【摘要】：廣州管圓線蟲是引起嗜酸粒細(xì)胞增多性腦膜炎和腦膜腦炎的重要病原體。人是廣州管圓線蟲的非適宜宿主,多因生食或半生食含有廣州管圓線蟲Ⅲ期幼蟲的中間宿主而感染。感染期幼蟲可穿過腸壁進(jìn)入血液循環(huán)系統(tǒng),隨血流在體內(nèi)移行,具有嗜神經(jīng)性,多侵犯中樞神經(jīng)系統(tǒng)。該病的特征為腦脊液中嗜酸性粒細(xì)胞升高,病變除了大腦和腦膜還可波及小腦、腦干、脊髓或眼球,主要的病變有腦充血、出血、腦組織機(jī)械性損傷及肉芽腫反應(yīng)。近些年來由于廣州管圓線蟲中間宿主福壽螺和褐云瑪瑙螺在我國南方地區(qū)大量繁殖,加上人們飲食上嗜好生猛鮮活與獵奇,導(dǎo)致廣州管圓線蟲病相繼在各地暴發(fā)或散發(fā)流行,已被列入新發(fā)感染性疾病。目前臨床上常用阿苯達(dá)唑驅(qū)蟲,但是蟲體死亡時(shí)崩解釋放大量的抗原引起的炎癥反應(yīng)會導(dǎo)致神經(jīng)系統(tǒng)癥狀的加重。因此,深入了解廣州管圓線蟲感染腦損傷的免疫應(yīng)答機(jī)制對于廣州管圓線蟲病的防治具有重要意義。研究表明中樞神經(jīng)系統(tǒng)和免疫系統(tǒng)以復(fù)雜的方式雙向相互作用。在理想的情況下,應(yīng)激條件下的炎癥和抗炎反應(yīng)是平衡的,有利于創(chuàng)傷愈合和遏制病原體,同時(shí)防止過高的炎癥反應(yīng)或嚴(yán)重的免疫抑制。然而,在沒有全身性炎癥的情況下,腦損傷局部炎性細(xì)胞因子擴(kuò)散觸發(fā)的抗炎反應(yīng)可能是有害的,因?yàn)檫@樣會下調(diào)機(jī)體的防御機(jī)制,使機(jī)體容易受到感染。有研究發(fā)現(xiàn),中樞神經(jīng)系統(tǒng)損傷對免疫功能產(chǎn)生深遠(yuǎn)的影響,如中風(fēng),創(chuàng)傷性腦損傷或脊髓損傷后的患者均表現(xiàn)出免疫功能受損,包括外周血淋巴細(xì)胞數(shù)量減少,脾臟和胸腺萎縮,T細(xì)胞活性受損等。進(jìn)一步研究發(fā)現(xiàn),急性中樞神經(jīng)系統(tǒng)受損患者循環(huán)單核細(xì)胞主要組織相容性復(fù)合體II類分子表達(dá)下調(diào),體外內(nèi)毒素刺激單核細(xì)胞,其產(chǎn)生促炎性細(xì)胞因子的能力大大降低。這種免疫抑制的臨床表現(xiàn)為在腦損傷后的不久高發(fā)全身性感染,特別是肺炎和尿路感染,而感染會阻礙神經(jīng)的恢復(fù)、增加患者死亡率。這些研究結(jié)果提示,腦損傷會導(dǎo)致外周免疫抑制。廣州管圓線蟲的幼蟲侵入人體后移行至大腦,可造成腦組織機(jī)械損傷,占位并釋放代謝產(chǎn)物損傷中樞神經(jīng)系統(tǒng)。基于腦部損傷與外周免疫抑制的密切聯(lián)系,我們提出如下假說：廣州管圓線蟲幼蟲侵入大腦損傷中樞神經(jīng)系統(tǒng)后,導(dǎo)致大腦局部炎性細(xì)胞因子擴(kuò)散刺激抗炎反應(yīng)引起外周免疫抑制,增加機(jī)體的繼發(fā)感染的機(jī)會。為了驗(yàn)證上述假說,在小鼠廣州管圓線蟲感染模型上,我們首先觀察了廣州管圓線蟲感染后免疫器官胸腺和脾臟的變化,同時(shí)對外周淋巴細(xì)胞亞群數(shù)量和功能的變化進(jìn)行了評估；其次,我們從細(xì)胞凋亡和細(xì)胞發(fā)育受阻兩方面探討了廣州管圓線蟲感染后外周淋巴細(xì)胞數(shù)量下降的原因；再次,我們研究了廣州管圓線蟲感染后腦組織炎性細(xì)胞因子、抗炎細(xì)胞因子和趨化因子的表達(dá),觀察中樞神經(jīng)系統(tǒng)與外周免疫系統(tǒng)聯(lián)系的下丘腦-垂體-腎上腺(HPA)軸的變化；最后,我們證實(shí)在廣州管圓線蟲感染中樞神經(jīng)系統(tǒng)損傷后,糖皮質(zhì)激素促進(jìn)了外周的免疫抑制。本研究獲得如下主要結(jié)果：1.廣州管圓線蟲感染誘導(dǎo)免疫器官萎縮和淋巴細(xì)胞數(shù)量減少感染后21天小鼠的胸腺和脾臟較對照組小鼠明顯萎縮,且單個(gè)核細(xì)胞數(shù)也減少,提示廣州管圓線蟲感染可影響小鼠的免疫器官。為了探討感染后胸腺和脾臟萎縮的原因,我們用流式細(xì)胞儀檢測了小鼠脾臟和外周血細(xì)胞亞群的變化。檢測結(jié)果表明,感染后脾臟和外周血中B細(xì)胞、T細(xì)胞、CD4+T細(xì)胞、CD8+T細(xì)胞、Thl細(xì)胞及NK細(xì)胞的數(shù)量較對照組顯著減少。2.廣州管圓線蟲感染降低細(xì)胞免疫和體液免疫功能為了證實(shí)在廣州管圓線蟲感染后,淋巴細(xì)胞的免疫功能也受感染的影響,我們進(jìn)一步研究了T淋巴細(xì)胞增殖功能、分泌細(xì)胞因子和B細(xì)胞產(chǎn)生抗體的能力。結(jié)果顯示,感染后小鼠T細(xì)胞的增殖能力較對照組小鼠降低；采用有絲分裂原刺激外周全血細(xì)胞,其培養(yǎng)上清用ELISA檢測的結(jié)果顯示,廣州管圓線蟲感染誘導(dǎo)低水平的IFN-γ、TNF-α和高水平的IL-4;用非特異性抗原OVA免疫廣州管圓線蟲感染小鼠和對照組小鼠,檢測OVA-IgGl抗體表達(dá)水平。檢測結(jié)果顯示,感染后小鼠體內(nèi)OVA-IgGl抗體水平較對照小鼠降低,提示廣州管圓線蟲感染降低了小鼠B細(xì)胞產(chǎn)生抗體的能力。為了進(jìn)一步證實(shí)這些結(jié)果,我們觀察了小鼠肺部的病理和采集小鼠的血液做菌血培養(yǎng)。結(jié)果顯示,感染后小鼠肺部有大量炎性細(xì)胞浸潤,表現(xiàn)為肺炎。同樣血液在血瓊脂平板上培養(yǎng)顯示感染后出現(xiàn)菌血癥,表明廣州管圓線蟲感染誘導(dǎo)了小鼠免疫抑制。3.廣州管圓線蟲感染增強(qiáng)NK細(xì)胞的殺傷功能 因?yàn)閺V州管圓線蟲感染降低了細(xì)胞免疫和體液免疫功能,導(dǎo)致小鼠的感染風(fēng)險(xiǎn)增加,所以我們進(jìn)一步評估了廣州管圓線蟲感染對NK細(xì)胞功能的影響。