死亡受体通路在E.tenalla宿主细胞凋亡中作用

发布时间：2018-07-10 15:51

本文选题：柔嫩艾美耳球虫 + 盲肠上皮细胞　；参考：《山西农业大学》2015年硕士论文

【摘要】：为探讨死亡受体凋亡通路在鸡柔嫩艾美耳球虫(E.tenella)诱导的宿主细胞凋亡中的作用以及感染E.tenella的细胞培养上清对未感染细胞凋亡的影响,采用E.tenella体外盲肠培养技术、流式细胞术、原位末端标记技术、酶联免疫分析法、HE染色等技术等,对Caspase-8抑制剂(Z-IETD-FMK)处理组和非处理组E.tenella宿主细胞凋亡率、DNA损伤情况、Caspase-8、Caspase-3、sFas、sFasL、mFas、mFasL的活性和虫体感染率进行动态测定；对不同时间段感染E.tenella与未感染E.tenella的细胞培养上清培养的盲肠上皮细胞凋亡率、Caspase-8、Caspase-9活性进行动态测定,结果表明：(1)感染E.tenalla 4h后,宿主细胞的早期凋亡率、晚期凋亡和坏死率低于空白对照组,但差异不显著(P0.05), DNA损伤率显著(P0.05)低于空白对照组；感染E.tenalla24h-72h、120h后,宿主细胞的早期凋亡率、晚期凋亡和坏死率极显著(P0.01)高于空白对照组；感染E.tenalla 24h-96h后,宿主细胞DNA损伤率极显著(P0.01)高于空白对照组；宿主细胞凋亡率随着E.tenalla感染时间的延长而增加。表明感染E.tenella早期,能抑制宿主细胞凋亡,后期能加快宿主细胞的凋亡。(2) Caspase-8抑制剂(Z-IETD-FMK)组在感染E.tenella早期(4h),宿主细胞早期凋亡率、晚期凋亡和坏死率、DNA损伤率以及虫体感染率高于接种不加药组,但差异不显著(P0.05)；Caspase-8抑制剂组在48h-96h,宿主细胞的早期凋亡率显著(P0.05)或极显著(P0.01)低于接种不加药组；在24h-120h,Caspase-8抑制剂组宿主细胞的晚期凋亡和坏死率均低于接种不加药组,且在72h、120h差异极显著(P0.01)；在24h-96h,Caspase-8抑制剂组宿主细胞的DNA损伤率均极显著(P0.01)或显著(P0.05)低于接种不加药组；在24h-120h,Caspase-8抑制剂组宿主细胞的虫体感染率均极显著(P0.01)高于接种不加药组。表明死亡受体通路参与了E.tenella引起的宿主细胞凋亡,抑制死亡受体通路有助于E.tenella子孢子的发育。(3)感染E.tenella早期(4h),宿主细胞Caspase-3的表达极显著(P0.01)或显著(P0.05)低于空白对照组；感染E.tenella后的24h-120h,宿主细胞Caspase-3、Caspase-8的表达极显著(P0.01)高于空白对照组；感染E.tenella早期(4h),接种并添加Caspase-8抑制剂(Z-IETD-FMK)组宿主细胞Caspase-8的表达显著(P0.05)高于接种不加药组；感染E.tenella后的24h-120h,接种并添加Caspase-8抑制剂组宿主细胞Caspase-3、Caspase-8的表达极显著(P0.01)低于接种不加药组。表明E.tenella可通过激活Caspase-8和Caspase-3的活性来调节宿主细胞的死亡受体通路。(4)接种E.tenella组,除48h外细胞培养上清中sFas表达均显著(P0.05)或极显著(P0.01)低于空白对照组,而细胞mFas表达均显著(P0.05)或极显著(P0.01)高于空白对照组；接种E.tenella组细胞培养上清中sFasL表达均显著(P0.05)或极显著(P0.01)低于空白对照组,除48h外细胞mFasL表达均显著(P0.05)或极显著(P0.01)高于空白对照组。表明Fas/FasL表达升高是E.tenalla引起的宿主细胞的凋亡机制之一。(5)含48h感染E.tenella的细胞培养上清液组培养的宿主细胞的早期凋亡率显著(P0.05)高于含48h不感染E.tenella的细胞培养上清液组；含12h感染E.tenella的细胞培养上清液组培养的宿主细胞的晚期凋亡和坏死率极显著(P0.01)低于含12h不感染E.tenella的细胞培养上清液组,含4h、24h-120h感染E.tenella的细胞培养上清液组培养的宿主细胞的晚期凋亡和坏死率高于含4h、24h-120h不感染E.tenella的细胞培养上清液组,且在24h、120h时差异极显著(P0.01)或显著(P0.05)。含感染E.tenella的细胞培养上清液培养的宿主细胞的Caspase-8的表达高于不感染E.tenella的细胞培养上清液培养组,但差异不显著(P0.05)；含4h、24h感染E.tenella的细胞培养上清液培养的宿主细胞的Caspase-9的表达显著(P0.05)高于含4h、24h不感染E.tenell的细胞培养上清液组,其余时间段差异不显著(P0.05)。表明宿主细胞感染E.tenella后,球虫的分泌物对未感染细胞的凋亡有影响,且主要通过线粒体通路引起宿主细胞的凋亡。
[Abstract]:In order to investigate the role of death receptor apoptotic pathway in the apoptosis of host cells induced by E.tenella and the effect of culture supernatant on apoptosis of non - infected cells , the apoptosis rate , DNA damage , Caspase - 8 , Caspase - 3 , sFas , sFasL , mFas , mFasL and the rate of infection of E . tenella host cells were measured by E . tenella .
