Crocker P R, Davies E V, Blackwell J M
Department of Tropical Hygiene, London School of Hygiene and Tropical Medicine, UK.
Parasite Immunol. 1987 Nov;9(6):705-19. doi: 10.1111/j.1365-3024.1987.tb00540.x.
Various macrophage populations isolated from mice (including congenic C57BL/10ScSn and B10.LLshr) bearing resistant or susceptible alleles for the natural resistance gene (Lsh) were infected with Leishmania donovani amastigotes in vitro and examined (a) for their ability to support growth of the amastigote population over 7 days of culture in vitro, and (b) for their ability to express Lsh gene controlled resistance and susceptibility in vitro. Resident macrophages from liver (Kupffer cells), spleen and lung, as well as 7-day bone marrow-derived macrophages and bone marrow macrophages obtained after 6 weeks of continuous culture in vitro, all supported growth of the amastigote population. Of these, significant differences in amastigote numbers in macrophages from Lshs and Lshr mice were observed after 48 h of infection in vitro for liver, lung and 7-day bone marrow macrophage populations only. Resident peritoneal macrophages grown in adherent or suspension cultures neither supported growth of the amastigote population nor showed any evidence of Lsh gene expression in vitro. Hence, multiplication of the parasite appeared to be a necessary but not sufficient condition for observation of Lsh gene activity against L. donovani in vitro. Use of tritiated thymidine incorporation and autoradiography to label dividing amastigotes showed equivalent multiplication of the parasite in liver macrophages from Lshs and Lshr mice between 24 h and 48 h after infection in vitro, with a dramatic difference observed thereafter. This was consistent with earlier observations of a 2-3 day delay in expression of Lsh gene controlled resistance in vivo. Comparison with studies using Salmonella typhimurium and Mycobacterium bovis suggests that the gene may be restricted in its action to a particular point in the parasite cell cycle, perhaps at the level of regulating DNA replication.
从携带天然抗性基因（Lsh）抗性或敏感等位基因的小鼠（包括同源C57BL/10ScSn和B10.LLshr）中分离出的各种巨噬细胞群体，在体外感染杜氏利什曼原虫无鞭毛体，并进行检测：（a）检测它们在体外培养7天期间支持无鞭毛体群体生长的能力；（b）检测它们在体外表达Lsh基因控制的抗性和敏感性的能力。来自肝脏（枯否细胞）、脾脏和肺的驻留巨噬细胞，以及体外连续培养6周后获得的7天骨髓来源巨噬细胞和骨髓巨噬细胞，均支持无鞭毛体群体的生长。其中，仅在体外感染48小时后，在肝脏、肺和7天骨髓巨噬细胞群体中，观察到Lshs和Lshr小鼠的巨噬细胞中无鞭毛体数量存在显著差异。在贴壁或悬浮培养中生长的驻留腹膜巨噬细胞既不支持无鞭毛体群体的生长，在体外也未显示出任何Lsh基因表达的证据。因此，寄生虫的增殖似乎是在体外观察Lsh基因对杜氏利什曼原虫活性的必要但不充分条件。使用氚标记胸腺嘧啶核苷掺入和放射自显影来标记分裂的无鞭毛体，结果显示在体外感染后24小时至48小时之间，Lshs和Lshr小鼠肝脏巨噬细胞中寄生虫的增殖相当，此后观察到显著差异。这与之前在体内观察到的Lsh基因控制的抗性表达延迟2 - 3天一致。与使用鼠伤寒沙门氏菌和牛分枝杆菌的研究相比，表明该基因的作用可能局限于寄生虫细胞周期的特定点，也许是在调节DNA复制的水平。
