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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 investigate the effect of bone morphogenetic protein (BMPs) on the expression of programmed death-ligand 1 (PD-L1) on dendritic cells (DCs) The impact of its regulation of immunity. Methods In vivo experiments: BALB / c mice bearing hypodermic and intraperitoneal CT26 colon cancer cells were randomly divided into control group, LDN193189 group and LDN193189 group and paclitaxel group. On the 8th day after tumor inoculation, the mice were divided into groups and given for 18 days. The tumor volume and abdominal circumference were measured. The percentage of DCs and macrophages in the tumor or ascites and the positive rate of PD-L1 on the surface were detected by flow cytometry. In vitro experiments: Dendritic cells (BMDCs) and macrophages (BMMs) isolated from bone marrow of normal BalB / c mice were treated as follows: 1 without treatment (control group); 2 with CT26 supernatant; 3 with CT26 Without contact with co-culture; 4 added CT26 supernatant combined with LDN193189; 5 CT26 non-contact co-culture, add LDN193189; 6 added CT26 supernatant combined LDN193189 and paclitaxel; 7 CT26 non-contact co-culture, add LDN193189 and paclitaxel. The expression of BMPs in CT26 culture supernatants was detected by ELISA. The positive rate of PD-L1 expression on BMDs and BMDs was detected by flow cytometry. The expression of interferon regulatory factor 1 (IRF-1) mRNA and protein expression. Results In vivo, LDN193189 group had the largest tumor volume or abdominal circumference with the lowest percentage of DCs and macrophages and the lowest positive rate of PD-L1 on the surface. In vitro experiments, the results of ELISA showed that BMPs were expressed in CT26 supernatant, The mass concentration was (0.59 ± 0.09) ng / mL. The positive rate of PD-L1 expression on the surface of BMDCs and BMMs after adding CT26 supernatant combined with LDN193189 and not co-cultured with CT26 and adding LDN193189 group was lower than that of control group. The results of RT-PCR and Western blot showed that the expression of IRF-1mRNA and protein in BMDCs and BMMs in the control group was lower than that in the CT26 supernatant group or in the CT26 supernatant group; LDN193189 was added to the supernatant with CT26 or co-cultured with CT26 Of BMDCs and BMMs, IRF-1mRNA and protein expression decreased; while two LDN193189 combined with paclitaxel group IRF-1mRNA and protein expression increased. Conclusion CT26 secreted BMPs enhance the expression of PD-L1 on the surface of dendritic cells and macrophages.