我們在加或不加外源性IL-12的條件下對NK細(xì)胞刺激培養(yǎng)24 h, ELISA檢測培養(yǎng)上清中IFN-γ和TNF-α的表達(dá)水平。結(jié)果表明,IL-12處理的感染小鼠NK細(xì)胞較對照小鼠產(chǎn)生更高水平的IFN-γ。無論是否存在外源性IL-12,廣州管圓線蟲感染小鼠和對照小鼠均只能誘導(dǎo)NK細(xì)胞產(chǎn)生低水平的TNF-β。進(jìn)一步我們評估了感染對NK細(xì)胞殺傷活性的影響,結(jié)果顯示,感染后的NK細(xì)胞較對照組小鼠有更強(qiáng)的殺傷功能。這些結(jié)果提示,廣州管圓線蟲感染雖然降低了NK細(xì)胞的數(shù)量,但是增強(qiáng)了NK細(xì)胞的殺傷功能。4.廣州管圓線蟲感染后B細(xì)胞和T細(xì)胞的減少不是由于凋亡所導(dǎo)致,但NK細(xì)胞在感染后出現(xiàn)大量的凋亡感染后B和T細(xì)胞數(shù)量急劇降低,為了進(jìn)一步明確細(xì)胞數(shù)量下降是否由凋亡所致,我們用流式檢測了這些細(xì)胞表面7AAD-AnnexinV+的比例。結(jié)果發(fā)現(xiàn),與對照組相比,感染后細(xì)胞表面7AAD" Annexin V+比例沒有增加。Caspase 3作為凋亡最終的執(zhí)行者,是檢測凋亡的一個(gè)重要指標(biāo)。為了進(jìn)一步證實(shí)這個(gè)結(jié)果,我們用磁珠分選的方法分選出B220-細(xì)胞和CD3-細(xì)胞,用estern blot的方法檢鋇Cleaved Caspase 3的表達(dá),結(jié)果表明感染后沒有檢測至Cleaved Caspase 3表達(dá)的增加,提示廣州管圓線蟲感染后B細(xì)胞和T細(xì)胞數(shù)量的下降不是由于凋亡所導(dǎo)致的。同樣地,為了明確NK細(xì)胞數(shù)量下降是否由凋亡所致,我們用流式檢測了NK細(xì)胞表面7AAD" Annexin V+的比例及用磁珠分選的方法分選出DX5+細(xì)胞,用estern blot的方法檢狽Cleaved Caspase 3的表達(dá)。結(jié)果發(fā)現(xiàn),與對照組相比,感染后NK細(xì)胞表面7AAD- Annexin V+和Cleaved Caspase 3的表達(dá)增加,提示廣州管圓線蟲感染導(dǎo)致NK數(shù)量的降低與NK細(xì)胞發(fā)生的凋亡有關(guān)。5.廣州管圓線蟲感染抑制骨髓中B細(xì)胞的發(fā)生和損傷T細(xì)胞在胸腺的發(fā)育廣州管圓線蟲感染后B細(xì)胞數(shù)量的降低不是由于細(xì)胞凋亡所引起的,我們推測在B細(xì)胞的發(fā)育的過程中,是否所有或部分發(fā)育B細(xì)胞亞群有損失。為了驗(yàn)證這一猜想,我們檢測了骨髓發(fā)育中B細(xì)胞的比例。結(jié)果所示,感染后骨髓發(fā)育中B細(xì)胞的比例較對照組降低,進(jìn)一步,我們分析了骨髓發(fā)育中B細(xì)胞的兩個(gè)亞群：pro-/pre-B細(xì)胞和不成熟B細(xì)胞。結(jié)果表明：感染后骨髓中pro-/pre-B細(xì)胞占發(fā)育中B細(xì)胞的比例較對照組顯著降低；而感染后骨髓中不成熟B細(xì)胞占發(fā)育中B細(xì)胞比例較對照組上升,提示廣州管圓線蟲感染導(dǎo)致骨髓發(fā)育中B細(xì)胞下降,其中受主要影響的是pro-/pre-B細(xì)胞。我們知道pro-/pre-B細(xì)胞在Ig H鏈和L鏈基因重排后,骨髓中的發(fā)育中B細(xì)胞進(jìn)入到不成熟B細(xì)胞階段,然后遷移到外周進(jìn)入到過渡期B細(xì)胞。進(jìn)一步我們檢測了脾臟中發(fā)育中B細(xì)胞和成熟B細(xì)胞的比例,結(jié)果顯示感染后21天,成熟B細(xì)胞降低。同時(shí),脾臟發(fā)育中B細(xì)胞的比例較對照組顯著降低。進(jìn)一步,我們分析了發(fā)育中B細(xì)胞的3個(gè)階段亞群：TR1、TR2和TR3亞群。結(jié)果表明,隨著感染時(shí)間的增加,TR1占發(fā)育中B細(xì)胞的比例逐步降低。TR2和TR3占發(fā)育中B細(xì)胞的比例隨著感染時(shí)間的增加而增加。提示廣州管圓線蟲感染導(dǎo)致脾臟發(fā)育中B細(xì)胞下降,其中受主要影響的是TR1細(xì)胞亞群。綜合以上結(jié)果,提示由于廣州管圓線蟲感染影響B(tài)細(xì)胞的發(fā)生,從而導(dǎo)致B細(xì)胞輸出下降。廣州管圓線蟲感染后T細(xì)胞數(shù)量的降低同樣不是由細(xì)胞凋亡所引起,并且在感染后胸腺出現(xiàn)嚴(yán)重的萎縮,我們推測是否T細(xì)胞在胸腺的發(fā)育過程受阻。為此我們用流式細(xì)胞儀檢測了胸腺T細(xì)胞亞群,結(jié)果顯示感染后CD4+CD8+T細(xì)胞的比例較對照組降低,與之相反的是隨著感染時(shí)間的增加,CD4-CD8-T細(xì)胞、CD4+CD8-T細(xì)胞和CD4-CD8+T細(xì)胞的比例較對照組顯著增高。進(jìn)一步我們觀察了胸腺各T細(xì)胞亞群的數(shù)量,結(jié)果顯示,感染后CD4CD8T細(xì)胞和CD4"CD8+T細(xì)胞的數(shù)量較對照組沒有差異；但是感染后CD4+CD8+T細(xì)胞的數(shù)量較對照組顯著降低。為了進(jìn)一步明確其細(xì)胞數(shù)量下降是否由凋亡所致,我們用流式檢測了CD4+CD8+T細(xì)胞表面7AAD" Annexin V+的表達(dá)。結(jié)果表明,與對照組相比,感染后CD4+CD8+T細(xì)胞表面7AAD-Annexin V+比例逐步增加。綜合以上結(jié)果,提示廣州管圓線蟲感染后胸腺細(xì)胞數(shù)量減少和外周T細(xì)胞數(shù)量的減少與CD4+CD8+T細(xì)胞凋亡后減少有關(guān)。6.廣州管圓線蟲感染導(dǎo)致的腦損傷引起淋巴細(xì)胞數(shù)量減少和下丘腦-垂體-腎上腺軸的活化廣州管圓線蟲幼蟲移行進(jìn)入腦內(nèi),侵犯神經(jīng)系統(tǒng),破壞腦組毛織。