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

发布时间：2018-08-04 22:10

【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1mL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15d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15d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(P0.05)、体质量下降(P0.05);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(P0.05);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 analyze the relationship between specific gene expression and virulence of Leptospira leishurus by detecting the difference of partial pathogenic gene expression of Leptospira Leptospira in continuous passage and in vitro culture. To understand the pathogenesis and pathogenesis of Leptospira leiensis. Methods 27 guinea pigs were divided into 3 groups (n = 9). 1mL was subcutaneously inoculated with Leptospira in vivo (in vivo culture group), Leptospira in vitro (in vitro culture group) or Leptospira medium (medium group). The concentration of Leptospira was 108/mL and was in logarithmic growth stage. The morbidity and mortality were observed within 15 days after inoculation, and the changes of body mass were measured. The surviving guinea pigs were killed on the 15th day after inoculation, and organ coefficients were measured to understand the virulence of Leptospira in different environments. Using the standard Leptospira leptospira strain DNA as the template, the selected specific gene was amplified by PCR as a probe. Leptospira RNA was isolated from several successive generations of Leptospira leptospira cultured in guinea pigs and cultured in EMJH medium. Cy5 and Cy3 were used to label Leptospira by reverse transcription and then hybridized with gene chip. The differential expression of virulence genes related to Leptospira leisepis was studied by analyzing the ratio of two fluorescence intensities. Results after inoculation, the survival rate of guinea pigs in vivo culture group was 0 and that in vitro group was 88.9. All the guinea pigs in the culture medium group survived, the body temperature increased (P0.05) and the body mass decreased (P0.05) in the in vivo culture group compared with the other two groups. The organ coefficient of kidney was larger than that of in vitro culture group and culture medium group (P0.05); after dissection, the pulmonary hemorrhage in vivo culture group was more serious than that in the other two groups. Hemolysin gene and other genes related to leptospirosis virulence showed different degrees of change. The hemolysin gene LA1027, LA1029, LA4004, LA3050, LA3540, LA0327, LA0378, LA1650, LA3937, LA2089, flagellin gene LA3576, outer membrane protein gene LA0011 and Loa22 gene were up-regulated. Conclusion the virulence of Leptospira is increased after continuous culture in vivo. There is a relationship between the virulence of Leptospira and the difference of its gene expression. It is important to understand the significance of the changes of these genes in elucidating the pathogenic mechanism of Leptospira.
【作者单位】：四川大学华西基础医学与法医学院感染免疫研究室;
【基金】：国家自然科学基金(No.30771908)资助
【分类号】：R514.4

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