Fractalkine、IP-10及不同信号通路抑制剂对肿瘤微环境中NK细胞的影响

Effect of fractalkine, IP-10 and different signal pathway inhibitors on NK cells in the tumor microenvironment

目的探讨Fractalkine(FKN)、干扰素诱导蛋白10(IP-10)以及不同信号通路抑制剂对肿瘤微环境自然杀伤(NK)细胞数目和功能的影响。方法免疫组化染色观察人乳腺癌组织中CD56和DAP10的分布情况。通过共培养小鼠巨噬细胞RAW 264.7与小鼠乳腺癌细胞4T1模拟体外肿瘤微环境(数目比例1:4),以小鼠NK细胞KY-1为研究对象。RTPCR检测肿瘤微环境中KY-1细胞表面活性受体CD16、NKG2D及NK1.1的表达;ELISA检测培养细胞上清CD107a的含量。在肿瘤微环境中加入10ng/ml FKN及10ng/ml IP-10,流式细胞术测定NK1.1+CD16+KY-1细胞的数量;趋化及黏附实验评价其迁移和黏附功能的变化。最后,以10nmol/L雷帕霉素、30μmol/L LY294002、500ng/μl穿心莲内酯、2μmol/L渥曼青霉素分别处理4T1细胞,收集上清分别孵育RAW 264.7细胞48h后,RT-PCR检测4T1、RAW 264.7 m RNA及两种混合m RNA中Rae1α、H60a的表达。结果肿瘤微环境中,KY-1细胞NK1.1+细胞比例、趋化率及黏附功能明显下降,加入FKN和IP-10后以上各项指标明显增高,不同信号通路阻滞剂使4T1细胞对NK细胞杀伤作用的敏感性均有不同程度提高,以雷帕霉素作用最为明显(P<0.05)。结论 FKN、IP-10可上调肿瘤微环境中NK细胞的数目和功能,雷帕霉素等通过诱导肿瘤细胞高表达NKG2D配体(Rae1、H60a),间接促进NK细胞的杀伤活性。

Objective To investigate the inducing effects of chemokines [fractalkine（FKN）, IP-10] and different signal pathway inhibitors on NK cells in the tumor microenvironment（TME）. Methods Immunohistochemistry was performed using antibodies for CD56 and DAP10 respectively on human breast carcinoma. Murine macrophages（RAW 264.7） and breast cancer cells（4T1） were co-cultivated at a 1：4 ratio to imitate the TME with NK cells（KY-1） set as the object. RT-PCR was used to determine the m RNA expressions of CD16, NKG2 D and NK1.1, and the content of CD107 a in the supernatants was determined by ELISA. 10ng/ml FKN and 10ng/ml IP-10 were added into the TME, NK1.1＋CD16＋KY-1 cells were counted with flow cytometry, migration and adhesion assays were used to assess the related function of KY-1 cells. 4T1 cells were incubated in 10nmol/L of rapamycin, 30μmol/L of LY294002, 500ng/μl of andrographolide and 2mmol/L of wortmannin, the 4T1 tumor supernatants（TSNs） were harvested separately and used to incubate RAW 264.7 for 48 h, then the expressions of Rae1α and H60 a m RNA in 4T1, RAW 264.7 and their mixture were determined by RT-PCR. Results The related indicators of KY-1 cells such as NK1.1＋ number, chemotaxis rate, and adhesion function decreased obviously in TME, and the above indices increased after the addition of FKN and IP-10, and some signal pathway inhibitors indirectly promoted NK cells＇ function in TME, and among them rapamycin was the most efficient one（P〈0.05）. Conclusion FKN and IP-10 may up-regulate the number and function of NK cells in TME, and rapamycin can promote NK cells＇ killing function by inducing high expression of NKG2DLs（Rae1, H60a） on tumor cells.