本文關(guān)鍵詞：氧含量對(duì)新生隱球菌形態(tài)學(xué)及毒力因子影響的初步研究　出處：《南京大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 新生隱球菌 氧含量 莢膜 黑素 尿素酶 磷脂酶

【摘要】：背景和目的 新生隱球菌(C.neoformans)在環(huán)境中廣泛存在,是單倍雌雄異株的擔(dān)子菌類機(jī)會(huì)性致病性真菌,它是一種重要的能導(dǎo)致系統(tǒng)感染的病原真菌,主要侵犯AIDS、器官移植、細(xì)胞毒藥物化療和皮質(zhì)類固醇長(zhǎng)期應(yīng)用等造成的免疫缺陷患者,也可感染免疫功能正常的人群,主要導(dǎo)致致命性的腦膜腦炎,感染死亡率很高,而且這種感染會(huì)造成慢性化或潛伏感染狀態(tài)。C.neoformans主要的致病因素為莢膜、產(chǎn)黑素、尿素酶和磷脂酶的毒性作用及其在宿主體溫環(huán)境生長(zhǎng)的能力等。 C.neoformans是專性需氧致病酵母菌,在實(shí)驗(yàn)室條件下,新生隱球菌在大氣氧含量(21%)條件下生長(zhǎng)最佳。吸入的C.neoformans可以通過血液播散到達(dá)中樞神經(jīng)系統(tǒng)主要導(dǎo)致致命性的腦膜腦炎。眾所周知,人腦組織的氧濃度明顯低于大氣氧含量(21%),不同的解剖部位也不同。此外,炎癥、血栓形成以及感染相關(guān)的壞死也導(dǎo)致組織氧含量下降。因此,C.neoformans在致病過程中必須面對(duì)并適應(yīng)宿主組織氧含量的變化。國(guó)內(nèi)外最新研究認(rèn)為C.neoformans這種專性需氧致病酵母菌可以適應(yīng)缺氧環(huán)境并生存,并且在中樞神經(jīng)系統(tǒng)的生長(zhǎng)在于調(diào)整其復(fù)制速率以適應(yīng)相應(yīng)的氧含量的變化,并描述了C.neoformans兩種可能感應(yīng)缺氧的途徑,即SREBP(甾醇調(diào)節(jié)因子連接蛋白家族)和Tco1途徑,但缺氧適應(yīng)對(duì)C.neoformans毒性因子的影響尚未被關(guān)注。我們前期研究中發(fā)現(xiàn)新生隱球菌在相對(duì)缺氧組織中莢膜生成缺陷,因此我們提出假設(shè)：缺氧可能導(dǎo)致新生隱球菌重要致病表型發(fā)生變化,使新生隱球菌在宿主中進(jìn)入慢性或潛伏感染狀態(tài),導(dǎo)致感染遷延不愈。 本研究通過建立不同氧含量的培養(yǎng)條件,探討氧含量變化對(duì)C.neoformans毒力因子的影響,以期為揭示缺氧在新生隱球菌導(dǎo)致潛伏感染或慢性化感染的機(jī)制中的作用提供新思路。 方法本研究以厭氧、微需氧和二氧化碳產(chǎn)氣袋建立不同氧含量培養(yǎng)條件,以大氣條件為正常氧含量培養(yǎng)條件,以新生隱球菌標(biāo)準(zhǔn)株5株(BLS71、BLS63、ATCC32609、ATCC3487、YD53,分別為血清型A、B、C、D、AD),臨床株1株為研究對(duì)象,利用形態(tài)學(xué)觀察培養(yǎng)基(YPD和YES)、產(chǎn)黑素培養(yǎng)基、尿素酶培養(yǎng)基和卵黃培養(yǎng)基等對(duì)不同菌株進(jìn)行培養(yǎng),通過觀測(cè)菌落直徑、莢膜厚度、產(chǎn)黑培養(yǎng)基和尿素酶培養(yǎng)基顏色變化情況以及卵黃培養(yǎng)基中的PZ值等指標(biāo),探討不同氧含量培養(yǎng)條件對(duì)新生隱球菌毒力因子的影響。 結(jié)果 1.形態(tài)學(xué)結(jié)果 各個(gè)菌株在不同氧含量條件下均有生長(zhǎng),正常氧含量條件下菌落生長(zhǎng)最好,受試菌株菌落生長(zhǎng)速度隨氧含量的遞減而減慢,所有菌株厭氧條件下生長(zhǎng)明顯減慢,未出現(xiàn)明顯的乳白色酵母樣菌落,菌落直徑比其他三組明顯減小。同種培養(yǎng)條件下各個(gè)菌株之間菌落直徑無(wú)明顯差異。YPD和YES兩種培養(yǎng)基對(duì)隱球菌生長(zhǎng)無(wú)明顯影響,菌落大小基本一致。 2.