The apoptosis rate , Caspase - 8 and Caspase - 9 activity of the cultured epithelial cells of E . tenella infected with E . tenella in different time periods were measured dynamically . The results showed that : ( 1 ) The early apoptotic rate , late apoptosis and necrosis rate of the host cells were lower than those in the blank control group after infection of E . tenella 4h , but the difference was not significant ( P0.05 ) .
The early apoptotic rate , late apoptosis and necrosis rate of the host cells were significantly higher than those in the blank control group after infection of E . tenalla24h - 72h , 120h .
After infection of E . tenalla 24h - 96h , the DNA damage rate of host cells was significantly higher than that in blank control group ( P0.01 ) .
( 2 ) Caspase - 8 inhibitor ( Z - IETD - FMK ) could inhibit the apoptosis of host cells in the early stage of infection .
The apoptosis rate of Caspase - 8 inhibitor was significantly higher ( P0.05 ) or very significant ( P0.01 ) .
In 24h - 120h , the apoptosis and necrosis rate of Caspase - 8 inhibitor group were significantly lower than those in the dose - free group , and the difference was significant at 72 h and 120 h ( P0.01 ) .
The DNA damage rate of the host cells of the caspase - 8 inhibitor group was significantly higher ( P0.01 ) or significant ( P0.05 ) at 24h - 96h , but lower than that in the administration group .
The results showed that the death receptor pathway was involved in the development of E . tenella . ( 3 ) The expression of Caspase - 3 in the host cell was significantly higher than that in the blank control group ( P0.05 ) .
The expression of Caspase - 3 and Caspase - 8 in the host cells was significantly higher than that in the blank control group ( P0.01 ) .
The expression of Caspase - 8 was significantly higher in the host cells infected with E . tenella ( 4h ) , and Caspase - 8 inhibitor ( Z - IETD - FMK ) was inoculated and added ( P0.05 ) .
The expression of Caspase - 3 and Caspase - 8 was significantly higher than that in control group ( P0.05 ) .
The expression of sFasL in culture supernatant of E . tenella group was significantly higher than that in blank control group ( P0.05 ) or very significant ( P0.01 ) .
The apoptosis and necrosis rate of the host cells cultured in the culture supernatant of E . tenella infected with E . tenella were significantly higher than those in the culture supernatant of E . tenella group ( P0.05 ) .
The expression of Caspase - 9 in the culture supernatant of E . tenella infected with E . tenella was significantly higher than that in culture supernatant ( P0.05 ) .
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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