Studies on the immunological and genetic properties of sensitive hosts for the pathogenic mycobacteria

小鼠天然耐药相关巨噬细胞蛋白NRAMP1对巨噬细胞的激活具有多种多效性作用，并调节细胞内病原体的生存。遗传分析现在已经将NRAMP1基因座及其周围的多态与人类对某些分枝杆菌物种的易感性联系在一起。采用Western印迹分析和实时定量聚合酶链式反应(RT-PCR)检测NRAMP-1蛋白和mRNA在人巨噬细胞系THP-1中的表达。使用佛波酯(PMA)分化的THP-1细胞进行研究。未经刺激(没有PMA)的THP-1细胞结构性地表达低水平的NRAMP1mRNA和蛋白。PMA和活菌刺激(M.avium)均能轻微促进mRNA的合成。重组人干扰素-γ(200U/ml)刺激THP-1细胞后，NRAMP1mRNA的量显著增加(约10倍)。PMA刺激THP-1细胞可抑制鸟型支原体的生长，而干扰素-γ不影响鸟型支原体的生长。这种体外感染模型似乎是研究人类NRAMP1功能的有用工具。接下来，我们研究了麻风分枝杆菌(MLM)感染小鼠中NRAMP-1的功能。虽然NRAMP1不影响小鼠对MLM感染的敏感性/抵抗力，但如果静脉注射10^8个细菌，雌性小鼠比雄性小鼠更敏感。最后，我们试图阐明NRAMP1和NK细胞之间的关系以及它们在感染鸟型支原体的小鼠中的作用。自然杀伤细胞缺乏的米色小鼠增加了它们对鸟型支原体的易感性。体内抗NK细胞抗体处理后，小鼠的NK细胞总数明显减少，产生干扰素-γ的能力明显降低。然而，对NRAMP1感染的自然抵抗力与NK细胞功能之间没有相关性。

The murine natural resistance-associated macrophage protein, NRAMP1 has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens. Genetic analyses have now linked polymorphisms in and around the NRAMP1 locus with susceptibility to some mycobacterial species in humans. In this time, the expression of NRAMP-1 proteins and mRNAs in the human macrophage cell line THP-1 was investigated by western blot analysis and real time-polymerase chain reaction(RT-PCR), respectively. Phorbol myristate acetate(PMA)- differentiated THP-1 cells were used in these studies. Unstimulated (without PMA) THP-1 cells constitutively expressed a low level of NRAMP1 mRNA and proteins. Both PMA and live bacteria stimulation (M.avium) could slightly accelerate the mRNA synthesis. The amount of NRAMP1 mRNA dramatically increased (>10 times) if THP-1 cells were stimulated with recombinant human IFN-γ(200U/ml). The growth of M.avium in THP-1 cells was inhibited if cells had been stimulated with PMA, and IFN-γ did not affect the growth of M.avium. This in vitro infection model seems to be a useful tool for studying the function of human NRAMP1. Next we examined the NRAMP-1 function in Mycobacterium lepraemurium(MLM) infected mice. Although NRAMP1 did not affect the susceptibility/resistance to the MLM infection among mouse strains, female mice were more sensitive than male mice if they were received with 10^8 bacteria intravenously. Finally, we tried to clarify the relationship between NRAMP1 and NK cells and their roles in M.avium infected mice. NK cell deficient beige mice increased their susceptibility to M.avium. Anti-NK cell antibody treatment in vivo decreased total number of NK cells in the mouse and reduced the ability to produce IFN-γ dramatically. However no relationship was observed between natural resistance to the infection by the NRAMP1 and NK cell function.