產(chǎn)黑素結(jié)果 在多巴培養(yǎng)基中,3天時(shí)正常氧、二氧化碳及微含氧培養(yǎng)條件下的所有受試菌株均使整個(gè)培養(yǎng)基變?yōu)樯詈稚?厭氧條件下培養(yǎng)基顏色未出現(xiàn)明顯變化,7天時(shí)培養(yǎng)基顏色變化程度比3天時(shí)加深。在咖啡酸培養(yǎng)基中,3天時(shí)除正常氧條件下所有受試菌株菌落及周圍出現(xiàn)咖啡色外,其余條件下均未出現(xiàn)顏色變化,7天時(shí)結(jié)果與3天基本一致。 3.莢膜測(cè)量結(jié)果 除菌株YD53(血清型AD)外,其余受試菌株均可形成明顯的莢膜。YD53在同樣培養(yǎng)條件下,大多以假菌絲樣形式生長(zhǎng)。 在非誘導(dǎo)培養(yǎng)條件下,菌株BLS71、BLS63、ATCC32609和臨床株在四種氧含量培養(yǎng)條件下莢膜大小差異有統(tǒng)計(jì)學(xué)意義。在誘導(dǎo)培養(yǎng)條件下,菌株BLS71、BLS63和臨床株在厭氧培養(yǎng)條件下的莢膜厚度比其余氧含量條件下的莢膜明顯變小。在所有氧含量培養(yǎng)條件下,菌株BLS71和臨床株誘導(dǎo)條件下莢膜比非誘導(dǎo)條件下莢膜大,差異有統(tǒng)計(jì)學(xué)意義。 4.尿素酶和磷脂酶活性 培養(yǎng)7天時(shí),正常氧含量培養(yǎng)條件下除菌株ATCC32609未出現(xiàn)顏色變化,其他菌株均出現(xiàn)不同程度的顏色變化,微含氧和厭氧培養(yǎng)條件下所有受試菌株均未出現(xiàn)顏色變化；將觀察時(shí)間延長(zhǎng)至14天,可見正常氧含量及二氧化碳培養(yǎng)條件下所有受試菌株都出現(xiàn)不同程度的顏色變化,而厭氧組培養(yǎng)條件下均未出現(xiàn)顏色變化。 培養(yǎng)7天時(shí),除厭氧培養(yǎng)條件下受試菌株未出現(xiàn)沉淀圈外,其余培養(yǎng)條件下的所有受試菌株均出現(xiàn)了特異性的白色沉淀圈。菌株BLS71和ATCC34874在正常氧培養(yǎng)條件下PZ值小于其他培養(yǎng)條件,差異有統(tǒng)計(jì)學(xué)意義。同一菌株培養(yǎng)條件隨氧含量的降低,PZ值有增大趨勢(shì)。同一種培養(yǎng)條件下受試菌株P(guān)Z值差異無(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論 新生隱球菌可以在厭氧條件下生長(zhǎng),低氧可能對(duì)新生隱球菌莢膜、產(chǎn)黑素、尿素酶和磷脂酶活性具有抑制作用。不同血清型的新生隱球菌菌株對(duì)氧含量變化的反應(yīng)可能不同。
[Abstract]:Background and purpose
Cryptococcus neoformans (C.neoformans) exists widely in the environment, is a heterothallic basidiomycete opportunistic pathogenic fungi, it is a kind of important fungal pathogens can lead to infection, mainly involving AIDS, organ transplantation, caused by cytotoxic chemotherapy and long-term corticosteroid application in immunodeficient patients, can be infected people with normal immune function, which lead to fatal encephalitis infection, the mortality rate is very high, and the infection can cause chronic or latent infection of the main pathogenic factors for.C.neoformans capsule, melanin production, toxicity of urease and phospholipase and temperature in the host environment for the growth of ability.
