本文關(guān)鍵詞：肝螺桿菌CdtB毒素致小鼠肝細(xì)胞損傷的初步研究　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 肝螺桿菌 細(xì)胞致死性腫脹毒素 細(xì)胞因子 凋亡 自噬

【摘要】：肝螺桿菌是一種重要的人獸共患病病原,涉及慢性活動(dòng)性肝炎、膽囊炎、盲腸炎、結(jié)腸炎、肝癌、膽石病、膽囊癌、乳腺癌等多種疾病,具有重要的公共衛(wèi)生意義。國(guó)際實(shí)驗(yàn)動(dòng)物組織及世界發(fā)達(dá)國(guó)家已將該類螺桿菌列為嚙齒類實(shí)驗(yàn)動(dòng)物必須排除的病原微生物;但我國(guó)對(duì)肝螺桿菌的危害和控制尚未引起高度關(guān)注,肝螺桿菌在我國(guó)嚙齒類實(shí)驗(yàn)動(dòng)物中保持著較高的感染率,其潛在威脅巨大。細(xì)胞致死性腫脹毒素(Cytolethal distending toxin,CDT)是目前革蘭氏陰性桿菌在引起致病過(guò)程中唯一已知的毒力因子,已有研究表明,肝螺桿菌CdtB可引發(fā)細(xì)胞凋亡,但其是否引起肝細(xì)胞炎癥及引發(fā)肝細(xì)胞凋亡的作用機(jī)制尚未得到深入闡述。本研究通過(guò)CdtB毒素處理小鼠原代肝細(xì)胞AML12,致力于探討肝螺桿菌細(xì)胞致死性腫脹毒素CdtB致小鼠肝損傷的作用機(jī)制。首先,用不同濃度的CdtB處理AML12細(xì)胞,我們發(fā)現(xiàn),隨著毒素濃度的增加,細(xì)胞出現(xiàn)生長(zhǎng)停滯、皺縮和破裂的現(xiàn)象;CCK-8測(cè)定細(xì)胞活力發(fā)現(xiàn),CdtB對(duì)AML12細(xì)胞增殖有抑制作用,且呈劑量-效應(yīng)關(guān)系;qRT-PCR檢測(cè)顯示,AML12細(xì)胞經(jīng)CdtB刺激后,IL-6、IL-lβ、TNF-α、IFN-αl、IFN-γ的表達(dá)水平顯著上升,免疫印跡檢測(cè)結(jié)果表明,STAT3被磷酸化,進(jìn)一步說(shuō)明了 CdtB可刺激AML12細(xì)胞釋放IL-6,經(jīng)JAK-STAT通路進(jìn)一步刺激細(xì)胞產(chǎn)生細(xì)胞因子引起炎癥;同時(shí),FACS的檢測(cè)結(jié)果顯示,CdtB可誘導(dǎo)AML12細(xì)胞產(chǎn)生劑量依賴性的凋亡效應(yīng)。為探討CdtB致AML12細(xì)胞凋亡的作用機(jī)制,我們用Western-blot法檢測(cè)了 Caspase、MAPK信號(hào)通路的相關(guān)蛋白,結(jié)果顯示,CdtB可以激活Caspase-9、Caspase-3、PARP、p38 和 Erk1/2 MAPK。以 Caspase 廣譜抑制劑 Z-VAD-FMK 和 Erk1/2 抑制劑 U0126、p38抑制劑SB203580預(yù)處理細(xì)胞后,再經(jīng)CdtB處理,結(jié)果發(fā)現(xiàn),凋亡率、Caspase-3及PARP的裂解在經(jīng)Z-VAD-FMK和SB203580處理后均顯著抑制,說(shuō)明Caspase及p38 MAPK參與了 CdtB引發(fā)的凋亡效應(yīng)。結(jié)合qRT-PCR結(jié)果,我們推測(cè)p38 MAPK通路在CdtB誘導(dǎo)的致AML12細(xì)胞炎性因子調(diào)控及凋亡中發(fā)揮著重要作用。自噬作為一種重要的保護(hù)機(jī)制,廣泛存在于所有真核生物中。為進(jìn)一步探討肝螺桿菌CdtB的作用機(jī)制,我們檢測(cè)了 AML12細(xì)胞的自噬水平,結(jié)果發(fā)現(xiàn)AML12細(xì)胞本身處于較高的自噬水平,CdtB可激活P13K/Akt/mTOR信號(hào)通路而抑制自噬。為進(jìn)一步驗(yàn)證自噬在AML12中的作用,我們使用自噬抑制劑氯喹(CQ)預(yù)處理細(xì)胞,然后再以CdtB處理細(xì)胞,結(jié)果顯示,經(jīng)CQ預(yù)處理細(xì)胞后,可顯著提高CdtB對(duì)AML12細(xì)胞的凋亡率,Western-blot結(jié)果同樣證實(shí),CQ可更加有效地激活Caspase-3和PARP,提示抑制自噬可加強(qiáng)肝螺桿菌CdtB誘導(dǎo)的肝細(xì)胞凋亡,這從分子的角度為解釋肝螺桿菌致病性提供了一些價(jià)值性線索。
[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- 偽, IFN- 偽 and IFN- 緯 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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378

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