过继骨髓细胞通过分化为NKT细胞并增加自身脂质含量减轻小鼠代谢功能障碍相关脂肪性肝病肝损伤

Adoptive bone marrow cells can reduce the liver injury of metabolic-dysfunctionassociated fatty liver disease in mice by differentiating into natural killer T(NKT)cells and increasing their own lipid content

目的 探讨骨髓细胞过继对于小鼠代谢功能障碍相关脂肪性肝病(MAFLD)的治疗作用,并探索起主要作用的细胞群体。方法 HE染色检测蛋氨酸-胆碱缺乏(MCD)诱导的C57BL/6小鼠MAFLD肝组织病变并通过检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平评估骨髓细胞过继对MAFLD的治疗作用。实时定量PCR检测T细胞、自然杀伤T(NKT)细胞、 Kupffer细胞等肝脏免疫细胞的低密度脂蛋白受体(LDLR)和白细胞介素4(IL-4) mRNA表达。羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)标记的骨髓细胞经小鼠尾静脉注射,采用冰冻切片观察肝组织CFSE+细胞比例,流式细胞术追踪CFSE+细胞在肝、脾组织的比例。流式细胞术检测CFSE+的过继细胞CD3、 CD4、 CD8、 NK1.1、 CD11b、粒细胞受体1(Gr-1)的表达确定其分化情况。尼罗红脂质染色评估肝组织NKT细胞胞内脂质含量。结果 骨髓细胞过继输入的MAFLD小鼠肝组织损伤明显减轻、血清ALT、 AST水平降低。同时,肝脏免疫细胞上调其IL-4和LDLR的表达。LDLR敲除小鼠给予MCD饮食后引发更严重的MAFLD。骨髓细胞过继细胞治疗效果显著并分化出更多的NKT细胞定殖在肝脏,同时该群NKT细胞胞内脂质显著增多。结论骨髓细胞过继治疗通过分化更多的NKT细胞,同时通过提高该群细胞的胞内脂质含量从而减轻MAFLD小鼠肝损伤。

Objective To investigate the therapeutic effect of bone marrow cell adoptive therapy on metabolic-dysfunctionassociated fatty liver disease(MAFLD)in mice and its possible cell population.Methods The staining was used to detect the liver lesions of MAFLD in C57BL/6 mice induced by methionine and choline deficiency diet(MCD)and the adoptive therapeutic effect of bone marrow cells on MAFLD was evaluated by detecting the levels of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST).The mRNA expressions of low density lipoprotein receptor(LDLR)and interleukin-4(IL-4)in liver immune cells(including T,NKT,Kupffer cells and other cell populations)were detected by real-time quantitative PCR.The bone marrow cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester(CFSE)were injected into the tail vein of mice.The proportion of CFSE positive cells in liver tissue was observed by the frozen section,and the proportion of labeled cells in the liver and spleen was tracked by flow cytometry.The expression of CD3,CD4,CD8,NK1.1,CD11b and Gr-1 in CFSE labeled adoptive cells was detected by flow cytometry.The intracellular lipid content of NKT cells in liver tissue was evaluated by Nile Red lipid staining.Results The injury of liver tissue and the levels of serum ALT and AST in MAFLD mice were significantly reduced.At the same time,liver immune cells up-regulated the expression of IL-4 and LDLR.LDLR knockout mice induced more severe MAFLD after giving MCD diet.Bone marrow adoptive cells had a significant therapeutic effect and differentiated more NKT cells to colonize the liver.At the same time,the intracellular lipids of these NKT cells increased significantly.Conclusion Bone marrow cell adoptive therapy can reduce liver injury in MAFLD mice by differentiating more NKT cells and increasing the intracellular lipid content of these cells.