卡氏肺孢子虫体外无细胞培养体系的建立与瑞香素抗卡氏肺孢子虫的初步研究
摘要
本研究通过对卡氏肺孢子虫(Pneumocystis carinii,Pc)体外无细胞系培养体系摸索，建立了Pc体外无细胞培养方法，并运用该培养体系研究了瑞香素在体外抗Pc的作用，获得如下结果和结论。
1. 建立了较为稳定的Pc体外无细胞培养方法。
通过对YPAD 、YEA、NPG、RPMI1640及IMDM多种基础培养基的筛选并反复摸索Pc培养条件，发现添加50μg/ml s-腺苷甲硫氨酸（SAM）、100mg/L N-乙酰氨基葡萄糖苷、80mg/L腐胺 、50mg/L L-半胱胺酸 、50mg/L L—谷胺酰胺、5×10-5mol/L2-巯基乙醇及15%新生小牛血清的IMDM培养基可支持Pc生长并可传代。在37℃，pH 8.0，5%CO2、95%O2条件下，接种新鲜分离的Pc包囊，培养10天，包囊可增殖8～9倍，滋养体可增殖11～12倍，在培养的前3天包囊生长较快，以后滋养体生长较快。经传代培养，包囊可稳定增殖5～6倍，滋养体可增殖8～9倍。冻存2月的Pc包囊，原代培养生长较差，传2 ～3倍代后与新鲜分离的Pc传代培养生长趋势相近。培养21天的Pc滋养体扫描电镜图象显示其表面有皱折，透射电镜图像显示其内部含有丰富的线粒体、内质网以及大量高密度颗粒。
2. 应用以上培养体系，建立了筛选抗Pc药物的方法。
运用建立的Pc体外无细胞培养方法，选用抗Pc的常用药物喷他脒1.0μg/ml、抗真菌的制霉菌素50μg/ml、抗细菌的庆大霉素400
μg/ml处理培养的Pc，检测它们对Pc生长的抑制率从而评估上述培养方法用于药物筛选的可行性。实验结果显示，喷他脒对包囊的抑制率为55.9%，对滋养体的抑制率为64.7%，与空白对照组比较有显著性差异(p<0.01)；而制霉菌素、庆大霉素对包囊的抑制率分别为8.1%、7.7%，对滋养体的抑制率为9.3%、5.6%，二者与空白对照组相比无显著性差异。实验结果与文献报道相符，说明用以上建立的培养方法筛选抗Pc的药物是可行的。
3. 应用以上培养体系、药物筛选方法，初步研究了瑞香素体外对Pc生长的抑制作用。
瑞香素抗Pc作用效应在1～20μmol/L浓度范围呈剂量依赖关系，10μmol/L瑞香素与1.0μg/ml喷他脒对Pc生长影响效果相当，它们对Pc包囊的抑制率分别为59.3%、56.7%，对滋养体抑制率分别为65.9%、62.3%。若预先将瑞香素与FeSO4按2：1比例混合后，瑞香素对Pc的生长抑制作用大大减弱；而预先与MgSO4以2：1混合后，瑞香素对Pc的生长抑制作用则不减弱，此结果说明瑞香素对Pc的抑制作用与其螯合了Pc生长代谢密切相关的铁离子有关。透射电镜观察，发现经10μmol/L瑞香素作用48h的虫体出现线粒体、内质网肿胀，核膜断裂，胞浆稀薄、空泡、溶解等超微结构改变，这些改变程度随药物剂量增加、时间延长而加重。
结 论 建立了Pc体外无细胞培养体系和药物筛选方法。瑞香素对Pc生长有一定抑制作用。
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 initial
culture, 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 cysts
and 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
1. 建立了较为稳定的Pc体外无细胞培养方法。
通过对YPAD 、YEA、NPG、RPMI1640及IMDM多种基础培养基的筛选并反复摸索Pc培养条件，发现添加50μg/ml s-腺苷甲硫氨酸（SAM）、100mg/L N-乙酰氨基葡萄糖苷、80mg/L腐胺 、50mg/L L-半胱胺酸 、50mg/L L—谷胺酰胺、5×10-5mol/L2-巯基乙醇及15%新生小牛血清的IMDM培养基可支持Pc生长并可传代。在37℃，pH 8.0，5%CO2、95%O2条件下，接种新鲜分离的Pc包囊，培养10天，包囊可增殖8～9倍，滋养体可增殖11～12倍，在培养的前3天包囊生长较快，以后滋养体生长较快。经传代培养，包囊可稳定增殖5～6倍，滋养体可增殖8～9倍。冻存2月的Pc包囊，原代培养生长较差，传2 ～3倍代后与新鲜分离的Pc传代培养生长趋势相近。培养21天的Pc滋养体扫描电镜图象显示其表面有皱折，透射电镜图像显示其内部含有丰富的线粒体、内质网以及大量高密度颗粒。
