肝脏自然杀伤细胞在小鼠急性肝衰竭中的作用

The functions of liver natural killer cells in murine fulminant hepatitis induced by murine hepatitis virus strain 3

目的探讨肝脏自然杀伤（NK）细胞在3型鼠肝炎病毒（MHV3）诱导的小鼠急性肝衰竭中的作用。方法取6～8周龄雌性Balb／cJ小鼠，腹腔注射100pfu MHV-3，采用流式细胞术检测感染MHV-30、24、48、70h后的Balb／cJ小鼠肝脏、脾脏、外周血和骨髓中NK细胞的百分率及肝脏NK细胞表面活化分子CD69表达的百分率。细胞内细胞因子染色法检测肝脏NK细胞分泌干扰素Y的水平。非放舯眭细胞毒试验检测肝脏NK细胞的杀伤活性。结果Balb／cJ小鼠感染MHV-3后，肝脏NK细胞的比例显著升高，在感染48h后达到峰值（43．9％2．3％），约为感染前的4倍，随后仍维持在较高水平至小鼠死亡；外周血NK细胞比例同样明显升高，在感染48h后达到峰值（18．0％±5．4％），但随后显著回落，至70h仅为1．3％±0．6％；脾脏和骨髓NK细胞比例均先明显减少后又有所上升。肝脏NK细胞在MHV3感染48h后其表面活化分子CD69表达明显上调，杀伤活性显著增强，同时分泌干扰素γ的水平也显著增加。结论Balb／cJ小鼠感染MHV-3后，来自骨髓和脾脏的NK细胞在肝脏迅速大量募集和活化，且杀伤活性显著增强，分泌干扰素γ水平也显著增加，表明肝脏NK细胞在MHV-3导致的急性肝衰竭中可能发挥着关键作用。

Objective To investigate the role of liver natural killer cells （NK cells） in murine hepatitis virus strain 3 （MHV-3） induced murine fulminant hepatitis. Method Balb/cJ mice （6-8 weeks, female） were intraperitoneally injected with 100 PFU MHV-3. The numbers of NK cells in their livers, spleens, blood and bone marrow and the expression of CD69 on liver NK cells at 0, 24, 48 and 70 h after MHV-3 infection were analyzed by flow cytometry. The cytotoxic activity of liver NK cells was detected by a nonradioactive cytotoxicity assay. The levels of IFN gamma produced by hepatic NK cells were detected by intraceUular cytokine staining. Result Following MHV-3 infection, the proportion of liver NK cells in the mice increased remarkably and reached the peak （43.9% ± 2.3%） at 48 h, then kept a high proportion until the mice were sacrificed. The proportion of NK cells in the peripheral blood also significantly increased and reached the peak （18.0% ± 5.4%） at 48 h. However, there were few NK cells in the peripheral blood at 70 h after infection; the ratio was only 1.3% ± 0.6%. In the spleens and bone marrow, the proportions of NK cells were both significantly decreased from 0 h to 48 h and then slightly increased. The expression of CD69 on liver NK cells was highly up-regulated after the infection and the cytotoxic activity of hepatic NK cells at 48 h was also significantly enhanced. In addition, an increase in IFN gamma production by hepatic NK cells was observed at 48 h. Conclusion After MHV-3 infection, NK cells were recruited to the liver quickly, probably from the spleen and bone marrow. Recruited NK cells remarkably express CD69, enhance cytotoxic activity and IFN gamma production, which correlate with the disease severity of fulminant viral hepatitis. Our results suggest that liver NK cells may play a pivotal role in the pathogenesis of fulminant viral hepatitis.