Influence Of Macrophage On The Viability And MRNA Expression Of Cryptococcus Neoformans

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 difference 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 J774.16Method: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...