2. 应用以上培养体系，建立了筛选抗Pc药物的方法。
运用建立的Pc体外无细胞培养方法，选用抗Pc的常用药物喷他脒1.0μg/ml、抗真菌的制霉菌素50μg/ml、抗细菌的庆大霉素400
μg/ml处理培养的Pc，检测它们对Pc生长的抑制率从而评估上述培养方法用于药物筛选的可行性。实验结果显示，喷他脒对包囊的抑制率为55.9%，对滋养体的抑制率为64.7%，与空白对照组比较有显著性差异(p<0.01)；而制霉菌素、庆大霉素对包囊的抑制率分别为8.1%、7.7%，对滋养体的抑制率为9.3%、5.6%，二者与空白对照组相比无显著性差异。实验结果与文献报道相符，说明用以上建立的培养方法筛选抗Pc的药物是可行的。
3. 应用以上培养体系、药物筛选方法，初步研究了瑞香素体外对Pc生长的抑制作用。
瑞香素抗Pc作用效应在1～20μmol/L浓度范围呈剂量依赖关系，10μmol/L瑞香素与1.0μg/ml喷他脒对Pc生长影响效果相当，它们对Pc包囊的抑制率分别为59.3%、56.7%，对滋养体抑制率分别为65.9%、62.3%。若预先将瑞香素与FeSO4按2：1比例混合后，瑞香素对Pc的生长抑制作用大大减弱；而预先与MgSO4以2：1混合后，瑞香素对Pc的生长抑制作用则不减弱，此结果说明瑞香素对Pc的抑制作用与其螯合了Pc生长代谢密切相关的铁离子有关。透射电镜观察，发现经10μmol/L瑞香素作用48h的虫体出现线粒体、内质网肿胀，核膜断裂，胞浆稀薄、空泡、溶解等超微结构改变，这些改变程度随药物剂量增加、时间延长而加重。
结 论 建立了Pc体外无细胞培养体系和药物筛选方法。瑞香素对Pc生长有一定抑制作用。
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 initial
culture, 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 cysts
and 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
引文
1. Simon H. Natur. Wissenschaften,1953;40:625—626.
2. Csillag A. Acta Microbiol,1957;4(1):1—8.
3. Csillag A, Brendstein L. Acta Microbiol,1954;1:525—529.
4. Pifer LL, Hughes WT, Murphy MJ Jr. Propagation of Pneumocystis carinii in vitro. Pediatr Res,1977;11:305—316..
5. Latorre CR,Sulzer AJ,Norman LG. Serial Propagation of Pneumocystis carinii in Cell Line Cultures.Appl Environ Microbiol. 1977;33:1204—1206
6. Cushion MT,Walzer PD.Growth and Serial Passage of Pneumocystis carinii in the A549 Cell Line. Infect Immun .1984;44:245—251.
7. Dei-Cas E. European Concerted Action on Pneumocystis carinii in vitro systems in Pneumocystis research. Parasitol Today. 1996;12:245—249.
8. Pifer LL,Woods D,Hughes WT.Propagation of Pneumocystis carinii in Vero cell Culture.
Infect Immun. 1978;20:66—68.
9. Pesanti EL. J Inf Dis,1980;141(6):775—780.
10.Tegoshi T,Yoshida Y. New system of in vitro cultivation of Pneumocystis carinii withoutfeedr cells. J. Protozool. 1989;36:29—31.
