自然杀伤细胞抑制血吸虫病肝纤维化作用的研究

Study on the inhibitory effect of natural killer cells on liver fibrosis of schistosomiasis

目的通过单抗敲低或过继自然杀伤(NK)细胞,探讨NK细胞在血吸虫病肝纤维化中的作用。方法24只C57BL/6健康小鼠取肝脏,制备NK细胞,用白细胞介素-15(IL-15)、IL-2、IL-18活化NK细胞。40只C57BL/6健康小鼠随机分为单抗组、IgG组、生理盐水组、NK细胞组、PBS组等5组,每组8只,每鼠经腹部贴片感染日本血吸虫尾蚴(20±1)条。单抗组、IgG组和生理盐水组小鼠于感染前1周开始,每周分别尾静脉注射1次抗NK1.1单抗(100μg/鼠)、IgG(100μg/鼠)、生理盐水,每次200μl/鼠;NK细胞组和PBS组小鼠于感染后4周开始,每周分别尾静脉注射1次活化的NK细胞(1×10^(5)/鼠)、PBS,每次200μl/鼠。感染后6和8周,分别取各组小鼠肝脏,流式细胞术检测小鼠肝NK细胞比例变化,Masson染色观察肝组织纤维化变化,荧光定量PCR检测Ⅰ型胶原蛋白(Ⅰ-C)、Ⅲ-C、Ⅳ-C、层黏连蛋白(LN)、纤维连接蛋白(FN)mRNA相对转录水平等肝纤维化指标。结果感染后6周,单抗组肝NK细胞比例为(2.56±0.47)%,低于IgG组的(4.75±0.62)%和生理盐水组的(5.35±0.40)%(F=31.59,P<0.01);感染后8周,单抗组、IgG组和生理盐水组NK细胞比例分别为(2.65±0.23)%、(3.43±0.46)%和(3.56±0.46)%,差异无统计学意义(F=4.48,P>0.05)。感染后6、8周,NK细胞组肝NK细胞比例分别为(5.58±0.30)%、(3.73±0.42)%,与PBS组的(5.51±0.27)%、(2.32±0.40)%差异均无统计学意义(t=0.25、2.64,P>0.05)。感染后6、8周,单抗组肝纤维化面积分别为(8.42±1.30)×10^(4)、(9.55±1.55)×10^(4)μm^(2),均大于IgG组的(6.40±1.40)×10^(4)、(6.11±1.10)×10^(4)μm^(2)和生理盐水组的(6.73±1.61)×10^(4)、(6.62±1.60)×10^(4)μm^(2)(F=13.63、30.33,P<0.01)。感染后6周,NK细胞组肝纤维化面积为(5.97±0.96)×10^(4)μm^(2),小于PBS组的(8.27±1.62)×10^(4)μm^(2)(t=4.85,P<0.01);感染后8周,NK细胞组、PBS组肝纤维化面积分别为(6.33±0.98)×10^(4)μm^(2)、(6.97±1.11)×10^(4)μm^(2),差异无统计学意义(t=1.80,P>0.05)。感染后6周,单抗组Ⅳ-C、LN、FN mRNA相对转录水平分别为1.81±0.47、1.63±0.38和1.41±0.16,较IgG组的1.00±0.35、0.81±0.29、0.79±0.16和生理盐水组的1.00±0.12、1.00±0.10、1.00±0.14均上调(F=8.30、8.25、14.40,P<0.01);感染后8周,单抗组Ⅳ-C mRNA相对转录水平为1.66±0.21,较IgG组的0.94±0.26和生理盐水组的1.00±0.09均上调(F=15.95,P<0.01)。感染后6周,NK细胞组Ⅰ-C、Ⅲ-C、LN mRNA相对转录水平分别为0.66±0.12、0.61±0.06、0.64±0.09,较PBS组的1.01±0.14、1.01±0.14、1.01±0.16均下调(t=3.36、4.41、3.59,P<0.05);感染后8周,NK细胞组Ⅰ-C、Ⅲ-C、LN mRNA相对转录水平分别为0.82±0.40、0.93±0.37、0.73±0.30,与PBS组的1.04±0.38、1.02±0.25、1.06±0.49相比,差异无统计学意义(t=0.55、0.27、0.81,P>0.05)。结论小鼠感染日本血吸虫后肝NK细胞在肝纤维化形成早期可发挥抑制肝纤维化的作用。

