本文選題：樹突狀細胞　切入點：巨噬細胞　出處：《四川大學(xué)學(xué)報(醫(yī)學(xué)版)》2017年01期

【摘要】：目的觀察小鼠結(jié)腸癌CT26細胞培養(yǎng)上清中骨形成蛋白(BMPs)對樹突狀細胞(DCs)和巨噬細胞表面程序性死亡分子配體1(programmed death-ligand 1,PD-L1)表達的影響,探討其對免疫的調(diào)控。方法體內(nèi)實驗:將皮下接種和腹腔接種CT26腸癌細胞的BALB/c荷瘤小鼠隨機分為對照組、BMPs抑制劑LDN193189組和LDN193189聯(lián)合紫杉醇組,腫瘤接種第8天,分組給藥18d。測量腫瘤體積和腹圍,流式細胞術(shù)分別檢測其瘤體或腹水中DCs和巨噬細胞百分比及其表面PD-L1陽性表達率。體外實驗:對正常BalB/c小鼠骨髓分離培養(yǎng)的樹突狀細胞(BMDCs)和巨噬細胞(BMMs)分別做以下處理:1不作處理(對照組);2加入CT26上清;3與CT26不接觸共培養(yǎng);4加入CT26上清聯(lián)合LDN193189;5與CT26不接觸共培養(yǎng),加入LDN193189;6加入CT26上清聯(lián)合LDN193189和紫杉醇;7與CT26不接觸共培養(yǎng),加入LDN193189和紫杉醇。ELISA檢測CT26培養(yǎng)上清中是否有BMPs的表達,流式細胞術(shù)檢測不同處理下BMDCs和BMMs表面的PD-L1表達陽性率,RT-PCR和Western blot檢測干擾素調(diào)節(jié)因子1(IRF-1)mRNA和蛋白的表達。結(jié)果體內(nèi)實驗中,LDN193189組腫瘤體積或腹圍最大,DCs和巨噬細胞百分比及其表面PD-L1陽性表達率最低;體外實驗中,ELISA檢測結(jié)果顯示,BMPs在CT26上清中有表達,其質(zhì)量濃度為(0.59±0.09)ng/mL。加入CT26上清聯(lián)合LDN193189和與CT26不接觸共培養(yǎng)并且加入LDN193189組BMDCs和BMMs表面的PD-L1表達陽性率較低,接近對照組水平。RT-PCR和Western blot檢測結(jié)果顯示,對照組BMDCs、BMMs中IRF-1mRNA和蛋白表達低于與CT26不接觸共培養(yǎng)或加入CT26上清組;LDN193189加入到加有CT26上清或與CT26共培養(yǎng)的BMDCs和BMMs中,IRF-1mRNA和蛋白表達下降;而兩個LDN193189聯(lián)合紫杉醇組,IRF-1mRNA和蛋白表達均增加。結(jié)論 CT26分泌的BMPs增強樹突狀細胞和巨噬細胞表面PD-L1的表達。
[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 鹵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.
【作者單位】： 四川大學(xué)華西醫(yī)院肺癌中心生物治療國家重點實驗室;
【分類號】：R392

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