CD24调节iNKT细胞介导的小鼠急性肝损伤的实验研究

CD24 Regulates acute liver injury through murine hepatic iNKT cells

目的:探讨小鼠急性肝损伤中CD24分子对不变的自然杀伤T(iNKT)细胞的调控作用。方法:小鼠腹腔注射α-半乳糖甘油酰胺(α-GalCer)特异活化iNKT细胞,诱导小鼠急性肝损伤。HE染色观察小鼠肝脏炎症损伤程度,血清学方法检测转氨酶变化,免疫荧光染色观察肝内iNKT细胞的数量变化,流式细胞术检测肝内总iNKT细胞、iNKT细胞亚型数量、比例及iNKT细胞活性的变化,定量-PCR(Q-PCR)检测肝组织中iNKT细胞相关细胞因子干扰素-γ(IFN-γ)和白细胞介素-4(IL-4)的表达。结果:注射α-GalCer后,CD24基因敲除(CD24-/-)小鼠肝脏炎性细胞浸润比野生型(WT)小鼠少(F=10.10,P<0.01),血清谷丙转氨酶(ALT)水平比WT小鼠低(F=10.11,P<0.01)。免疫荧光染色结果表明,正常生理状态及α-GalCer模型中,CD24-/-小鼠肝内iNKT细胞数量显著低于WT小鼠(F=13.27,P<0.01)。流式细胞术分析发现,正常生理状态下,CD24-/-小鼠肝内总iNKT细胞和其亚型的数量显著低于WT小鼠(F=6.841,P<0.05);注射α-GalCer后,小鼠肝内总iNKT细胞数量显著增多(F=33.01,P<0.001);进一步分析iNKT细胞亚型发现,NKT1、NKT2细胞数明显增多(F=37.12、40.55,均P<0.05),但CD24-/-小鼠仍低于WT小鼠(F=40.07、12.53,均P<0.05),NKT17细胞无变化(P>0.05)。Q-PCR结果表明,注射α-GalCer后,CD24-/-小鼠肝组织中IFN-γ和IL-4 mRNA表达水平明显低于WT小鼠(F=14.34、19.77,均P<0.01),流式细胞术检测胞内细胞因子结果表明,α-GalCer模型中,CD24-/-小鼠iNKT细胞分泌的IFN-γ和IL-4明显低于WT小鼠(F=25.600、5.574,均P<0.05)。结论:肝内iNKT细胞与小鼠急性肝损伤密切相关,CD24分子可以通过调节肝内iNKT细胞的数量和活性影响急性肝损伤进程。

Objective:To investigate the regulation of CD24 on invariant NKT cells(iNKT)in murine acute liver injury.Methods:In-traperitoneal injection ofα-galactosylceramide(α-GalCer)was used to activate hepatic iNKT cells and induce acute liver injury in mice.Levels of alanine amino transferase(ALT)in serum were detected and liver sections were stained with hematoxylin and eosin(HE)staining to assess the severity of liver injury.Changes of the number of iNKT cells in the liver were observed by immune-fluorescence staining.Flow cytometric analysis was used to detect the changes of the number and proportion of total iNKT cells,subtypes and iNKT cells activity in the liver.Q-PCR was used to detect the expression of iNKT cell-related cytokines including interferon-γ(IFN-γ)and interleukin-4(IL-4)in mice liver tissues.Results:After injection ofα-GalCer,CD24 knockout(CD24-/-)mice had less hepatic inflammatory cell infiltration and lower serum ALT levels when compared with wild-type(WT)mice(F=10.10,10.11,both P<0.01).Immune-fluorescence staining results showed that the number of intrahepatic iNKT cells was significantly lower in CD24-/-mice than in WT mice in the normal physiological state or in theα-GalCer induced acute liver injury model(F=13.27,P<0.01).Flow cytometry analysis showed that the number of total iNKT and subtypes in the liver of CD24-/-mice was lower than that of WT mice under normal physiological conditions(F=6.841,P<0.05).Afterα-GalCer injection,the total number of iNKT cells in the liver of mice was signifi-cantly increased(F=33.01,P<0.001).Further analysis of iNKT cell subtypes showed thatα-GalCer injection significantly increased the numbers of NKT1 and NKT2 cells(F=37.12,40.55,both P<0.05),but CD24-/-mice remained lower than WT mice(F=40.07,12.53,both P<0.05),and NKT17 cells were unchanged(P>0.05).Q-PCR showed that the expression levels of IFN-γand IL-4 mRNA were significantly lower in the liver tissues of CD24-/-mice than WT mice afterα-GalCer injection(F=14.37,19.77,both P<0.01).Flow cytometry analysis showed that the secretion of IFN-γand IL-4 were significantly lower in iNKT cells of CD24-/-mice when compared with WT mice inα-GalCer induced liver injury model(F=25.600,5.574,both P<0.05).Conclusion:iNKT cells in the liver are closely related to acute liver injury in mice,CD24 can affect the process of acute liver injury in mice by regulating the number and activi-ty of hepatic iNKT cells.