為了明確廣州管圓線蟲感染導(dǎo)致的淋巴細(xì)胞數(shù)量的降低是否由于腦損傷導(dǎo)致,我們采用阿苯達(dá)唑殺蟲實(shí)驗(yàn)觀察感染后殺蟲對外周淋巴細(xì)胞亞群的影響。結(jié)果表明,殺蟲處理組在感染后21天胸腺和脾臟大小較正常對照組沒有顯著的差異。感染后殺蟲脾臟淋巴細(xì)胞亞群B細(xì)胞、T細(xì)胞、CD4+T細(xì)胞、CD8+T細(xì)胞和NK細(xì)胞的比例較正常對照組也沒有顯著差異,提示廣州管圓線蟲感染后外周出現(xiàn)免疫抑制發(fā)生在幼蟲富集于腦組織導(dǎo)致CNS損傷之后,也即是感染導(dǎo)致的腦損傷誘導(dǎo)了外周的免疫抑制。為了明確廣州管圓線蟲腦損傷后的外周免疫抑制是否與下丘腦-垂體-腎上腺軸的活化從而引起糖皮質(zhì)激素分泌增加有關(guān),我們檢測了下丘腦-垂體-腎上腺軸活化的指標(biāo)。結(jié)果發(fā)現(xiàn),感染后血漿中皮質(zhì)酮含量的表達(dá)較對照組相比顯著增加；小鼠海馬、下丘腦室旁核、垂體和腎上腺c-fos mRNA的表達(dá)水平顯著增加；下丘腦室旁核中促腎上腺皮質(zhì)激素釋放激素和腎上腺中腎上腺酪氨酸羥化酶的mRNA的表達(dá)水平較對照組表達(dá)水平顯著增加。以上結(jié)果提示,廣州管圓線蟲感染會導(dǎo)致下丘腦-垂體-腎上腺軸的活化。7.廣州管圓線蟲感染后阻斷糖皮質(zhì)激素受體可部分逆轉(zhuǎn)B細(xì)胞發(fā)育的停止,但不能逆轉(zhuǎn)受損的胸腺T細(xì)胞的發(fā)育為了進(jìn)一步明確下丘腦-垂體-腎上腺軸活化后釋放的糖皮質(zhì)激素對外周免疫細(xì)胞的發(fā)育,我們用糖皮質(zhì)激素受體阻斷劑阻斷下丘腦-垂體-腎上腺軸并觀察感染后B細(xì)胞和胸腺T細(xì)胞的發(fā)育。我們先觀察了骨髓中B細(xì)胞的發(fā)育,結(jié)果發(fā)現(xiàn),糖皮質(zhì)激素受體阻斷劑RU486處理感染組骨髓發(fā)育中B細(xì)胞的比例較溶劑處理感染組增加。進(jìn)一步,我們分析了發(fā)育中B細(xì)胞的兩個(gè)亞群：pro-/pre-B細(xì)胞和不成熟B細(xì)胞。結(jié)果表明,RU486處理感染組pro-/pre-B細(xì)胞占發(fā)育中B細(xì)胞的比例較溶劑處理感染組顯著增加；RU486處理感染組不成熟B細(xì)胞占發(fā)育中B細(xì)胞比例較溶劑處理感染組無顯著差異。進(jìn)一步觀察脾臟中B細(xì)胞的發(fā)育,結(jié)果顯示,RU486處理感染組脾臟發(fā)育中B細(xì)胞的比例較溶劑處理感染組增加。以上結(jié)果提示廣州管圓線蟲感染導(dǎo)致下丘腦-垂體-腎上腺軸活化釋放的糖皮質(zhì)激素,與B細(xì)胞發(fā)育受阻從而導(dǎo)致B細(xì)胞輸出下降有關(guān)。除了評價(jià)阻斷下丘腦-垂體-腎上腺軸對B細(xì)胞發(fā)育的影響,我們同時(shí)檢測RU486阻斷下丘腦-垂體-腎上腺軸對胸腺T細(xì)胞發(fā)育的影響。結(jié)果發(fā)現(xiàn), RU486處理感染組胸腺CD4+CD8+T比例較溶劑處理感染組無顯著差異。提示RU486阻斷下丘腦-垂體-腎上腺軸并不能逆轉(zhuǎn)CD4+CD8+T比例的降低,胸腺T細(xì)胞發(fā)育受阻可能是下丘腦-垂體-腎上腺軸活化之外的機(jī)制所導(dǎo)致。綜上所述,本研究證實(shí)了廣州管圓線蟲感染后引起的腦損傷會對免疫系統(tǒng)產(chǎn)生抑制效應(yīng),并初步闡明下丘腦-垂體-腎上腺軸活化在其中的效應(yīng)機(jī)制。研究結(jié)果進(jìn)一步豐富了對廣州管圓線蟲病致病機(jī)制的認(rèn)識,也對指導(dǎo)廣州管圓線蟲病的臨床治療,提供了重要的參考依據(jù)。
[Abstract]:Guangzhou tube nematode is an important pathogen causing eosinophilic meningitis and meningoencephalitis. Human is an unsuitable host of the Guangzhou tube nematode, which infects the intermediate host of the third stage larva of the Guangzhou tube nematode. The infective larvae can pass through the intestinal wall and enter the blood circulation system and move with the blood flow in the body. A neurotropic, multiple invasion of the central nervous system. The disease is characterized by a rise in eosinophils in the cerebrospinal fluid, with the exception of the brain and meninges in the cerebellum, brain stem, spinal cord, or eyeball. The main lesions are cerebral congestion, bleeding, mechanical damage to the brain and granuloma in the brain. In recent years, the central Lodge of the Guangzhou nematode nematode The main species of snail and agate in the southern part of China are propagated in the southern part of our country, and