Objective: To compare the growth and reproduction of the promastigotes of Leishmania isolates from various endemic areas of visceral leishmaniasis in China in various culture media, so as to provide experimental evidence for selecting an appropriate medium for the culture of Leishmania.
Methods: A total of 3 × 105 promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates were inoculated into 1 mL NNN medium, 1 mL M199 medium supplemented with 20% fetal bovine serum medium, 1 mL M199 medium supplemented with 20% horse serum medium, and 1 mL brain heart infusion medium containing heme, respectively. All media were placed at 22 ℃ under a sterile condition, and the number of promastigotes was counted continuously for 8 days under a microscope. The growth curve was plotted for the three Leishmania isolates.
Results: The promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates all grew and reproduced in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium. The number of promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates was all significantly higher in the NNN medium than in the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05), and the number of promastigotes of the KS-2 isolate was all significantly greater than that of the Cy and JIASHI-5 isolates in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05). In ad dition, the promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates failed to grow and reproduce in the brain heart infusion medium.
Conclusions: The growth and reproduction of the promastigotes of various Leishmania isolates from various endemic areas of visceral leishmaniasis in China vary in the same culture medium, and the growth and reproduction of a Leishmania isolate vary in different culture media. The NNN medium best fits for the culture of Leishmania isolates in the endemic areas of visceral leishmaniasis in China.
[摘要] 目的 比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况, 为选择 合适培养基用于利什曼原虫培养提供实验依据。方法 将 3 × 105 个KS-2、Cy、JIASHI-5 株利什曼原虫前鞭毛体分别接种 至 1 mL NNN 培养基、1 mL M199 + 20% 胎牛血清培养基、1 mL M199 + 20% 马血清培养基及 1 mL 脑心浸液培养基 (含血红素) 中, 22 ℃温箱中无菌静置培养, 显微镜下连续观察计数 8 d, 绘制 3 株利什曼原虫前鞭毛体的生长曲线。结果 KS-2、Cy、JIASHI-5 株利什曼原虫前鞭毛体均能在 NNN 培养基、M199 + 20% 胎牛血清培养基和 M199 + 20% 马血清培养基中生长繁殖, 在 NNN 培养基中培养不同时间后的 3 株利什曼原虫前鞭毛体计数均显著高于 M199 + 20% 胎牛血清培养基和 M199 + 20% 马血清培养基 (P 均 < 0.05), 在这 3 种培养基中培养不同时间后的 KS-2 株利什曼原虫前鞭毛体计数均显著 高于 Cy 和 JIASHI-5 株 (P 均 < 0.05)。KS-2、Cy、JIASHI-5 株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论 分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异, 同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN 培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。.
Keywords: Culture medium; In vitro culture; Leishmania; Promastigote; Visceral leishmaniasis.