摘要
研究背景：
新生隐球菌是临床上重要的致病真菌，它可引起致命的系统性感染，目前对其致病机理还不很清楚，且尚无非常有效的治疗方法。现研究表明巨噬细胞是抗新生隐球菌第一线反应细胞，对新生隐球菌有吞噬、杀菌、抗原提呈等作用，而新生隐球菌本身存在着很强的毒性因子，起到逃避宿主免疫的作用，因而能在巨噬细胞内寄生。但新生隐球菌逃避宿主免疫反应的机制是复杂的，研究人员仍在努力探索新生隐球菌潜在的可能毒性因子，以期对新生隐球菌的致病机制和临床上治疗新生隐球菌病有个重大突破。本课题共分四部分来研究新生隐球菌在被巨噬细胞吞噬前后的存活力及基因表达差异，探讨新生隐球菌被巨噬细胞吞噬后的出芽繁殖能力、凋亡率和细胞周期的变化、基因表达差异，这将有助于探讨巨噬细胞对新生隐球菌的吞噬、杀菌作用机制，以及新生隐球菌逃避宿主免疫的可能机制，寻找潜在的可能毒性基因，了解新生隐球菌的致病机制。
研究目的：
第一部分：分析巨噬细胞J774.16对新生隐球菌野生株B3501、荚膜突变株Cap60及白化株Mel-的存活力和出芽繁殖能力的影响。
第二部分：分析巨噬细胞J774.16对新生隐球菌野生株B3501、荚膜突变株Cap60及白化株Mel-的凋亡和细胞周期的影响。
第三部分：研究巨噬细胞对新生隐球菌野生株B3501的基因表达影响。
第四部分：研究巨噬细胞对新生隐球菌野生株B3501的主要毒性基因表达影响。
研究方法：
第一部分：将对数生长期的J774.16细胞分别与新生隐球菌野生株B3501、荚膜缺陷株Cap60、白化株Mel-按细胞：菌=1：10共孵育，通过Giemsa染色观察J774.16细胞在共孵育时间为1，2，4，8小时对新生隐球菌的吞噬指数变化，以及新生隐球菌在上述共孵育时间在J774.16细胞内的出芽率：通过电子显微镜观察新生隐球菌在J774.16内的形态。
博士论文
摘要
第二部分:将对数生长期的J774.16细胞分别与新生隐球菌野生株B3501、
荚膜缺陷株Cap60、白化株Mel一按细胞:菌=1:10共孵育，通过流式细胞仪检测
J774.16内、共孵育的J774.16细胞外及对照组新生隐球菌的凋亡率和细胞增殖指
数变化。
第三部分:将对数生长期的J774.16细胞与新生隐球菌野生株B3501共孵育
至4小时，收集被J774.16吞噬的B3501作为实验组，提取实验组和37℃，5%C02
单独培养的对照组B3501的RNA，采用酿母基因芯片598进行mRNA转录差异
筛选。
第四部分:将对数生长期的J774.16细胞分别与新生隐球菌野生株B3501共
孵育至4小时，收集被J774.16吞噬的B3501作为实验组，提取实验组和37℃，
5%CO:单独培养的对照组B3501的RNA，采用实时荧光定量PCR技术，检测
J774.16细胞内和对照组B35OI的CNLACI、CAP60、UREI、NMT表达的差异。
结果:
第一部分:经Giemsa染色，可清楚看到J774.16细胞内吞有被染呈紫蓝色
的新生隐球菌。Giemsa染色和电镜结果均发现一个J774.16细胞一般仅吞噬一个
新生隐球菌野生株B3501，而一个J774.16细胞可吞噬多个Cap60和Mel一突变株;
随共孵育时间的延长，J774.16巨噬细胞对B3501和Cap60的吞噬指数没有明显
的提高(P>0.5)，吞噬指数分别在5.67士1.29%一8.76士3.09%和31.62士 12.70%一
41.86士9.74%之间。Mel一株在共孵育4hrs时内，吞噬率迅速提高(P<0.05)，后
趋于稳定(P>0.05)，J774.16对细胞Mel一株的吞噬指数在0.36士0.05一1.24士0.21
之间。
B3501菌在J774.16细胞内的出芽率较高，可达46.85士6.63%，从共孵育时
间达4 hrs后(P<0.01)，出芽率开始下降;C叩60和Mel一株的出芽率较低，最高
分别仅为10.73士0.39%和8.44士1.28%，并且出芽率在共孵育时间分别为Zhrs和
4hrs后开始下降
第二部分:各新生隐球菌对照组、细胞外和细胞内菌体的凋亡率分别为
Bl=0 .5%、BZ=1 0.7%和B3=1 2.8%:CI=8.1%、CZ=1 1 .1%和C3=35.6%:Ml=15.4%、
MZ=25.6%和M3=38.2%。增殖指数分别为BI=79.4%、BZ=73.5%和B3=64.7%;
博士论文
摘要
CI=74.0%、CZ=61.4%和C3=60.0%;MI=72.0%、MZ=55.1%和M3=20.9%。
第三部分:被巨噬细胞吞噬后，新生隐球菌上调的基因有25条，下调的基
因有100多条;上调的基因中主要有腺昔激酶(ADKI)、腺普环化酶(CYRI)、
PeP4p蛋白酶抑制剂(PAI3)、Cl一四氢叶酸合成酶(MSll)等基因，前两者与细
胞周期和增殖有关;下调的基因主要有Manganese一traffieking protein(ATXZ)、
High一affinity zine transport Protein(ZRTI)及Putative low一affinity eopper transport
protein(CTRZ)基因，这三者分别与锰(Mn)、锌(Zn)和铜(Cu)离子的转运
有关。与能量代谢有关的putative hexose permease(HXT12)、磷酸丙酮酸水合
酶(ENOI)、丙酮酸脱梭酶(PDCI)、乙醇脱氢酶(ADHI)表达也下降。还有
与酵母出芽繁殖有关的Meiosis一speeifie protein required for spore formation(xselo)
基因也表达下降。另外，还有一些功能未尚明确的基因表达也发生变化。
第四部分:实验组中新生隐球菌的CAP60、CNLACI、NMT及UREI基因
的mRNA在每百万看家基因(GAPDH基因)中的平均含量分别为2.70E+04、
l.slE+03、2.27E+04和4.O4E+04;而?