people are addicted to living and hunting, resulting in the outbreak or epidemic of the disease in Guangzhou. The disease has been included in the new infectious disease. The inflammatory response can lead to the aggravation of the nervous system symptoms. Therefore, it is of great significance to understand the immune response mechanism of the Guangzhou tube nematode infection to the disease of Guangzhou. The study shows that the central nervous system and the immune system interact in a complex way. In the ideal case, the stress bar is ideal. Inflammatory and anti-inflammatory responses are balanced, conducive to healing and containment of the pathogen, while preventing excessive inflammation or severe immunosuppression. However, in the absence of systemic inflammation, the anti-inflammatory response triggered by local inflammatory cytokine proliferation may be harmful in the absence of systemic inflammation, as this will reduce the body's prevention. It has been found that the central nervous system injury has a profound effect on the immune function, such as stroke, traumatic brain injury or spinal cord injury, all of the patients show impaired immune function, including the decrease in the number of lymphocytes in the peripheral blood, the atrophy of the spleen and thymus, and the damage of T cell activity. It was found that the expression of major histocompatibility complex II molecules in the circulating monocytes was downregulated in the patients with acute central nervous system damage, and the ability to stimulate monocytes in vitro was greatly reduced. The clinical manifestation of this immunosuppression was the high incidence of systemic infection, especially in the lungs, after the injury of the brain. The results of these studies suggest that brain damage can lead to peripheral immunosuppression. The results of these studies suggest that brain damage can lead to peripheral immunosuppression. The larvae of Guangzhou's tube nematode can be moved to the brain after invasion to the brain, causing mechanical damage to brain tissue, occupying and releasing metabolites to damage the central nervous system. Based on brain damage and The close link of peripheral immunosuppression, we propose the following hypothesis: Guangzhou tube nematode larvae invade the brain damage to the central nervous system and cause local inflammatory cytokine diffusion to stimulate the peripheral immune suppression and increase the opportunity for secondary infection of the body. In order to verify the hypothesis, the mouse Guangzhou tube nematode In the infection model, we first observed the changes in the thymus and spleen of the immune organs of Guangzhou, and