本文選題：問號鉤端螺旋體 + 鞘磷脂酶類溶血素��； 參考：《浙江大學(xué)》2011年碩士論文

【摘要】：目的了解問號鉤端螺旋體(簡稱鉤體)感染細(xì)胞前后sph2基因表達(dá)水平變化,確定鞘磷脂酶類溶血素Sph2誘導(dǎo)人和鼠單核-巨噬細(xì)胞、肝細(xì)胞凋亡的活性及其機(jī)制。 方法采用實(shí)時熒光定量PCR檢測問號鉤體賴株感染小鼠單核-巨噬細(xì)胞J774A.1、小鼠肝細(xì)胞IAR20和人肝細(xì)胞L-02、人單核-巨噬細(xì)胞THP-1后sph2基因mRNA水平變化。采用PCR從黃疸出血群賴型賴株鉤體基因組DNA中擴(kuò)增全長sph2基因片段并構(gòu)建sph2基因原核表達(dá)系統(tǒng),采用SDS-PAGE檢查重組Sph2(rSph2)的表達(dá)情況,Ni-NTA親和層析法提純r(jià)Sph2。采用綿羊血平板溶血試驗(yàn)及血紅蛋白分光光度法測定rSph2的溶血活性；采用流式細(xì)胞術(shù)檢測重組表達(dá)的Sph2(rSph2)誘導(dǎo)J774A.1、IAR20、L-02和THP-1細(xì)胞凋亡活性。采用FITC標(biāo)記rSph2(FITC-rSph2),檢測FITC-rSph2內(nèi)化進(jìn)入細(xì)胞的情況。 結(jié)果問號鉤體賴株感染細(xì)胞后0.25-2 h,其sph2基因mRNA水平開始上升,最高時可達(dá)感染前的4-8倍(P0.05),然后逐漸下降。與GenBank中sph2基因比較,所克隆的sph2基因序列相似性為100%。所構(gòu)建的原核表達(dá)系統(tǒng)能高效表達(dá)rSph2。rSph2以濃度依賴方式溶解綿羊紅細(xì)胞。10μg/mL rSph2可誘導(dǎo)J774A.1、IAR20、THP-1和L-02細(xì)胞凋亡,凋亡率峰值分別為23.96%、32.92%、17.52%和24.13%。FITC-rSph2與J774A.1、IAR20、THP-1和L-02細(xì)胞共孵育15-30 min后即可內(nèi)化進(jìn)入細(xì)胞內(nèi)。 結(jié)論問號鉤體sph2基因呈宿主細(xì)胞接觸式瞬時表達(dá),提示其表達(dá)產(chǎn)物Sph2鞘磷脂酶類溶血素在問號鉤體致病過程中發(fā)揮作用。rSph2不僅可損傷單核-巨噬細(xì)胞和肝細(xì)胞細(xì)胞膜,還可誘導(dǎo)上述細(xì)胞發(fā)生凋亡,故Sph2是問號鉤體重要的毒力因子,與鉤體病時黃疸和肺出血密切相關(guān)。rSph2誘導(dǎo)細(xì)胞凋亡機(jī)制可能與其水解細(xì)胞膜鞘磷脂產(chǎn)生神經(jīng)酰胺、損傷線粒體膜導(dǎo)致多種凋亡或促凋亡因子釋放、激活細(xì)胞凋亡線粒體和神經(jīng)酰胺途徑有關(guān)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R377.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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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本文編號：1894949