中国麻风皮肤病杂志 ›› 2022, Vol. 38 ›› Issue (9): 583-588.doi: 10.12144/zgmfskin202209583

• 论著 • 上一篇    下一篇

黑素瘤小鼠脾脏淋巴细胞抗肿瘤效应及机制的初步探究

邹锐涛1,2,蔡桂月1,2,陈晓旋1,肖铿2,樊志金2,陈嵘祎1,2   

  1. 1广东医科大学，广东广州， 523024；
    2南方医科大学皮肤病医院，广东广州，510091
  • 出版日期:2022-09-15 发布日期:2022-07-13

Preliminary study on antitumor effect and mechanism of spleen lymphocytes in melanoma mice

ZOU Ruitao1,2, CAI Guiyue1,2, CHEN Xiaoxuan1, XIAO Keng2, FAN Zhijin2, CHEN Rongyi1,2   

  1. 1 Guangdong Medical University,Guangdong-Guangzhou 523024, China;
    2 Dermatology Hospital,Southern Midecal University,Guangdong-Guangzhou 510019, China
  • Online:2022-09-15 Published:2022-07-13

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摘要： 目的：探究B16黑素瘤小鼠脾脏淋巴细胞的抗肿瘤效应与机制，为过继性免疫治疗提供新的思路。方法：B16黑素瘤小鼠模型构建，B16细胞分别与正常小鼠脾脏淋巴细胞（Spleen lymphocytes of normal mice，NL）和荷瘤小鼠脾脏淋巴细胞（Splenic lymphocytes of tumor-bearing mice，ML）按照1:20共培养，并加入刀豆蛋白A(ConA)刺激24 h，CCK8检测各组细胞活力，Calcein/PI荧光染色同步检测；流式细胞术检测NL/ML中CD4+、CD8+比例占比；ConA刺激NL/ML 24 h，Western blot检测PD-1、颗粒酶B、穿孔素的表达水平，ELISA检测TNF-α、IFN-γ表达水平。结果：CCK8以及Calcein/PI荧光染色结果提示ML具有更明显的抑瘤效果；与NL相比，在ML中CD8+表达比例增加，PD-1表达下调（P<0.05）；ConA刺激后，ML中颗粒酶B、穿孔素以及TNF-α、IFN-γ表达较NL上调（P<0.05）。结论：黑素瘤小鼠脾脏淋巴细胞较正常小鼠脾脏淋巴细胞对黑素瘤具有更明显的抑瘤效应。

关键词: 黑素瘤, 荷瘤小鼠, PD-1, 过继性细胞疗法

Abstract: Objective: To explore the antitumor effect and mechanism of spleen lymphocytes in mice with B16 melanoma, and provide new ideas for adoptive immunotherapy. Methods: Spleen lymphocytes of normal mice were extracted from B16 cells (NL) and splenic lymphocytes of tumorbearing mice (ML) were co-cultured according to 1:20, and ConA was added to stimulate cells for 24h. CCK8 was used to detect cell viability of all groups, and Calcein/PI fluorescent staining was performed simultaneously. The proportion of CD4+ and CD8+ in NL/ML was detected by flow cytometry. ConA stimulated NL/ML for 24h, the expression levels of PD-1,Granase B and perforin were detected by Western blot, and the expression levels of TNF-α and IFN-γ were detected by ELISA. Results: CCK8 and Calcein/PI fluorescence staining indicated that ML had more obvious tumor suppressive effect. Compared with NL, the level of CD8+ expression proportion was higher in ML, and PD-1 expression was lower (P<0.05). After ConA stimulation, the expression of Granase B, perforin, TNF-α, IFN-γ in ML was higher than that in NL (P<0.05). Conclusion: Compared with normal mice, spleen lymphocytes of B16 cells have more obvious antitumor effect on melanoma.

Key words: melanoma, Tumor-bearing mice, PD-1, adoptive cell therapy