evaluated the changes in the number and function of the peripheral lymphocyte subsets. Secondly, we explored the number of peripheral lymphocytes after the two aspects of the apoptosis and the obstruction of the cell development in Guangzhou. Again, we studied the expression of inflammatory cytokine, anti-inflammatory cytokine and chemokine in the brain tissue of Guangzhou, and observed the changes in the hypothalamus pituitary adrenal (HPA) axis of the central nervous system and the peripheral immune system. Finally, we confirmed that the central nervous system of Guangzhou tube nematode was infected with the central nervous system. The main results were as follows: 1. the thymus and spleen of mice were obviously atrophied and the number of mononuclear cells decreased in 21 days after the infection induced atrophy of the immune organs and the number of lymphocytes in Guangzhou, and the number of mononuclear cells was also reduced, suggesting the infection of Guangzhou tube nematode. The immune organs of mice were affected. In order to investigate the causes of the atrophy of the thymus and spleen after infection, we detected the changes in the spleen and peripheral blood cell subsets in mice by flow cytometry. The results showed that the number of B cells, T cells, CD4+T cells, CD8+T, Thl and NK cells in the spleen and peripheral blood after infection was significantly higher than that of the control group. .2. Guangzhou tube nematode infection reduces cellular and humoral immunity in order to prove that the immune function of lymphocyte is also affected by the infection of T. We further study the proliferation of T lymphocytes and the ability to secrete cytokines and B cells to produce antibodies. The results show that the infected mouse T cells are infected after infection. The ability of proliferation was lower than that of the control group; using mitogen stimulated peripheral blood cells, the result of ELISA detection in the culture supernatant showed that the infection of Guangzhou tube nematode induced low level of IFN- gamma, TNF- alpha and high level IL-4, and the non specific antigen OVA immunized the infected mice and the control mice of the Guangzhou tube nematode, and detected the OVA-Ig. The level of Gl antibody expression. The results showed that the level of OVA-IgGl antibody in the mice after infection was lower than that of the control mice. It suggested that the infection of Guangzhou tube nematode decreased the ability to produce antibodies in mouse B cells. In order to further confirm the results, we observed the pathology of the lungs and the blood culture of mice. The results showed that the results showed that the mice were cultured. A