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  

Bone Morphogenetic Proteins from CT26 Regulate the Expression of PD-L1 in Murine Dendritic Cells and Macrophages

作　　者：赵茂媛 王喻义 杜驰[1] 罗锋[1] 

机构地区：[1]四川大学华西医院肺癌中心生物治疗国家重点实验室,成都610041

出　　处：《四川大学学报（医学版）》2017年第1期33-40,共8页Journal of Sichuan University(Medical Sciences)

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。Objective To analyze the influence of bone morphogenetic proteins （BMPs） from CT26 on PD-L1 of dendritic cells and macrophages. Methods In vivo, we respectively inoculated CT26 colon cancer cells subcutaneously and intraperitoneally to BALB/c mice.The mice were randomly assigned to three groups and treated with ① normal saline; ② BMPs inhibitor LDN193189; ③ BMPs inhibitor LDN193189 combined with paclitaxel, respectively. The treatments started on the eighth day after inoculating, when the tumor volume reached 150 mm3 or the abdominal circumference was greater than 6 cm. After 2 weeks of treatments, the mice were sacrificed.The counts of dendritic cells and macrophages and the expression of PD-L1 in tumors or ascites were detected by flow cytometry （FCM）.In vivo, the dendritic cells and macrophages from normal BALB/c mice bone marrow were exposed to： ① no treatment; ② CT26 supernatant; ③ CT26; ④ CT26 supernatant and LDN193189; ⑤ CT26 and LDN193189; ⑥ CT26 supernatant, LDN193189 and paclitaxel; ⑦ CT26, LDN193189 and paclitaxel. BMPs from CT26 was detected by ELISA.The counts of dendritic cells and macrophages and their PD-L1 expressions were detected by FCM. IRF-1 expression was detected by real-time （RT）-PCR and Western blot. Results In vivo, LDN193189 treated mice had the greatest tumor size or abdominal circumference, with least dendritic cells andCM（155.3mm]macrophages and expressions of PD-L1.In vivo, ELISA test results showed that the concentration of BMPs inCT26 supernatant was （0.59±0.09） ng/mL. FCM, RT-PCR and Western blot showed that dendritic cells and macrophages exposed to CT26 supernatant and LDN193189 or CT26 and LDN193189 expressed the least PD-L1 and IRF-1, which was close to those without treatment. While added the PTX to the above treatment, the expressions of PD-L1 and IRF 1 increased in the test results. Conclusion BMPs from CT26 up-regulate the expression of PD-L1 in murine dendritic cells and macrophages.

关 键 词：树突状细胞 巨噬细胞 PD-L1 BMPS 

分 类 号：R392[医药卫生—免疫学]