小鼠肝脏内自然杀伤细胞功能和数量与鼠龄变化的关系

Age-related changes of the function and amount of natural killer cells in the liver:experiment with mice

目的研究不同鼠龄小鼠肝脏内自然杀伤细胞的功能和数量变化的规律。方法应用4小时51Cr释放法测定2周龄、8周龄小鼠(CL57BL/6)肝脏和脾脏内NK细胞的杀伤活性;应用流式细胞仪测定2周龄、8周龄小鼠肝脏和脾脏内NK细胞的百分率和数量,测定NK细胞表面CD69,M ac-1和Ly49C/I的表达水平;应用RT-PCR法测定肝脏单核细胞内穿孔素的表达强度。结果2周龄和8周龄小鼠肝脏内NK细胞占全部淋巴细胞的百分比大致相同(11.9%±1.7%vs 9.9%±1.6%,P>0.05),但是单位质量肝脏内所含的NK细胞数在2周龄鼠明显多于8周龄鼠[(11.6±2.5)×105/g vs(3.4±0.8)×105/g,P<0.05]。2周龄小鼠肝内NK细胞具有极强的细胞杀伤活性(71.0%±5.5%vs 23.8%±4.4%,P<0.05)。2周龄小鼠肝内NK细胞表型主要是CD69增高M ac-1、Ly49C/I减低,而8周龄鼠肝内NK细胞主要是CD69减低,M ac-1、Ly49C/I增高。2周龄小鼠肝脏单核细胞内高度表达穿孔素。结论2周龄小鼠肝内NK细胞具有极强的杀伤能力和特殊的表型。这提示幼龄期鼠肝脏具有特殊的微环境以及肝脏内NK细胞特殊的发育方式。

Objective To investigate the age-related changes of the function and amount of natural killer （NK） cells in the liver. Methods C57BL/6 mice aged 2 weeks and 8 weeks were killed and their livers and spleens were taken out. The percentages and absolute values of the NK cells in the livers and spleens were examined by flow cytometry. Using YAG-1 cells as the target cells, the cytotoxicity of the NK cells was detected. Using thymocytes of the homologous mice as the target cells, the eytotoxieity of the NK T cells was detected. The cytotoxicity of the cytotoxic T lymphocytes （CTLs） was detected with the EL-4 cells as the target cells. RT-PCR was used to detect the mRNA expression of perforin. Results The NK cell percentage in the liver of the 2-week-old mice was 11.9% ± 1.7%, not significantly different from that of the 8-week-old mice （9.9% ± 1.6%, P〉0.05）. The NK cell amount of per weight liver tissue of the 2- wwek-old mice was （11.6 ±2.5） x 10^5/g, significantly higher than that of the 8-week-old mice, （3.4 ± 0.8） × 10^5/g, （P 〈0.05）. The cytotoxicity of the liver NK cells of the 2-week-old mice was 71.0% ±5. 5 %, significantly higher than that of 8-week-old mice （23.8 % ± 4.4%, P 〈 0.05）. The cytotoxicity levels of the NK T cells and CTLs were not significantly different between the mice of different age. The cytotoxicity level of the spleen NK cells was not significantly different between the 2-week-old and 8-week-old mice as well. Phenotypic characterization showed that the NK cells in the liver of the 2-wwek-old mice were CD69^high MAC-1^low LY49C/1^low, whereas those in the liver of the 8-week-old mice were inverse, i.e.e. , CD69^low MAC-1^high LY49C/1^high. The level of perforin mRNA was remarkably higher in the lever of the 2-week-old mice than in the liver of the 8-week-old mice. Conclusion The liver NK cells of the young mice have a higher level of cytotoxicity and a unique phenotype. The liver of young mice has its unique mi and their liver NK cells have a special development pathway.