肝螺杆菌CdtB毒素致小鼠肝细胞损伤的初步研究

发布时间：2018-01-12 00:15

本文关键词：肝螺杆菌CdtB毒素致小鼠肝细胞损伤的初步研究　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：肝螺杆菌是一种重要的人兽共患病病原,涉及慢性活动性肝炎、胆囊炎、盲肠炎、结肠炎、肝癌、胆石病、胆囊癌、乳腺癌等多种疾病,具有重要的公共卫生意义。国际实验动物组织及世界发达国家已将该类螺杆菌列为啮齿类实验动物必须排除的病原微生物;但我国对肝螺杆菌的危害和控制尚未引起高度关注,肝螺杆菌在我国啮齿类实验动物中保持着较高的感染率,其潜在威胁巨大。细胞致死性肿胀毒素(Cytolethal distending toxin,CDT)是目前革兰氏阴性杆菌在引起致病过程中唯一已知的毒力因子,已有研究表明,肝螺杆菌CdtB可引发细胞凋亡,但其是否引起肝细胞炎症及引发肝细胞凋亡的作用机制尚未得到深入阐述。本研究通过CdtB毒素处理小鼠原代肝细胞AML12,致力于探讨肝螺杆菌细胞致死性肿胀毒素CdtB致小鼠肝损伤的作用机制。首先,用不同浓度的CdtB处理AML12细胞,我们发现,随着毒素浓度的增加,细胞出现生长停滞、皱缩和破裂的现象;CCK-8测定细胞活力发现,CdtB对AML12细胞增殖有抑制作用,且呈剂量-效应关系;qRT-PCR检测显示,AML12细胞经CdtB刺激后,IL-6、IL-lβ、TNF-α、IFN-αl、IFN-γ的表达水平显著上升,免疫印迹检测结果表明,STAT3被磷酸化,进一步说明了 CdtB可刺激AML12细胞释放IL-6,经JAK-STAT通路进一步刺激细胞产生细胞因子引起炎症;同时,FACS的检测结果显示,CdtB可诱导AML12细胞产生剂量依赖性的凋亡效应。为探讨CdtB致AML12细胞凋亡的作用机制,我们用Western-blot法检测了 Caspase、MAPK信号通路的相关蛋白,结果显示,CdtB可以激活Caspase-9、Caspase-3、PARP、p38 和 Erk1/2 MAPK。以 Caspase 广谱抑制剂 Z-VAD-FMK 和 Erk1/2 抑制剂 U0126、p38抑制剂SB203580预处理细胞后,再经CdtB处理,结果发现,凋亡率、Caspase-3及PARP的裂解在经Z-VAD-FMK和SB203580处理后均显著抑制,说明Caspase及p38 MAPK参与了 CdtB引发的凋亡效应。结合qRT-PCR结果,我们推测p38 MAPK通路在CdtB诱导的致AML12细胞炎性因子调控及凋亡中发挥着重要作用。自噬作为一种重要的保护机制,广泛存在于所有真核生物中。为进一步探讨肝螺杆菌CdtB的作用机制,我们检测了 AML12细胞的自噬水平,结果发现AML12细胞本身处于较高的自噬水平,CdtB可激活P13K/Akt/mTOR信号通路而抑制自噬。为进一步验证自噬在AML12中的作用,我们使用自噬抑制剂氯喹(CQ)预处理细胞,然后再以CdtB处理细胞,结果显示,经CQ预处理细胞后,可显著提高CdtB对AML12细胞的凋亡率,Western-blot结果同样证实,CQ可更加有效地激活Caspase-3和PARP,提示抑制自噬可加强肝螺杆菌CdtB诱导的肝细胞凋亡,这从分子的角度为解释肝螺杆菌致病性提供了一些价值性线索。
[Abstract]:Helicobacter hepatica is an important zoonotic pathogen, involving chronic active hepatitis, cholecystitis, cecositis, colitis, liver cancer, cholelithiasis, gallbladder cancer, breast cancer and many other diseases. It has important public health significance. The international laboratory animal organization and developed countries have listed this kind of helicobacterium as the pathogenic microorganism which must be excluded from rodent laboratory animal. However, the harmfulness and control of Helicobacter hepatis in China has not attracted much attention, and the infection rate of Helicobacter hepatis in rodent laboratory animals in China remains high. The potential threat is enormous. Cytolethal distending toxin. CdtB is the only known virulence factor in the pathogenesis of Gram-negative bacilli. It has been shown that Helicobacter hepatica CdtB can induce apoptosis. However, the mechanism of inducing hepatocyte inflammation and hepatocyte apoptosis has not been elucidated. In this study, CdtB toxin was used to treat mouse primary hepatocyte AML12. In order to explore the mechanism of liver injury induced by lethal swelling toxin CdtB of Helicobacter hepatis in mice. Firstly, we found that AML12 cells were treated with different concentrations of CdtB. With the increase of toxin concentration, cell growth arrest, shrinkage and rupture; CCK-8 assay showed that CdtB inhibited the proliferation of AML12 cells in a dose-effect manner. QRT-PCR analysis showed that the expression of IL-6, IL-l 尾, TNF- 伪, IFN- 伪 and IFN- 纬 in AML12 cells stimulated by CdtB was significantly increased. Western blot analysis showed that STAT3 was phosphorylated, which suggested that CdtB could stimulate the release of IL-6 from AML12 cells. JAK-STAT pathway further stimulates the production of cytokines and causes inflammation. At the same time, the results of FACS showed that CDTB could induce the apoptosis of AML12 cells in a dose-dependent manner. In order to investigate the mechanism of apoptosis induced by CdtB in AML12 cells. We used Western-blot method to detect the related proteins of Caspase- MAPK signaling pathway. The results showed that caspase- CDTB could activate Caspase-9. Caspase-3 and PARP. P38 and Erk1/2 MAPK. Caspase broad-spectrum inhibitor Z-VAD-FMK and Erk1/2 inhibitor U0126 were used. After pretreatment with p38 inhibitor SB203580 and then treated with CdtB, apoptosis rate was found. The cleavage of Caspase-3 and PARP was significantly inhibited by Z-VAD-FMK and SB203580. The results showed that Caspase and p38 MAPK were involved in the apoptosis induced by CdtB, and combined with the results of qRT-PCR. We speculate that p38 MAPK pathway plays an important role in the regulation and apoptosis of AML12 inflammatory cytokines induced by CdtB. Autophagy is an important protective mechanism. In order to further study the mechanism of the action of Helicobacter hepatis CdtB, we detected the autophagy level of AML12 cells. The results showed that the AML12 cells themselves were at a higher autophagy level. CdtB can activate the P13K / Akt / mTOR signaling pathway and inhibit autophagy. To further verify the role of autophagy in AML12, we pretreated the cells with chloroquine. Then the cells were treated with CdtB. The results showed that CQ pretreatment could significantly increase the apoptosis rate of AML12 cells induced by CdtB. The Western-blot results also confirmed that the cells were pretreated with CQ. CQ can activate Caspase-3 and par more effectively, suggesting that inhibition of autophagy can enhance hepatocyte apoptosis induced by CdtB. This provides some valuable clues to explain the pathogenicity of Helicobacter hepatis from a molecular perspective.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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