溶血素基因以及其他与钩体生存致病能力相关的基因呈现出不同程度的改变,溶血素基因LA1027、LA1029、LA4004、LA3050、LA3540、LA0327、LA0378、LA1650、LA3937、O抗原连接酶基因LA2089、鞭毛动力蛋白基因LA3576、外膜蛋白基因LA0011及Loa22基因上调。结论 赖型钩体经连续体内培养后毒力增高,钩体毒力变化与其基因表达差异存在联系,了解这些基因的变化意义对阐明钩体的致病机制有重要意义。  
    Abstract: Objective To find the change of virulent gene expression and to analyze the relevance between the virulent change and the gene expression. Methods Grouped guinea pigs were inoculated with 1 mL Leptospira cultured in vivo, Leptospira cultured in vitro and the Leptospira culture medium through abdominal subcutaneous respectively. The survival rate, body mass and temperature change of guinea pigs in different groups were measured within 15 d after the inoculation, then the survived guinea pigs were scarified, and the organ coefficient was also measured to know the virulence of Leptospira cultured in different environment. The amplified gene segments from Leptospira were used as probes and wrote the microarray. The total RNA was extracted from Leptospirastandard strain cultured in culture medium and guinea pigs. After reverse transcription to cDNA, they were labeled with Cy3 and Cy5 respectively. Labeled cDNA was mixed and hybridized with the microarray. The hybridized mircroarray was scanned and analysed. Results The survival rate of inoculated guinea pig was different from group to group (in vivo group: 0%; n vitro group: 88.9%; culture medium group: 100%). The guinea pigs in vivo group had a higher temperature (P <0.05), lighter body mass (P <0.05), larger organ coefficient (P <0.05) and a more serious hemorrhage in lung. The genes from Leptospira: LA1027, LA1029, LA4004, LA3050, LA3540, LA0327, LA0378, LA1650, LA3937, LA2089, LA2144, LA3576, LA0011 and gene of Loa22 were up regulation after continuously cultured in guinea pigs. Conclusion The pathogenic ability of Leptospira cultured in different environment is different and the gene expression of Leptospirais different between in vivo and in vitroas well. The understanding of the meaning of this change might help to know the pathogenecity of Leptospira.  
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