Background:
Cryptococcus neoformans is important pathogenic fungus that could cause fatal systemic disease (i.e. cryptococcosis) in clinic. At present, the pathogenesis of cryptococcosis is still not clear, and there is not a very effective treatment for cryptococcosis. We knew that the macrophage is the first line immune cell for anti-cryptococcosis. It could phagocytise and kill Cryptococcus neoformans cells, and present the antigen to the other effective immune cells. But the Cryptococcus neoformans posseses strong virulence factors that could help the pathogen escape and survive from the host immune reaction. At another side, the anti-kill mechanism of Cryptococcus neoformans is very sophisticated. So it is very important to study of the potential virulence factors of Cryptococcus neoformans for understanding and treatment for cryptococcosis. The four parts of our experiment tried to detect the change of viability and mRNA expression of Cryptococcus neoformans after phagocytosis by macrophage, to implore the differe
nce of budding, apoptosis, cell cycle, mRNA expression of Cryptococcus neoformans. This study could help us understand the mechanism of phagocytising, killing Cryptococcus neoformans of macrophage, escaping from host immune system, and finding of the potential virulence factors.
Objective:
Part I: Study of the effect of mouse macrophage line J774.16 on the survival and budding of Cryptococcus noeformans wild strain B3501, capsule depletion strain Cap60, and melanin depletion strain Mel-.
Part II: Study of the effect of mouse macrophage line J774.16 on the apoptosis and cell cycle of Cryptococcus noeformans wild strain B3501, capsule depletion strain Cap60, and melanin depletion strain Mel-.
Part : Study of the difference of mRNA expression of Cryptococcus neoformans wild strain B3501 after phagocytosis by macrophage J774.16.
Part IV: Study of the difference of main virulence genes mRNA expression of Cryptococcus neoformans wild strain B3501 after phagocytosis by macrophage J774.16
Method:
Part I: Co-cultured the logarithmic growing J774.16 with Cryptococcus noeformans wild strain B3501, capsule depletion strain Cap60, and melanin depletion strain Mel- at the rate of macrophage : yeast=1:10. respectively. Detected the phagocytic index of the macrophage J774.16 to Cryptococcus neoformans and the budding rate of the Cryptococcus neoformans inside the macrophage J774.16 by Giemsa staining at co-cultured time 1.2, 4, Shours. Inspected the ultrastructure of the yeasts inside the macrophage J774.16 by electron microscopy.
Part : Co-cultured the logarithmic growing J774.16 with Cryptococcus noeformans wild strain B3501, capsule depletion strain Cap60, and melanin depletion strain Mel- at the ratio of macrophage : yeast=1:10, respectively. Detected the apoptosis and proliferating index (cell cycle) of the macrophage J774.16 ingested yeasts, un-ingested (but co-cultured) macrophage J774.16 yeasts, and control group (solely cultured at 37, 5%CO2) yeasts.
Part : Co-cultured the logarithmic growing J774.16 with Cryptococcus noeformans wild strain B3501. collected the macrophage J774.16 ingested yeast at co-cultured 4 hours and control group(solely cultured at 37, 5%CO2) yeast. Then their total RNA was extracted for screening the difference of the mRNA expression by gene microarray analysis.
Part : Co-cultured the logarithmic growing J774.16 with Cryptococcus noeformans wild strain B3501. collected the macrophage J774.16 ingested yeast at co-cultured 4 hours and control group(solely cultured at 37, 5%CO2) yeast. Then their total RNA was extracted for screening the difference of main virulence factor gene CNLAC1, CAP60, URE, and NMT mRNA expression by real-time PCR.
Results:
Part I: Royal blue yeast is very clear inside the macrophage J774.16 cell by Giemsa staining. The finding of Giemsa and electron microscopy both showed that on macrophage J774.16 cell phagocytsied on B3501 strain yeast cell, and one macrophage J774.16 cell could phagocytise multiple strain Cap60 and Mel- yeasts' cells. Elongating
引文
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