南方医科大学学报

南方医科大学学报 ›› 2018, Vol. 38 ›› Issue (01): 20-.

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sPD-1过表达增强衰老肿瘤细胞疫苗抗小鼠乳腺癌作用

陈泽宏，林惠文，胡康，苏如雄，赖楠，杨子科，康世均   

  • 出版日期:2018-01-20 发布日期:2018-01-20

Soluble PD-1 over-expression enhances the anti-tumor effect of senescence tumor cell vaccine against breast cancer cell growth in tumor-bearing mice

  • Online:2018-01-20 Published:2018-01-20

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摘要： 目的探讨可溶性PD-1（sPD-1）过表达后对衰老肿瘤细胞疫苗（STCV）抗小鼠乳腺癌的增强作用。方法干扰素γ（IFN-γ） 刺激小鼠乳腺癌细胞4T1，流式细胞术检测PD-L1表达；sPD-1过表达慢病毒感染4T1，显微镜观察增强绿色荧光蛋白表达情况； CCK8实验比较4T1、4T1/sPD-1增殖活力；qRT-PCR、Western blot 从mRNA、蛋白质水平验证sPD-1表达；干扰素γ预处理4T1细 胞，添加4T1/sPD-1培养上清，孵育后流式细胞术检测PD-1阳性细胞比例；X射线照射联合Veliparib处理4T1、4T1/sPD-1细胞， 衰老相关β-半乳糖苷酶（SA-β-gal）染色，观察蓝染细胞比例；Balb/c小鼠右后腿皮下种植4T1，左后腿注射PBS、4T1 STCV、4T1/ sPD-1 STCV，观察各组小鼠成瘤率。结果IFN-γ能导致4T1细胞PD-L1上调（P<0.001），且浓度越高，PD-L1表达上调越明显， 最高达（84.80±1.03）%；病毒感染4T1细胞后，显微镜下可见绿色荧光；CCK8实验中4T1、4T1/sPD-1细胞增殖曲线无明显差异 （P>0.05）；4T1/sPD-1细胞在mRNA和蛋白质上均可检测到sPD-1表达产物，4T1细胞则均未能检测到；干扰素γ预处理的4T1细 胞，PD-1阳性比例（6.893±0.271）%，添加4T1/sPD-1细胞培养液处理后，PD-1阳性细胞比例升高达（55.450±0.555）%（P<0.001）； 4T1、4T1/sPD-1在联合处理后，镜下见大量蓝染变形的衰老细胞；小鼠荷瘤实验中，预防肿瘤发生实验，PBS组所有小鼠出现肿 瘤生长，4T1 STCV组有28.787%小鼠未发生肿瘤，4T1/sPD-1 STCV组近55.556%小鼠未发现肿瘤发生；治疗肿瘤实验中，观察 期内PBS组小鼠均发生肿瘤，4T1 STCV、4T1/sPD-1 STCV组无瘤小鼠比例分别为11.111%和38.89%。结论衰老肿瘤细胞疫 苗对小鼠乳腺癌具有防治作用，且sPD-1过表达后能够增强衰老肿瘤细胞疫苗的抗肿瘤作用。

Abstract: Objective To investigate whether soluble PD-1 overexpression in 4T1 senescence tumor cells enhances the antitumor effect of senescence tumor cell vaccine (STCV) against breast tumor cells in a tumor-bearing mouse model. Methods 4T1 cells were treated with interferon-γ (IFN-γ) and the expression of PD-L1 was detected by flow cytometry. CCK8 assay was used to compare the cell proliferation activity between 4T1 cells and 4T1 cells infected by a lentiviral vector of sPD-1 (4T1/sPD- 1 cells), and the expressions of sPD-1 mRNA and protein in 4T1/sPD-1 cells were detected using qPCR and Western blotting. The culture supernatant of 4T1/sPD-1 cells was added in 4T1 cells pre-treated with IFN-γ, and PD-1-positive 4T1 cells were detected with flow cytometry. Senescence β-galactosidase staining kit was used to detect the senescent 4T1 and 4T1/sPD-1 cells following exposure to X- ray radiation and Veliparib. Balb/c mice bearing subcutaneous 4T1 tumor xenografts were treated with injections of PBS, 4T1 STCV, or 4T1/sPD-1 STCV, and tumor growth was observed. Results Stimulation with IFN-γ concentration-dependently up-regulated PD-L1 expression by as much as (84.80 ± 1.03)% in 4T1 cells (P<0.001). sPD-1 overexpression in 4T1 cells did not significantly affect the cell proliferation. Treatment of 4T1 cells with 4T1/sPD-1 cell culture supernatant significantly increased the proportion of PD-1 + cells from (6.893 ± 0.271)% to (55.450 ± 0.555)% (P<0.001). X-ray irradiation combined with Veliparib caused obvious senescence in 4T1 and 4T1/sPD-1 cells. In the tumor-preventing experiment, tumor formation occurred in all the mice in PBS group; 28.787% of the mice in 4T1 STCV group and 55.556% in 4T1/sPD-1 STCV group showed no tumor formation. In the tumor treatment experiment, tumor formation occurred in all the mice in PBS groups while in 4T1 STCV and 4T1/sPD-1 STCV groups, 11.111% and 38.89% of the mice were tumor-free during the observation period, respectively. Conclusions Senescence tumor cells vaccine has antitumor effect against breast cancer in mice, and sPD-1 overexpression can enhance this effect of the vaccine.