The Experimental Study Of Indomethacin Inhibiting The Hypha Formation Of Candida Albicans

Candida albicans is an opportunistic dimorphic fungus that inhabits the normal human mucosal surfaces. Recently, some researches identified that the morphogenesis of Candida albicans is close correlative with its pathogenicity. Hypha can not only enhance the ability of adhesives and invasion to the mucosa , but also help it succeed in escaping the attack of human immune system. The experiment in vitro already confirmed that many enviroment factors can induce hypha formation through the different or congenerous pathway . In the condition of inducing hypha formation, seeking an drug of inhibiting the hypha formation will be a hot spot of studying anti-candida-albicans drug.Part I The Condition of Hypha Formation of Candida Albicans and the Quantu Analysis Method of Hypha FormationIn part I, RPMI 1640 liquid medium contained 10% calf serum and corn-tween 80 solid medium was used to induce hypha formation of candida albicans ; to observe the hypha formation from morphology through lacto-phenol cotton blue-stains; to quantu analysis hypha formation through crystal-violet hypha stained. The results showed that masses of hypha formed in serum-induced RPMI1640 liquid medium and above 96% hypha formed( quantu ); in the corn-tween80 solid medium, chlamydospore and pseudomycelium formation was evident.Part II PGE₂ Induces hypha formation of candida albicans and anti-PGE₂ Antibody repress its'effectIn this part , To measure the hypha formation in the RPMI1640 liquid medium contained and uncontained 10% calf-serum when added in exogenous PGE₂; and when added in anti-PGE₂ antibody. The result indicated that PGE₂ can enhance hypha formation; and when added in 300 pg/ml, its action is correspondent with 10% calf-serum,while added in anti-PGE₂ antibody simultaneously, it repressed the effect of PGE₂ on hypha formation of candida albicans, and the rate of hypha formation reduced 40%; It is more important that anti-PGE₂ antibody also repressed the effect of serum factors(non PGE₂) on the hypha formation, and the rate of hypha formation reduced 42%. It implies that PGE₂ may be one of the serum factors.Part III The Effect of Indomethacin on Hyphal Formation of Candida Albicans and Its Mechanism ResearchExperiment one The Effect of Indomethacin on the Hypha Formation in the Liquid MediumIn this experiment, to observe the hypha formation of three strain candida albicans after different concentration Indomethacin was added in RPMI1640 contained 10% calf serum in microscope (x400), and to analysis the percent rate of hypha formation with crystal-violet germ tube assay. The result showed Indomethacin inhibited the formation of germ tube and hypha of candida albicans by dose dependence: indomethacin can inhibit 50% germ tube formation at 0.75mM level, while it can fully inhibit germ tube formation at 2.0mM level.Experiment two The Effect of Indomethacin on the Hypha Formation in the chlamydospores'testThis experiment is to investigate the Chlamydospores and pseudomycelium after different concentration Indomethacin was added in corn-tween80. The results indicated that indomethacin can inhibit the formation of pseudomycelium and chlamydospores at 1.0mM level, and no pseudomycelium formed at 2.0mM level.Experiment three The Effect of Indomethacin on Hypha Formation when Exogenous PGE₂ Is PresentIn this experiment, to study the effect of Indomethacin on hypha formation after Indomethacin (2.0mM) and exogenous PGE₂ (300pg/ml) were added in RPMI1640 with crystal violet-based germ tube assay and to further interpret the mechanism of Indomethacin inhibiting hypha formation. The results showed that Indomethacin can still inhibited the hypha formation in the present of exogenous PGE₂.Brief summaryThe results of the above-mentioned three part suggested : Indomethacin can inhibit the hypha formation of candida albicans in serum-RPMI1640 medium(cAMP-Efg1 pathway) ,corn-tween80 medium(CZF1 pathway) and in the present of exogenous PGE₂. This results showed that the mechanism of Indomethacin inhibiting hyphal formation is multielement, except inhibiting the production of PGE₂ and then reducing the signal molecul of enhancing hyphal formation. It is more important that Indomethacin can inhibit the hyphal formation directly. So it is potential that this drug will be used to therapy the vaginal candidiasis .