α-半乳糖基神经酰胺静脉注射对小鼠肝脏恒定性自然杀伤T细胞及亚群的影响

Effect of intravenous injection ofα-galactosylceramide on the frequency and subpopulation of iNKT cells in mouse liver

目的通过采用静脉注射α-半乳糖基神经酰胺(α-GalCer),观察其对小鼠肝脏恒定性自然杀伤T细胞(iNKT)频率及亚群的影响。方法研究时间2021年11月至2022年2月,选取C57BL/6J健康小鼠,采用随机数字表法分为观察组和对照组,观察组静脉注射α-GalCer,对照组静脉注射生理盐水,在注射后的第2、4、6、8、10天取小鼠肝脏,采用流式细胞术检测小鼠肝脏的iNKT细胞及亚群变化情况;采用酶联免疫吸附测定(ELISA)检测外周血血清中白细胞介素-4(IL-4),干扰素-γ(IFN-γ),白细胞介素-17A(IL-17A)的表达水平。结果与对照组相比,静脉注射α-GalCer可引起肝脏iNKT细胞频率及亚群频率变化,且差异有统计学意义(P<0.05)。观察组肝脏iNKT细胞频率明显升高,但随时间呈逐渐降低趋势,自第6天恢复至与正常水平相当[观察组第2、4、6、8、10天分别为(20.87±2.15)%、(5.61±0.51)%、(2.65±0.26)%、(2.62±0.64)%、(2.51±0.11)%,对照组分别为(2.72±0.19)%、(2.72±0.19)%、(2.93±0.24)%、(3.06±0.45)%、(3.14±0.35)%];注射α-GalCer后,iNKT1亚群频率低于对照组,于第10天恢复至正常水平[观察组第2、4、6、8、10天分别为(1.04±0.37)%、(0.48±0.12)%、(1.99±1.04)%、(0.60±0.11)%、(3.25±0.53)%,对照组分别为(3.68±1.02)%、(2.74±0.21)%、(2.81±0.33)%、(2.93±0.29)%、(3.12±0.11)%];iNKT2亚群频率高于对照组,后逐渐降低[观察组第2、4、6、8、10天分别为(75.13±3.48)%、(33.83±9.08)%、(17.20±5.37)%、(12.00±1.99)%、(6.26±1.28)%,对照组分别为(38.03±1.70)%、(40.13±3.20)%、(40.16±3.15)%、(38.03±1.70)%、(36.37±1.18)%];iNKT1亚群频率总体上低于iNKT2亚群;iNKT17亚群频率低于对照组,第4天亚群频率最高,后又降至正常水平以下;与对照组相比,血清中IL-4水平在注射第2天无明显变化(P>0.05),注射后第6天,IL-4水平明显降低(P<0.05);IFN-γ水平自注射α-GalCer后明显升高,第6天又降低(P<0.05),IL-17A水平均低于正常水平(P<0.05)。结论静脉注射α-GalCer可诱导小鼠肝脏iNKT细胞的增殖,影响其亚群的分化。

Objective To explore the effect of intravenous injection ofα-galactosylceramide(α-GalCer)on the frequency and subpopulation of iNKT cells in mouse liver.Methods C57BL/6J healthy mice were selected and randomly divided into observation group and control group,andα-GalCer and normal saline were intravenously injected respectively from November 2021 to February 2022.The livers of mice were taken on days 2,4,6,8 and 10 after injection,flow cytometry was used to detect the changes of iNKT cells and subpopulations in mouse liver,and the expression levels of IL-4,IFN-γand IL-17A in peripheral blood serum were detected by ELISA.Results Compared with the control group,intravenous injection ofα-GalCer could change the frequency and subpopulation frequency of liver iNKT cells,and the difference was statistically significant(P<0.05).The frequency of iNKT cells in the liver of the observation group was significantly increased,but it gradually decreased over time,and returned to a level comparable to normal level from the 6^(th) day[observation group(20.87±2.15)%,(5.61±0.51)%,(2.65±0.26)%,(2.62±0.64)%,(2.51±0.11)%,control group(2.72±0.19)%,(2.72±0.19)%,(2.93±0.24)%,(3.06±0.45)%,(3.14±0.35)%on days 2,4,6,8 and 10 after injection].After injection of α-GalCer,the frequency of the iNKT1 subpopulation was lower than that of the control group,and it returned to normal level on the 10th day[observation group(1.04±0.37)%,(0.48±0.12)%,(1.99±1.04)%,(0.60±0.11)%,(3.25±0.53)%,control group(3.68±1.02)%,(2.74±0.21)%,(2.81±0.33)%,(2.93±0.29)%,(3.12±0.11)%on days 2,4,6,8 and 10 after injection].The frequency of iNKT2 subpopulation was higher than that of the control group,and then gradually decreased[observation group(75.13±3.48)%,(33.83±9.08)%,(17.20±5.37)%,(12.00±1.99)%,(6.26±1.28)%,control group(38.03±1.70)%,(40.13±3.20)%,(40.16±3.15)%,(38.03±1.70)%,(36.37±1.18)%on day 2,4,6,8 and 10 after injection].The overall frequency of iNKT1 subpopulation was lower than that of the iNKT2 subpopulation.The frequency of the iNKT17 subgroup was lower than that of the control group,and the frequency of the subpopulation on day 4 was the highest.Then it dropped to below the normal level.Compared with the control group,the level of IL-4 in serum had no significant change on the second day of injection(P>0.05),on the 6th day after injection,the IL-4 level decreased significantly(P<0.05),the IFN-γlevel increased significantly after the injection ofα-GalCer,decreased again on day 6(P<0.05),and the level of IL-17A was lower than normal(P<0.05).Conclusion Intravenous injection ofα-GalCer can induce the proliferation of iNKT cells in mouse liver and affect the differentiation of their subpopulations.