问号钩端螺旋体鞘磷酯酶类溶血素Sph2原核表达及诱导细胞凋亡活性研究

发布时间：2018-05-16 02:06

  本文选题：问号钩端螺旋体 + 鞘磷脂酶类溶血素　； 参考：《浙江大学》2011年硕士论文

【摘要】：目的了解问号钩端螺旋体(简称钩体)感染细胞前后sph2基因表达水平变化,确定鞘磷脂酶类溶血素Sph2诱导人和鼠单核-巨噬细胞、肝细胞凋亡的活性及其机制。 方法采用实时荧光定量PCR检测问号钩体赖株感染小鼠单核-巨噬细胞J774A.1、小鼠肝细胞IAR20和人肝细胞L-02、人单核-巨噬细胞THP-1后sph2基因mRNA水平变化。采用PCR从黄疸出血群赖型赖株钩体基因组DNA中扩增全长sph2基因片段并构建sph2基因原核表达系统,采用SDS-PAGE检查重组Sph2(rSph2)的表达情况,Ni-NTA亲和层析法提纯rSph2。采用绵羊血平板溶血试验及血红蛋白分光光度法测定rSph2的溶血活性；采用流式细胞术检测重组表达的Sph2(rSph2)诱导J774A.1、IAR20、L-02和THP-1细胞凋亡活性。采用FITC标记rSph2(FITC-rSph2),检测FITC-rSph2内化进入细胞的情况。 结果问号钩体赖株感染细胞后0.25-2 h,其sph2基因mRNA水平开始上升,最高时可达感染前的4-8倍(P0.05),然后逐渐下降。与GenBank中sph2基因比较,所克隆的sph2基因序列相似性为100%。所构建的原核表达系统能高效表达rSph2。rSph2以浓度依赖方式溶解绵羊红细胞。10μg/mL rSph2可诱导J774A.1、IAR20、THP-1和L-02细胞凋亡,凋亡率峰值分别为23.96%、32.92%、17.52%和24.13%。FITC-rSph2与J774A.1、IAR20、THP-1和L-02细胞共孵育15-30 min后即可内化进入细胞内。 结论问号钩体sph2基因呈宿主细胞接触式瞬时表达,提示其表达产物Sph2鞘磷脂酶类溶血素在问号钩体致病过程中发挥作用。rSph2不仅可损伤单核-巨噬细胞和肝细胞细胞膜,还可诱导上述细胞发生凋亡,故Sph2是问号钩体重要的毒力因子,与钩体病时黄疸和肺出血密切相关。rSph2诱导细胞凋亡机制可能与其水解细胞膜鞘磷脂产生神经酰胺、损伤线粒体膜导致多种凋亡或促凋亡因子释放、激活细胞凋亡线粒体和神经酰胺途径有关。
[Abstract]:Objective to investigate the changes of sph2 gene expression before and after Leptospira interrogans (Leptospira interrogans) infection, and to determine the activity and mechanism of apoptosis of human and mouse mononuclear macrophages induced by sphingomyelin lysin Sph2. Methods Real-time quantitative PCR was used to detect the changes of sph2 gene mRNA level in monocyte macrophage J774A.1, IAR20 and L-02 hepatocytes of mice infected with Leptospira interrogans and THP-1 of human monocyte-macrophage. The full-length sph2 gene fragment was amplified by PCR from the genomic DNA of Leptospira japonicus and the prokaryotic expression system of sph2 gene was constructed. The expression of recombinant Sph2 rSph2) was purified by Ni-NTA affinity chromatography. The hemolytic activity of rSph2 was measured by hemolytic test and hemoglobin spectrophotometry, and the apoptotic activity of J774A.1IAR20AL-02 and THP-1 cells was detected by flow cytometry. FITC labeled rSph2 FITC-rSph2 was used to detect the internalization of FITC-rSph2 into the cells. Results the mRNA level of sph2 gene of Leptospira interrogans strain began to rise at 0.25 h after infection, and reached 4-8 times of P0.05 before infection, and then decreased gradually. Compared with the sph2 gene in GenBank, the sequence similarity of the cloned sph2 gene was 100%. The constructed prokaryotic expression system could efficiently express rSph2.rSph2 and dissolve sheep erythrocytes. 10 渭 g/mL rSph2 in a concentration-dependent manner could induce apoptosis of THP-1 and L-02 cells in J774A.1IIAR20N. The peak apoptotic rate was 23.96A 32.92% and 17.52%, respectively. After co-incubating with J774A.1IAR20THP-1 and L-02 cells for 15-30 min, J774A.1IAR20THP-1 and L-02 cells could internalize into the cells. Conclusion the transient expression of sph2 gene of Leptospira interrogans in the host cells suggests that Sph2 sphingolipase lysins play an important role in the pathogenesis of Leptospira interrogans. RSph2 can not only damage monocyte-macrophage and hepatocyte membrane. Therefore, Sph2 is an important virulence factor of Leptospira interrogans, which is closely related to jaundice and pulmonary hemorrhage induced by Leptospira disease. The mechanism of apoptosis induced by rSph2 may be related to the production of ceramide by phospholipid in membrane sheath of Leptospira. Mitochondrial membrane damage may induce apoptosis or promote the release of apoptotic factors, which is related to activation of apoptotic mitochondria and ceramide pathway.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R377.5

【参考文献】

相关期刊论文 前1条

1 ;Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai[J];Acta Pharmacologica Sinica;2005年04期

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本文编号：1894949