NLRP3炎性复合体表达水平与小鼠异基因造血干细胞移植肝损伤的关系研究

The relationship between NLRP3 inflammsomes expression and liver damage after allogeneic hematopoietic stem cell transplantation in mice model

目的 探索NOD样受体蛋白3（NLRP3）炎性复合体表达与小鼠异基因造血干细胞移植（allo-HSCT）肝损伤的关系.方法 建立allo-HSCT小鼠模型,流式细胞术检测受鼠骨髓嵌合率;通过HE染色、免疫组化染色及Masson染色观察肝组织病理形态,通过病理损伤评分评价肝损伤程度;用Western blot法检测肝脏组织炎性细胞及NLRP3炎性复合体表达水平;用实时荧光定量PCR法检测肝组织NLRP3炎性复合体组分相关基因及细胞因子IL-1β、IL-18 mRNA表达水平.结果 allo-HSCT后第10天（＋10 d）,供鼠造血干细胞基本上完全植入,嵌合率大于97％,同时肝脏病理形态显示有肝损伤,＋15d肝损伤最重,随后逐渐减轻;在肝损伤过程中伴有IL-1β、IL-18 mRNA表达水平增高,＋15d时二者表达量分别为正常组的（1.19±0.40）倍和（1.64±0.76）倍;在肝损伤最严重时伴有caspase-1mRNA表达水平增高,为正常组的（3.51±0.46）倍;移植后肝组织细胞NLRP1、NLRP3、NLRC4、NLRP5mRNA均有一定程度的表达,其中NLRP3表达水平最高,其mRNA及蛋白表达水平与肝损伤程度同步,＋15d时mRNA表达量为正常组的（2.91±0.41）倍.结论 NLRP3在allo-HSCT小鼠表达水平明显增高,可能为移植后肝脏炎症性损伤的因素之一.

Objective To explore the function ofnucleotide-binding domain （NOD）-like receptor protein 3 （NLRP3） inflammsomes in liver damage after allogeneic hematopoietic stem cell transplantation （allo-HSCT）.Methods The study presented a murine （BALB/c-based） model of allo-HSCT.Chimera rate was measured by flow cytometry.The hematoxylin-eosin,Masson＇ s trichrome,immunohistochemistry staining were used to observe the pathology changes in liver,then measured the degree of liver damage.Inflammation cells and NLRP3 were measured by Western blot,cytokines IL-1β,IL-18 and NLRP3 related genes were tested with real-time quantitative polymerase chain reaction （q-PCR）.Results Hematopoietic stem cells had been successfully transplanted,the chimera rate was geater than 97％ on the 10th day.Liver damage occurred after allo-HSCT and suffered infiltration of inflammation cells,which reached the peak on day 15,then moved to moderate; the cytokines IL-1β,IL-18 had the similar trend with liver injury,and reached the highest level on day 15,their mRNA expressions increased by （1.19±0.40） fold and （1.64±0.76） fold,respectively; Meanwhile,caspase-1 had the similar trend,its mRNA expression increased by （3.51±0.46） fold on day 15; the inflammasomes NLRP1,NLRP3,NLRC4 and NLRP5 expressed in liver on day 15 of post-allo-HSCT,and NLRP3 inflammasome expressed highest among them.The mRNA and protein level of NLRP3 inflammasomes were kept with the serious degree of the liver damage,its mRNA expression increased by （2.91±0.41） fold on day 15.Conclusion NLRP3 inflammsome expressed in liver injury during allo-HSCT in mice,and may be one of the important factors contributed to liver injury.