| In this study, the various media without feeder cells were investigated to establish a system of continuous axenic culture of P.carinii. A culture system was achieved and applicated for assessing the effect of daphnetin on the growth of P. carinii in vitro. Some conclusions can be deducted as following.1. A continous axenic culture of P. carinii in vitro has been achieved. Having investigated the media,such as YPAD, YEA, NPG, RPMI1640, IMDM and tested various conditions of supporting growth of P. carinii, we found that the growth medium based on IMDM with Earle's salt, which is supplemented with s-adenosyl-L-methionine (50μg/ml), N-acetylglucosamine (100mg/L), putrescine(80mg/L), L-cysteine(50mg/L), L-glutamine(50 mg/L), 2-mercapto ethanol(5x10-5mol/L), and 15% newborn bovine serum.The incubation is in room air at 37℃, 5% CO2, 95% O2, the optimal pH at 8.0. In this culture system, P. carinii organisms isolated freshly grew much better than those frozen for 2 months did. In 10 day initial culture, the freshly isolated cysts and trophoziotes got an increase of 8-to 9- fold , 11-to 12-fold respectively, but the cysts grew the faster than the trophoziotes did during the initial 3 days incubation. The cysts and trophoziotes in subculture, however, can only increased steadly 5-to 6- fold, 8-to 9- fold respectively.The P. carinii organisms frozen for 2 months grew poorly in initialculture, but they could grow as well as the unfrozen organisms did after 2- to 3- following subculture. The morphology of organisms cultured for 21 days in stained smears and in transmission electron micrographs was that of P. carinii. The organisms metabolized lively, which were confirmed indirectly by the abudant of mitochondria, endoplasmic reticulum and heavy density particles in transmission electron micrographs. 2. This culture system has been applicated for screening the drug against P. carinii in vitro. Three drugs were evaluated to investigate the utility of the axenic system for drug screening. Pentamidine(1.0μg/ml),a common effective drug against P. carinii, was used at concentrations shown to reduce the proliferation of P. carinii. Nystatin (50μg/ml ) and Qingdaimeisu(400μg/ml ) ,which are effictive on fungi and bacteria respectively but noneffective on P. carinii ,were used at concentrations shown effective to fungi and bacteria.The results showed that the percentage of suppression of Pentamidine, Nystatin and Qingdaimeisu to the cysts and trophoziotes were 55.9% and 64.7%, 8.1% and 9.3%, 7.7% and 5.6% respectively. The difference was significant between Pentamidine group and blank control group(p<0.01), but no notable difference were found in Nystatin and Qingdaimeisu group compared with the blank control group. It has been suggested, therefore, that it was practicable to applicate the culture system for screening drug against P. carinii in vitro. 3. The effects of Daphnetin on the growth of P. carinii in vitro was assessed. Daphnetin suppressed the growth of P. carinii in vitro in a dose-dependent manner in the range concentration of 1～20μmol/L. No significant difference was found between the activity of 10μmol/L Daphnetin and that of 1.0μg/ml Pentamidine. The percentages of suppression of Daphnetin and Pentamidine on cystsand trophoziotes were 59.3% and 65.9%, 56.7% and 62.3% respectively. The inhibitory activity of Daphnetin on growth of P. carinii in vitro was removed by previous chelation of Daphnetin with FeSO4 in a 2:1 molar ratio, but not removed by mixing of the chelator with MgSO4. This implies that a chelator of iron, rather than a direct toxic effect, is responsible for the inhibitory activity. Incubation with 10μmol/L Daphnetin for 48h, the changs of the ultrastructures in most organisms were apparent, such as swollen mitochondrion and endoplasmic reticulum, empty vacuoles, lysised plasma, broken nuclear membrane and so on. But these changes were not seen to be localized in any one celluar compartment; rather, as time passed, more and more organisms lost definit...
|
PDF Full Text Request