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目的分析米托蒽醌体外抗锥虫的作用机制并考察其体内抗锥虫活性。方法通过倍比稀释合并alamar Blue染色测定米托蒽醌体外抗血流型布氏锥虫427(T.brucei BF427)的量-效和时-效曲线,并与抗锥虫药物喷他脒和美拉胂醇进行比较;通过DAPI染色、细胞周期表型分析考察米托蒽醌的抗锥虫作用机制;采用血虫症小鼠考察米托蒽醌的体内抗锥虫活性,米托蒽醌经腹腔给药,生理盐水为对照组;每天取血计数血虫密度;用药7d后停药,计算小鼠存活率。结果量-效曲线测得米托蒽醌对体外培养锥虫(起始密度为1×10~6个/m L)24h MIC为9.7μM;时-效曲线显示米托蒽醌和喷他脒都可显著抑制锥虫生长、持续降低锥虫密度;而经美拉胂醇处理后的锥虫密度在12 h内仍缓慢增长、随后出现明显降低;因此,在MIC作用浓度下米托蒽醌的体外抗锥虫活性好于美拉胂醇、与喷他脒相似。对照组小鼠的血虫密度迅速增加,感染6d时全部死亡;米托蒽醌明显抑制小鼠血虫增长,停药后2d小鼠出现死亡,最长存活14d,存活时间明显延长。DAPI染色和细胞周期表型分析显示锥虫核内(N)基因组DNA的复制受到抑制、而动基体(K)DNA的复制不受影响,表现为1N2K细胞型的比例明显上升,由10.8%上升至17.5%,2N2K细胞型的比例明显下降,由6.3%下降至3.0%。结论米托蒽醌具有体内抗锥虫活性,抑制锥虫核内基因组DNA的复制并使其产生损伤是作用机制之一。
Objective To analyze the mechanism of mitoxantrone in vitro against Trypanosoma and investigate its anti-trypanosome activity in vivo. Methods The dose-response and time-response curves of mitoxantrone-resistant T.brucei BF427 in vitro were determined by fold dilution combined with alamar Blue staining and compared with the anti-trypanosomes pentamidine and the United States Rat Danslalcohol was compared; DAPI staining, cell cycle phenotype analysis of mitoxantrone anti-trypanosome mechanism of action; the use of blood-borne mice mitoxantrone in vivo anti-trypanosome activity mitoxantrone Intraperitoneal administration, normal saline as the control group; daily blood count of bloodworm density; drug withdrawal after 7d, calculate the survival rate of mice. Results The dose-response curve of mitoxantrone against cultured Trypanosomes (initial density of 1 × 10 ~ 6 cells / m L) 24h MIC was 9.7μM; time-response curve showed mitoxantrone and pentamidine Can inhibit the growth of trypanosomes significantly and decrease the density of trypanosomes continuously. However, the density of trypanosomes after treatment with melasolol still increased slowly within 12 h and then decreased significantly. Therefore, mitoxantrone The anti-trypanosome activity in vitro is better than melasolol, similar to pentamidine. In the control group, the density of blood-borne insects increased rapidly and died on the 6th day after infection. Mitoxantrone significantly inhibited the growth of blood-borne insects in mice, and the mice died 2 days after the drug was stopped. The longest survival time was 14 days and the survival time was significantly prolonged. DAPI staining and cell cycle phenotype analysis showed that the replication of intranuclear (N) genomic DNA in trypanosomes was inhibited, while the replication of the motile (K) DNA was not affected, showing a marked increase in the proportion of 1N2K cell types from 10.8% To 17.5%, the proportion of 2N2K cell type decreased significantly from 6.3% to 3.0%. Conclusion Mitoxantrone has the anti-trypanosome activity in vivo and inhibits the replication of genomic DNA in Trypanosoma brucei nucleus and causes its damage.