SOCS1沉默DC联合CIK细胞体外抗三阴乳腺癌作用及机制研究

Preliminary study on the antitumor activity and mechanism of SOCS1 silenced-dendritic cells and cytokine-induced killer cells against triple negative breast cancer in vitro

目的树突状细胞(dendritic cell,DC)联合细胞因子诱导杀伤细胞(cytokine-induced killer,CIK)免疫治疗是一种肿瘤治疗新模式。近年来发现,细胞因子信号抑制因子1(suppressor of cytokine signaling 1,SOCS1)是一种重要的抑制DC活化的负调控因子。本研究旨在初步探讨SOCS1沉默DC联合CIK对三阴乳腺癌细胞MDA-MB-231的体外杀伤作用和机制。方法选取2015-12-01-2017-12-01于广西壮族自治区人民医院住院的晚期三阴乳腺癌患者,采集患者外周血,利用细胞因子分别诱导培养DC和CIK细胞,RNA干扰技术沉默DC中SOCS1,实验分为空白对照(Blank)组、阴性对照(NC)组以及SOCS1沉默〔SOCS1(-)〕组,收集3组DC分别与CIK细胞共培养7d,获得3组DC-CIK细胞。实时荧光定量PCR法(real-time quantitative PCR,Realtime qPCR)检测DC中SOCS1siRNA沉默效果,流式细胞术检测3组DC及CIK细胞表型。CCK8试剂盒检测3组DC-CIK细胞对三阴乳腺癌细胞MDA-MB-231体外杀伤作用。酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)分析3组DC-CIK细胞上清中γ-干扰素(interferon-γ,IFN-γ)分泌水平。流式细胞术检测3组DC-CIK细胞对三阴乳腺癌细胞MDA-MB-231凋亡影响,蛋白质印迹法进一步检测MDA-MB-231细胞中凋亡通路蛋白、B细胞淋巴瘤/白血病-2(B-cell lymphoma-2,Bcl-2)及Bcl-2相关蛋白X(Bcl-2-associated X protein,Bax)表达水平的变化。结果体外成功诱导出DC和CIK细胞,SOCS1siRNA序列可下调DC中SOCS1表达水平(0.07±0.01、1.00±0.15和0.89±0.11),均P<0.001。流式细胞术检测发现,SOCS1(-)组DC表面分子CD80、CD83和HLA-DR的阳性表达率均上升,分别达到(67.70±1.77)%、(55.63±2.77)%和(79.50±1.31)%,F值分别为48.59、58.22和70.84,均P<0.001。同时,SOCS1(-)组DC-CIK细胞中CD3+CD8+、CD3+CD56+阳性细胞比例分别上调至(60.43±1.02)%和(37.63±3.53)%,F值分别为38.79和87.41,均P<0.001。SOCS1(-)组DC-CIK细胞对三阴乳腺癌细胞MDA-MB-231具有更强的体外杀伤作用,F=85.78,P<0.001;其上清中IFN-γ干扰素分泌水平升高至(228.30±5.85)pg/mL,F=125.66,P<0.001。SOCS1(-)组DC-CIK细胞作用的靶细胞MDA-MB-231凋亡率增加到(18.00±1.80)%,F=93.04,P<0.001;MDA-MB-231中Bcl-2蛋白的表达量降低,F=178.07,P<0.001;Bax蛋白的表达量升高,F=742.14,P<0.001。结论沉默DC的SOCS1后,可以提高DC-CIK细胞对三阴乳腺癌细胞MDA-MB-231体外杀伤作用,同时DC-CIK细胞上清中IFN-γ干扰素分泌量增加。此外,DC-CIK可能通过诱导三阴乳腺癌细胞的凋亡,发挥抗肿瘤作用。

OBJECTIVE Immunotherapy of dendritic cell(DC)combined with cytokine-induced killer(CIK)is a new model of tumor therapy.Suppressor of cytokine signaling 1(SOCS1)is an important negative regulator inhibiting DC activation.The purpose of this study was to preliminarily explore the killing effect and mechanism of SOCS1 silencing DC combined with CIK against triple-negative breast cancer cells MDA-MB-231 in vitro.METHODS Patients with advanced triple-negative breast cancer admitted to Guangxi Zhuang Autonomous Region People’s Hospital from December 1,2015 to December 1,2017 were collected.Gathering the peripheral blood of patients,DC and CIK cells were generated from peripheral blood mononuclear cells(PBMCs)by using cytokines,and SOCS1 siRNA was applied to knockdown SOCS1 expression in DC.The experiment was divided into blank control group(Blank group),negative control group(NC group)and SOCS1 silent group[SOCS1(-)group],then three groups DC and CIK were co-cultured for 7 days respectively,to obtain three groups of DC-CIK cells.Real-time quantitative PCR(Realtime qPCR)was used to analyze the silencing effect of SOCS1 siRNA in DC,and flow cytometry was used to detect the phenotypes of DC and CIK cells in three groups.The killing effect of three group DC-CIK cells on MDA-MB-231 breast cancer cells in vitro was detected by CCK8 kits.The secretion of IFN-γinterferon in the supernatant of DC-CIK cells in three groups was measured by ELISA kits.Flow cytometry was used to detect the effects of DC-CIK cells in the three groups on the apoptosis of MDA-MB-231 breast cancer cells.Western blot(WB)was performed to further detect the apoptosis pathway protein,b-cell lymphoma/lymphoma 2(Bcl-2)and Bcl-2-related protein X(Bax)in MDA-MB-231 cells.RESULTS DC and CIK cells were obtained successfully in vitro,and the expression of SOCS1 was knocked down statistically by SOCS1 siRNA(0.07±0.01,1.00±0.15,0.89±0.11,both P<0.001).Flow cytometry showed that the positive expression rates of CD80,CD83 and HLA-DR,the surface molecules of DC,in the SOCS1(-)group were increased,reaching(67.70±1.77)%,(55.63±2.77)% and(79.50±1.31)%,respectively(F=48.59,P<0.001;F=58.22,P<0.001;F=70.84,P<0.001).Meanwhile,the proportion of CD3+CD8+and CD3+CD56+positive cells in DC-CIK cells of the SOCS1(-)group was increased to(60.43±1.02)% and(37.63±3.53)% respectively(F=38.79,P<0.001;F=87.41,P<0.001).DC-CIK cells in SOCS1(-)group had stronger killing effect against triple negative breast cancer cells MDA-MB-231 in vitro(F=85.78,P<0.001),and the secretion level of IFN-γinterferon in its supernatant was significantly increased to(228.30±5.85)pg/mL,F=125.66,P<0.001.The apoptosis rate of MDA-MB-231 cells induced by SOCS1(-)group DC-CIK was significantly increased to(18.00±1.80)%,F=93.04,P<0.001.The expression of Bcl-2 protein in MDA-MB-231 was decreased(F=178.07,P<0.001),but the expression of Baxin MDA-MB-231 was increased(F=742.14,P<0.001).CONCLUSIONS Silencing SOCS1 in DC could improve the killing effect of DC-CIK cells against triple negative breast cancer cells MDA-MB-231 invitro.The secretion of IFN-γin the supernatant of DC-CIK cells is increased.Furthermore,DC-CIK may play an anti-tumor role by inducing apoptosis of triple negative breast cancer cells.