熊胆粉联合环磷酰胺用药通过调控肿瘤微环境抑制结直肠癌肝转移作用的研究

Study on inhibitory effect of combined administration of bear bile powder and cyclophosphamide on colorectal cancer liver metastasis by regulating tumor microenvironment

目的:探讨熊胆粉联合环磷酰胺(cyclophosphamide,cytoxan,CTX)用药通过调节肿瘤炎症微环境对小鼠结直肠癌(colorectal carcinoma,CRC)肝转移模型的增免抑瘤抗转移作用。方法:脾脏原位注射SL4细胞制作小鼠CRC肝转移模型。随机分5组:模型组、CTX组(80mg·kg-1)和CTX联合熊胆粉高、中、低剂量组(300,150,75mg·kg-1)。术后口服灌胃给药12d,于第13天取材,解剖肝脾称重,HE染色,免疫荧光分析;外周血、脾和肝转移瘤进行流式细胞免疫表型分析。结果:CTX组及CTX联合熊胆粉各组肝脾质量明显低于模型组(P<0.05);HE染色和免疫荧光分析表明,正常组织中淋巴细胞浸润较多,肿瘤组织周围有以巨噬细胞为主的大量炎细胞浸润;流式细胞检测结果表明:外周血中,联合治疗组与CTX组相比,T淋巴细胞显著升高(P<0.05),CD4/CD8增加;脾细胞悬液中,联合治疗组与CTX组比较,淋巴细胞总数增加,以B细胞增加较为显著(P<0.05),CD11b,F4/80细胞明显降低(P<0.05);肝转移瘤中,联合治疗后比单独CTX治疗单核巨噬细胞下降(P<0.05)。结论:熊胆粉联合CTX治疗不仅能保肝增免,还可通过调节肿瘤微环境,减少对单核巨噬细胞等的募集起到抗炎进而抗肿瘤转移作用,其中熊胆粉低剂量与CTX联合用药组降低炎症细胞浸润的作用最为显著。

Objective： To explore the inhibitory effect of combined administration of bear bile powder （BBP） and cyclophosphamide （Cytoxan, CTX） on colorectal cancer liver metastasis by regulating tumor promotion inflammation microenvironment. Method ： The CRC liver metastasis mode in mice was established through in situ spleenic injection of SL4 tumor cells into spleens. The mice were randomly divided into 5 groups ： the model group, the CTX （ 80 mg · kg- 1 ） treatment group, the CTX ＋ BBP high dose （ 300 mg · kg-1） group, the CTX ＋ BBP middle dose （ 150 mg ~ kg 1 ） group and the CTX ＋ BBP low dose （75 mg · kg-1 ） group. Mice were orally administered with drugs for 12 days, and sacrificed on the 13th day for weighing their spleens and lives, HE staining, and immunofluorescence analysis. Their peripheral blood, and metastatic tumor in spleens and lives were analyzed with fiow cytometry. Result： Spleen and liver weights of the CTX treatment group and other doses groups were significantly lower than that of the model group. HE staining and immunofluorescence analysis showed that lymphocyte infiltration was detected in normal tissues, and macrophages infiltration was observed around the tumor tissues. Flow cytometry analysis showed that the number of T-lymphocytes in peripheral blood of different doses groups were much higher than that of the CTX treatment group （ P 〈 0.05 ） , with the rise in the ratio of CIM/CD8 ; the total number of lymphocytes in spleen cell suspension increased in different doses groups, compared to the CTX treatment group, with notable increase in B cells （P 〈0.05） and significant decrease in CDllb, F4/80 cells （P 〈0.05）. The combined treatment showed less monocyte macrophages in liver metastasis than that of the CTX treatment group. Conclusion： The combined treatment of bear bile powder and cyclophosphamide has the effect in not only protecting liver and increase immunity, but also in anti-inflammation and antitumor by regulating tumor microenvironment and reducing the collection of mononuclear macrophages. Particularly, the combined administration of low dose of bear bile powder and CTX shows the most significant effect in reducing inflammatory cell infiltration.