Objective Using monoclonal antibodies to deplete or passively transfer natural killer(NK)cell,to explore the role of NK cells in schistosomiasis liver fibrosis.Methods The livers of 24 C57BL/6 healthy mice were collected to isolate NK cells,and the NK cells were activated with interleukin-15(IL-15),IL-2 and IL-18.Forty C57BL/6 mice were randomly divided into five groups,namely monoclonal antibody group,IgG group,normal saline group,NK cell group and PBS group,with 8 mice in each group.Each mouse was infected with Schistosoma japonicum cercariae(20±1)via an abdominal patch.The mice in the monoclonal antibody group,IgG group and normal saline group were injected with anti-NK1.1 monoclonal antibody(100μg/mouse),IgG(100μg/mouse),and normal saline through the tail vein at a dose of 200μl/mouse each time,once a week,started one week before the infection.Four weeks after infection,the mice in the NK cell group and PBS group were transferred with activated NK cells(1×10^(5)/mouse)and PBS through the tail vein weekly at dose of 200μl/mouse each time.At six and eight weeks after infection,flow cytometry was used to detect changes in the proportion of NK cells in the liver of the mice.Masson staining was used to observe liver tissue lesions,and Real-time PCR was used to detect indicators of liver fibrosis,including mRNA relative transcription levels of Ⅰ-collagen(Ⅰ-C),Ⅲ-C,Ⅳ-C,fibronectin(FN),and laminin(LN).Results The proportion of liver NK cells at six weeks after infection in the monoclonal antibody group was(2.56±0.47)%,which was lower than that in the IgG group(4.75±0.62)% and normal saline group(5.35±0.40)%(F=31.59,P<0.01).At 8 weeks post-infection,the proportion of liver NK cells were(2.65±0.23)%,(3.43±0.46)%,and(3.56±0.46)% for the monoclonal antibody group,the IgG group and the normal saline group,respectively,and the difference was not statistically significant(F=4.48,P>0.05).The proportion of liver NK cells in the NK cell group was(5.58±0.30)% and(3.73±0.42)% for 6 and 8 weeks after infection,respectively.There was no statistically significant difference between 6 weeks(5.51±0.27)%and 8 weeks(2.32±0.40)%after infection for the PBS group(t=0.25,2.64,P>0.05).The area of liver fibrosis in the monoclonal antibody group was(8.42±1.30)×10^(4)μm^(2) at 6 weeks after infection,which was larger than that in the IgG group(6.40±1.40)×10^(4)μm^(2) and normal saline group(6.73±1.61)×10^(4)μm^(2)(F=13.63,P<0.01).The area of liver fibrosis for the monoclonal antibody group was(9.55±1.55)×10^(4)μm^(2) at 8 weeks after infection,which was larger than that in the IgG group(6.11±1.10)×10^(4)μm^(2) and normal saline group(6.62±1.60)×10^(4)μm^(2)(F=30.33,P<0.01).The area of liver fibrosis in the NK cell group was(5.97±0.96)×10^(4)μm^(2) at 6 weeks after infection,which was smaller than that in the PBS group(8.27±1.62)×10^(4)μm^(2)(t=4.85,P<0.01).However,at 8 weeks after infection,the area of liver fibrosis for the NK cell group and the PBS group were(6.33±0.98)×10^(4)μm^(2) and(6.97±1.11)×10^(4)μm^(2),respectively.The difference was not statistically significant(t=1.80,P>0.05).The relative transcription levels of Ⅳ-C,FN,and LN mRNA in the monoclonal antibody group were 1.81±0.47,1.63±0.38 and 1.41±0.16,which are higher than that in the IgG group(1.00±0.35,0.81±0.29,0.79±0.16)and the normal saline group(1.00±0.12,1.00±0.10,1.00±0.14)(F=8.30,8.25,14.40,P<0.01).At 8 weeks after infection,the relative transcription level of Ⅳ-C mRNA in the monoclonal antibody group was 1.66±0.21,which is higher than that in the IgG group(0.94±0.26)and the saline group(1.00±0.09)(F=15.95,P<0.01).At 6 weeks after infection,the relative transcription levels for Ⅰ-C,Ⅲ-C and LN mRNA in the NK cell group were 0.66±0.12,0.61±0.06,0.64±0.09,respectively.The relative transcription levels were much higher compared with the PBS group(1.01%±0.14,1.01±0.14,1.01±0.16)(t=3.36,4.41,3.59,P<0.05).No statistical significant differences were observed at 8 weeks after infection for the NK cell group(0.82±0.40,0.93±0.37,0.73±0.30)and the PBS group(1.04±0.38,1.02±0.25,1.06±0.49)(t=0.55,0.27,0.81,P>0.05).Conclusion It was shown that after being infected with S.japonicum,NK cells from mouse liver might inhibit liver fibrosis in the early stage of its formation.