C.neoformans is an obligate aerobic pathogenic yeast, under laboratory conditions, Cryptococcus neoformans in atmospheric oxygen content (21%) under the conditions of growth of the best. Inhalation of C.neoformans can reach the central nervous system through the blood spread mainly cause life-threatening meningoencephalitis. As everyone knows, the oxygen concentration of brain tissue was significantly lower than the atmospheric oxygen content (21%), anatomy the different parts are different. In addition, inflammation, thrombosis and necrosis associated with infection also leads to decreased oxygen content. Therefore, C.neoformans must face and adapt to changes in the oxygen content of the host tissue in the pathogenic process at home and abroad. According to the latest research from C.neoformans this obligate aerobic pathogenic yeast can adapt to hypoxic environment and survival, and is adjust the replication rate to adapt to changes in the oxygen content of the corresponding growth in the central nervous system, and describes two possible C.neoformans Way of induction of hypoxia, namely SREBP (sterol regulatory element junction protein family) and Tco1 pathway, but the effect of preconditioning on C.neoformans virulence factors have not been concerned. Cryptococcus neoformans in relative hypoxia tissue defects generated in our previous study found that the capsule, so we hypothesized that hypoxia may lead to important pathogenic phenotype of Cryptococcus neoformans the change of Cryptococcus neoformans in the host into chronic or latent infection, leading to delayed healing of infection.
This study through the establishment of different culture conditions on oxygen content, oxygen content effect on C.neoformans virulence factors, in order to reveal hypoxia in Cryptococcus neoformans cause latent infection or chronic infection mechanism to provide new ideas.
Methods in this study, the anaerobic, microaerophilic and carbon dioxide gas generating bag to establish culture conditions with different oxygen content, with atmospheric conditions for cultivating conditions of normal oxygen content, with 5 strains of Cryptococcus neoformans strains (BLS71, BLS63, ATCC32609, ATCC3487, YD53, respectively, serotype A, B, C, D, AD), clinical 1 strains as the research object, using the morphological observation of culture medium (YPD and YES), producing melanin medium, urease medium and egg yolk medium of different strains were cultured by observing colony diameter, the thickness of the capsule, black culture medium and culture medium and color change of urease yolk medium PZ value, to investigate the effect of culture conditions on Cryptococcus neoformans virulence factors with different oxygen content.
Result
1. morphological results
All the strains in the conditions of different oxygen contents were normal growth, oxygen content under the condition of colony growth, the tested strains colony growth rate with decreasing oxygen content decreased, the growth slowed down all the strains under anaerobic conditions, no obvious white yeast like colonies, the colony diameter was significantly reduced than in the other three groups of the same culture. Under the condition of each strain between colony diameter had no significant difference in.YPD and YES two kinds of medium on the growth of Cryptococcus neoformans had no obvious effect on colony size is basically the same.
2. production of melanin
In DOPA medium, normal oxygen for 3 days, carbon dioxide and oxygen micro culture all isolates were the medium to dark brown color of the medium conditions, there was no significant change in culture under anaerobic conditions, the 7 day training degree based color change 3 days than in the coffee acid burn. The medium for 3 days in addition to the normal oxygen condition of all the subjects appear Brown colony and around, under other conditions there were no color change, 7 days and 3 days were basically the same.
3. capsular measurement results
Except for the bacteria strain YD53 (serotype AD), the other tested strains could form a distinct capsule.YD53 under the same culture conditions, mostly in the form of false mycelium like growth.
Under non inducing conditions, strain BLS71, BLS63, ATCC32609 and clinical isolates in four kinds of oxygen content in culture has significant difference in size of capsular condition. Under the condition of strain BLS71 cultured BLS63 and clinical strains in anaerobic culture conditions than the rest of the thickness of the capsule of oxygen under the condition of the capsule was significantly smaller in all culture conditions. The oxygen content, strain BLS71 and strain induced clinical conditions than non capsular inducing conditions capsule, the difference was statistically significant.
4. activity of urease and phospholipase
When cultured for 7 days, normal oxygen culture conditions except strain ATCC32609 without color change, other strains have different degrees of color change, containing micro aerobic and anaerobic culture conditions of all subjects were not changed color strains; the observing time is extended to 14 days, all the tested strains have different degrees of the color change under normal oxygen and carbon dioxide visible and anaerobic culture conditions, group culture conditions showed no color change.
When cultured for 7 days, in addition to anaerobic culture conditions tested strains did not appear precipitation outside the ring, the culture conditions of all tested strains showed a white precipitate ring specific. Strains BLS71 and ATCC34874 culture conditions, PZ value is less than the other culture conditions in normal oxygen, statistically significant differences between the same strain. Culture conditions with oxygen content decreased, PZ value increased. The same culture conditions tested strains PZ were no significant difference.
conclusion
Cryptococcus neoformans can grow under anaerobic conditions. Hypoxia may inhibit the production of melanin, urease and phospholipase activity in Cryptococcus neoformans. The reactions of different serotypes of Cryptococcus neoformans to oxygen content may be different.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R379

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本文編號(hào)：1358573