large number of inflammatory cells were infiltrated in the lungs of the mice after infection, showing pneumonia. The same blood on the blood agar plate showed infection after infection, indicating that the infection of Guangzhou tube nematode induced the immune suppression of.3. Guangzhou tube nematode infection enhanced NK cell killing function because Guangzhou tube nematode infection reduced cell immunity The immune function of pestilence and humoral immunity increased the risk of infection in mice, so we further evaluated the effect of Guangzhou tube nematode infection on NK cell function. We stimulated 24 h by adding or without exogenous IL-12, and ELISA was used to detect the expression level of IFN- gamma and TNF- alpha in the culture supernatant. The results showed that IL-12 treatment was the result of IL-12 treatment. The infected mouse NK cells produced a higher level of IFN- gamma than the control mice. No matter whether there were exogenous IL-12 or not, the infected mice and the control mice of the Guangzhou tube could only induce the low level of TNF- beta in the NK cells. Further we evaluated the effect of infection on the cytotoxicity of NK cells. The results showed that the infected NK cells were compared with the control group. These results suggest that the infection of Guangzhou tube nematode, although the number of NK cells decreased, increased the killing function of NK cells,.4., and the decrease of B and T cells after the infection of Guangzhou tube nematode is not caused by apoptosis, but the number of B and T cells after a large number of apoptotic infection after the infection of NK cells is the number of NK cells. In order to further clarify whether the cell number decline was caused by apoptosis, we detected the proportion of 7AAD-AnnexinV+ on the surface of these cells by flow cytometry. The results showed that, compared with the control group, the 7AAD "Annexin V+ ratio" on the surface of the infected cells did not increase.Caspase 3 as the ultimate executor of apoptosis, and was a heavy detection of apoptosis. To further confirm the results, B220- cells and CD3- cells were sorted with magnetic beads. The expression of barium Cleaved Caspase 3 was detected by estern blot. The results showed that the expression of Cleaved Caspase 3 was not detected after infection, and the number of B cells and T cells decreased after the infection of Guangzhou tube nematode. It was not caused by apoptosis. Similarly, to determine whether the number of NK cells decreased by apoptosis, we detected the proportion of 7AAD "Annexin V+" on the surface of NK cells and the separation of DX5+ cells by magnetic beads, and the expression of Cleaved