M1巨噬细胞回输治疗小鼠肝癌的研究

Study on M1 macrophage transfusion in the treatment of hepatocellular carcinoma in mice

目的研究回输M1型骨髓来源的巨噬细胞(BMDMs)对原位肝肿瘤小鼠模型的治疗作用。方法①分离野生小鼠的骨髓细胞并体外诱导成BMDMs(即M0型巨噬细胞),在IFN-γ+LPS诱导下极化为M1型巨噬细胞。通过肝内接种Luciferase-Hepa1-6细胞建立原位小鼠肝癌模型。在接种肿瘤7 d后,经尾静脉回输PBS(对照组)、M0型和M1型巨噬细胞,通过小动物活体成像技术检测小鼠肝脏肿瘤的生长状态。接种21 d后,分离并观察肝脏肿瘤大小及称取质量,确定回输不同类型巨噬细胞对小鼠肝脏肿瘤进展的影响。利用流式细胞仪分析肝脏肿瘤癌旁组织内巨噬细胞亚群变化。②分离GFP转基因小鼠的骨髓细胞,诱导分化成M0型和M1型巨噬细胞后经尾静脉回输至肝癌小鼠体内,利用流式细胞仪及免疫荧光等方法检测回输1、3、7 d后肝脏内GFP阳性的BMDMs细胞数。结果与对照组相比,回输M0型和M1型BMDMs均促进了肝肿瘤的生长(P<0.05)。回输M0型或M1型BMDMs后,癌旁组织中枯否细胞增多,骨髓单核来源的炎性巨噬细胞增多(P<0.05)。经尾静脉回输1×10^(6)个M0型或M1型GFP+BMDMs,1 d后约3×10^(3)个细胞驻留于肝脏中,并随着时间增长逐渐减少。结论回输M1型巨噬细胞并未能达到治疗肝肿瘤的目的,反而促进肝肿瘤的生长。

Objective To investigate the therapeutic effect of M1 bone marrow-derived macrophages(BMDMs)on the mouse model of orthotopic liver tumor.Methods①Bone marrow cells isolated from wild mice were induced into BMDMs(M0 macrophages)in vitro,which were polarized into M1 macrophages under IFN-γ+LPS induction.An orthotopic mouse model of hepatocellular carcinoma was established by intrahepatic inoculation of Luciferase-Hepa1-6 cells.Seven days after tumor inoculation,PBS(control group),M0 and M1 macrophages were transfused through the tail vein,and the growth status of mouse liver tumors was detected by in vivo small-animal imaging.Twenty-one days after inoculation,liver tumor size and mass were isolated and observed to determine the effects of different types of macrophages on the progression of liver tumors in mice.Flow cytometry was used to analyze the changes of macrophage subsets in liver tumor-adjacent tissues.②Bone marrow cells of GFP transgenic mice were isolated and induced to differentiate into M0 and M1 macrophages,and then transfused into hepatocellular carcinoma mice via the tail vein.The number of GFP positive BMDMs in the liver was detected by flow cytometry and immunofluorescence at 1,3 and 7 d after transfusion.Results Compared with the control group,both M0 and M1 BMDMs transfusion promoted liver tumor growth(P<0.05).After transfusion of M0 or M1 BMDMs,Kupffer cells and inflammatory macrophages increased in paracancerous tissues(P<0.05).After 1×10^(6) M0 or M1 GFP+BMDMs were transfused via the tail vein,about 3×10^(3) cells remained in the liver after 1 d,and gradually decreased with time.Conclusion M1 macrophage transfusion does not achieve the purpose of treating liver tumors,but promotes the growth of liver tumors.