11.Cushion MT ,Ebbets D Growth and metabolism of Pneumocystis carinii in Axenic Culture. Journal of Clinical Microbiology, 1990,28(6),1385.
12. Merali S，Frevert u, Williams JH, et al. Continuous axenic cultivation of Pneumocystis carinii . Proc.Natl.Acad.Sci.uSA,1999;96:2402—2407.
13. 韩龙志，颜晓箐，安春丽. 卡氏肺孢子虫肺炎的预防和治疗研究进展. 国外医学寄生虫病分册，2000；27（4）：155—160.
14.Weinberg GA. Iron chelators as therapentic agents against Pneumocystis carinii. Antimicrob Agents Chemocher, 1994;38(5):997-1003.
15.刘云光, 倪奕昌. 一类新抗疟药--铁螯合剂的研究进展. 中国寄生虫学与寄生虫病杂志, 1999;26(5):193-196.
16. Polster VJ,Schwenk M. Zur spectroskopischen analyse von metalkoplesgleichge-whichten am beispil hnetin und FeCl3. Z Physik Chem Neue Golge, 1986,150S:87-96.
17.郑建靖 石森林. 瑞香素的药理研究进展 浙江中医学院学报.1999.23(4).-50-51
18.Wang QM,Ni YC,xu YQ,et al.The schizontocidal activity of daphnetin against malaria parasites in vitro and in vivo.Chin J Parasitol Parait Dis.2000;18:204-206.
2. Csillag A. Acta Microbiol,1957;4(1):1—8.
3. Csillag A, Brendstein L. Acta Microbiol,1954;1:525—529.
4. Pifer LL, Hughes WT, Murphy MJ Jr. Propagation of Pneumocystis carinii in vitro. Pediatr Res,1977;11:305—316..
5. Latorre CR,Sulzer AJ,Norman LG. Serial Propagation of Pneumocystis carinii in Cell Line Cultures.Appl Environ Microbiol. 1977;33:1204—1206
6. Cushion MT,Walzer PD.Growth and Serial Passage of Pneumocystis carinii in the A549 Cell Line. Infect Immun .1984;44:245—251.
7. Dei-Cas E. European Concerted Action on Pneumocystis carinii in vitro systems in Pneumocystis research. Parasitol Today. 1996;12:245—249.
8. Pifer LL,Woods D,Hughes WT.Propagation of Pneumocystis carinii in Vero cell Culture.
Infect Immun. 1978;20:66—68.
9. Pesanti EL. J Inf Dis,1980;141(6):775—780.
10.Tegoshi T,Yoshida Y. New system of in vitro cultivation of Pneumocystis carinii withoutfeedr cells. J. Protozool. 1989;36:29—31.
11.Cushion MT ,Ebbets D Growth and metabolism of Pneumocystis carinii in Axenic Culture. Journal of Clinical Microbiology, 1990,28(6),1385.
12. Merali S，Frevert u, Williams JH, et al. Continuous axenic cultivation of Pneumocystis carinii . Proc.Natl.Acad.Sci.uSA,1999;96:2402—2407.
13. 韩龙志，颜晓箐，安春丽. 卡氏肺孢子虫肺炎的预防和治疗研究进展. 国外医学寄生虫病分册，2000；27（4）：155—160.
14.Weinberg GA. Iron chelators as therapentic agents against Pneumocystis carinii. Antimicrob Agents Chemocher, 1994;38(5):997-1003.
15.刘云光, 倪奕昌. 一类新抗疟药--铁螯合剂的研究进展. 中国寄生虫学与寄生虫病杂志, 1999;26(5):193-196.
16. Polster VJ,Schwenk M. Zur spectroskopischen analyse von metalkoplesgleichge-whichten am beispil hnetin und FeCl3. Z Physik Chem Neue Golge, 1986,150S:87-96.
17.郑建靖 石森林. 瑞香素的药理研究进展 浙江中医学院学报.1999.23(4).-50-51
18.Wang QM,Ni YC,xu YQ,et al.The schizontocidal activity of daphnetin against malaria parasites in vitro and in vivo.Chin J Parasitol Parait Dis.2000;18:204-206.