Caspase 3 with estern blot. The results showed that the infection was compared with the control group. The expression of 7AAD- Annexin V+ and Cleaved Caspase 3 on the surface of the post NK cells increased, suggesting that the decrease of NK number was associated with the decrease of NK number and the apoptosis of NK cell, which inhibited the occurrence of B cells in the bone marrow and the damage of T cells in the thymus gland, and the decrease of the number of B cells after the development of the thymus in the thymus gland. It is due to cell apoptosis, we speculate whether or not all or part of the developing B cell subsets are lost during the development of B cells. In order to verify this conjecture, we detected the proportion of B cells in the development of bone marrow. The results showed that the proportion of B cells in the development of bone marrow after infection was lower than that of the control group. Further, we analyzed it. Two subgroups of B cells in the development of bone marrow: pro-/pre-B cells and immature B cells. The results showed that the proportion of pro-/pre-B cells in the bone marrow after infection was significantly lower than that of the control group, while the proportion of immature B cells in the bone marrow after infection accounted for the increase of the proportion of B cells in the developmental B cells, suggesting that the infection of Guangzhou tube nematode was caused by infection. B cells decline in the development of bone marrow, which is mainly affected by pro-/pre-B cells. We know that after the rearrangement of the Ig H chain and the L chain gene, the developing B cells in the bone marrow enter the immature B cell stage and then migrate to the peripheral B cells in the transitional period. We have detected the B cells and maturation of the spleen in the development of the spleen. The proportion of B cells showed that the mature B cells decreased at 21 days after infection. At the same time, the proportion of B cells in the development of spleen was significantly lower than that of the control group. Further, we analyzed the 3 subgroups of the developing B cells: TR1, TR2 and TR3 subgroups. The results showed that the proportion of TR1 in the developing B cells gradually reduced.TR2 with the increase of the infection time. The proportion of TR3 to the developing B cells increased with the increase of the time of infection. It suggests that the infection of Guangzhou tube nematode leads to the decrease of B cells in the development of the spleen, which is mainly affected by the TR1 cell subgroup. The above results suggest that the output of B cells is affected by the infection of the Guangzhou tube nematode, which