CT26细胞分泌的BMPs对小鼠树突状细胞和巨噬细胞表面PD-L1表达的影响

发布时间：2018-03-29 23:20

本文选题：树突状细胞　切入点：巨噬细胞　出处：《四川大学学报(医学版)》2017年01期

【摘要】：目的观察小鼠结肠癌CT26细胞培养上清中骨形成蛋白(BMPs)对树突状细胞(DCs)和巨噬细胞表面程序性死亡分子配体1(programmed death-ligand 1,PD-L1)表达的影响,探讨其对免疫的调控。方法体内实验:将皮下接种和腹腔接种CT26肠癌细胞的BALB/c荷瘤小鼠随机分为对照组、BMPs抑制剂LDN193189组和LDN193189联合紫杉醇组,肿瘤接种第8天,分组给药18d。测量肿瘤体积和腹围,流式细胞术分别检测其瘤体或腹水中DCs和巨噬细胞百分比及其表面PD-L1阳性表达率。体外实验:对正常BalB/c小鼠骨髓分离培养的树突状细胞(BMDCs)和巨噬细胞(BMMs)分别做以下处理:1不作处理(对照组);2加入CT26上清;3与CT26不接触共培养;4加入CT26上清联合LDN193189;5与CT26不接触共培养,加入LDN193189;6加入CT26上清联合LDN193189和紫杉醇;7与CT26不接触共培养,加入LDN193189和紫杉醇。ELISA检测CT26培养上清中是否有BMPs的表达,流式细胞术检测不同处理下BMDCs和BMMs表面的PD-L1表达阳性率,RT-PCR和Western blot检测干扰素调节因子1(IRF-1)mRNA和蛋白的表达。结果体内实验中,LDN193189组肿瘤体积或腹围最大,DCs和巨噬细胞百分比及其表面PD-L1阳性表达率最低;体外实验中,ELISA检测结果显示,BMPs在CT26上清中有表达,其质量浓度为(0.59±0.09)ng/mL。加入CT26上清联合LDN193189和与CT26不接触共培养并且加入LDN193189组BMDCs和BMMs表面的PD-L1表达阳性率较低,接近对照组水平。RT-PCR和Western blot检测结果显示,对照组BMDCs、BMMs中IRF-1mRNA和蛋白表达低于与CT26不接触共培养或加入CT26上清组;LDN193189加入到加有CT26上清或与CT26共培养的BMDCs和BMMs中,IRF-1mRNA和蛋白表达下降;而两个LDN193189联合紫杉醇组,IRF-1mRNA和蛋白表达均增加。结论 CT26分泌的BMPs增强树突状细胞和巨噬细胞表面PD-L1的表达。
[Abstract]:Objective to investigate the effects of bone morphogenetic protein (BMPs) on the expression of dendritic cells (DC) and 1(programmed death-ligand 1 (PD-L1), a molecular ligand on the surface of murine colon carcinoma (CT26) cells cultured in vitro. Methods: BALB/c bearing mice inoculated subcutaneously and intraperitoneally with CT26 intestinal cancer cells were randomly divided into control group (LDN193189 group) and LDN193189 combined with paclitaxel group. The tumor volume and abdominal circumference were measured. Flow cytometry was used to detect the percentage of DCs and macrophage and the positive expression rate of PD-L1 on the surface of tumor or ascites. In vitro, the dendritic cells (BMDCs) and macrophages (BMMs) isolated from bone marrow of normal BalB/c mice were treated with BMMs. The control group added CT26 supernatant 3 and CT26 non-contact co-culture and added CT26 supernatant combined with LDN19395 to co-culture without contact with CT26. CT26 supernatant combined with LDN193189 and paclitaxel 7 were added to LDN193189H6 to co-culture CT26 and LDN193189 and paclitaxel .ELISA were added to detect the expression of BMPs in the supernatant of CT26 culture. Flow cytometry was used to detect the positive rate of PD-L1 expression on the surface of BMDCs and BMMs under different treatments. RT-PCR and Western blot were used to detect the expression of IFN- regulatory factor 1(IRF-1)mRNA and protein. The score ratio and the positive expression rate of PD-L1 on the surface were the lowest. The results of Elisa in vitro showed that BMPs were expressed in the supernatant of CT26 with a mass concentration of 0.59 卤0.09 ng / mL. The positive rate of PD-L1 expression on the surface of BMDCs and BMMs was lower in the group of adding CT26 supernatant combined with LDN193189 and non-contact with CT26 and adding LDN193189 to the surface of BMDCs and BMMs. Close to the control level. RT-PCR and Western blot analysis showed that the expression of IRF-1mRNA and protein in BMDCshBMMs in control group was lower than that in control group without contact with CT26 or in CT26 supernatant group. LDN193189 was added to BMDCs and BMMs with CT26 supernatant or co-cultured with CT26, and the expression of IRF-1 mRNA and protein in BMDCs and BMMs co-cultured with CT26 was lower than that in control group. The expression of IRF-1 mRNA and protein was increased in both LDN193189 and paclitaxel groups. Conclusion BMPs secreted by CT26 can enhance the expression of PD-L1 on dendritic cells and macrophages.
【作者单位】：四川大学华西医院肺癌中心生物治疗国家重点实验室;
【分类号】：R392

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