leads to the decline of the output of B cells. Guangzhou tube The decrease in the number of T cells after the infection of the nematode is also not caused by apoptosis, and there is a serious atrophy in the thymus after infection. We speculate whether the T cells are blocked in the development of the thymus. Therefore, we detected the thymus T cell subgroup by flow cytometry, and the result showed that the proportion of CD4+CD8+T cells after infection was lower than that of the control group. On the contrary, the proportion of CD4-CD8-T cells, CD4+CD8-T cells and CD4-CD8+T cells increased significantly with the increase of infection time. Further we observed the number of T cell subgroups in the thymus. The results showed that the number of CD4CD8T cells and CD4 "CD8+T cells" after infection was not different from that of the control group; but after infection, CD4+CD was found to be CD4+CD. The number of 8+T cells was significantly lower than that in the control group. In order to further clarify whether the number of cells decreased by apoptosis, we detected the expression of 7AAD "Annexin V+" on the surface of CD4+CD8+T cells by flow cytometry. The results showed that the proportion of 7AAD-Annexin V+ on the surface of CD4+CD8+T cells after infection was gradually increased compared with the control group. The results of the above results suggest that the ratio of 7AAD-Annexin V+ to the surface of CD4+CD8+T cells after infection is gradually increased. The decrease of the number of thymus cells and the number of peripheral T cells and the decrease of the number of peripheral T cells and the decrease of CD4+CD8+T cell apoptosis after the infection of the Caenorhabditis elegans in Guangzhou, the decrease of the number of lymphocytes and the activation of the hypothalamus pituitary adrenal axis in the brain, and the invasion of the nervous system by the activation of the larva of the hypothalamus pituitary adrenal axis of the Guangzhou tube nematode. In order to determine whether the decrease of the number of lymphocytes caused by the infection of the nematode in Guangzhou was caused by brain damage, we used albendazole to observe the influence of the peripheral lymphocyte subsets after infection. The results showed that the size of the thymus and spleen in the treatment group was more than that of the normal control group 21 days after the infection. There was no significant difference. The proportion of B lymphocyte, T cell, CD4+T cell, CD8+T cell and NK cell of spleen lymphocyte subgroup after infection were higher.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R532.1;R741
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本文編號：2119022