肝癌荷瘤小鼠调节性T细胞数量的改变及其与肿瘤生长的关系

目的研究肝癌荷瘤小鼠调节性T细胞数量的改变及其与肿瘤生长的关系。方法采用小鼠肝癌细胞系H22接种BALB／c小鼠,建立肝癌模型;采用流式细胞术方法检测CD4^＋ CD25^＋T／CD4^＋T细胞的比例;以RT-PCR和流式细胞术检测Foxp3基因的表达。以免疫磁珠分选法纯化CD4^＋CD25^＋T和CD4^＋CD25^-T细胞;在体外,用3H-TdR掺入法检测T细胞的增殖情况;在体内,观察荷瘤小鼠来源的CD4^＋CD25^＋T细胞对肿瘤生长的作用。结果（1）荷瘤小鼠在引流淋巴结中,CD4^＋CD25^＋T细胞占CD4＋T细胞（18．80％±0．06％）比例增高,与对照组（9．50％±0．03％）相比,差异有统计学意义（P〈0．01）;在非引流淋巴结（LN）和脾脏（SP）中,荷瘤小鼠CD4^＋CD25^＋T／CD4^＋T比例分别为16．28％±0．02％和17．28％±0．06％,与对照组9．50％±0．03％和11．08％±0．04％相比,差异有统计学意义（P〈0．01,P〈0．05）;同时,调节性T细胞特异性标志Foxp3 mRNA的表达也升高。在同一只荷瘤小鼠中,引流淋巴结中CD4^＋CD25^＋T细胞数量（18．8％±0．06％）较对侧非引流淋巴结（16．28％±0．02％）略有升高,但差异无统计学意义（P〉0．05）。（2）从荷瘤小鼠中纯化的CD4^＋CD25^＋T细胞,在体外对抗CD3单抗的刺激无反应,但能抑制CD4^＋CD25^-T细胞的增殖。（3）CD4^＋CD25^＋T／CD4＋T比例与肿瘤大小呈正相关,并且可以抑制CD4^＋CD25^-T细胞的抗肿瘤效应。结论肝癌细胞在小鼠体内的生长可以提高调节性T细胞的数量,其数量的高低与肿瘤的大小呈正相关,提示清除调节性T细胞将是肿瘤免疫治疗的策略之一。

Changes of regulatory T cell number in hepatocellular carcinoma-bearing mice and its relationship with tumor growth

OBJECTIVE: To study the relationship between the change of regulatory T cell number in CD4+ T subset and the growth of tumor in H22 hepatocellular carcinoma-bearing mice. METHODS: Tumor-bearing mice were established by subcutaneous inoculation of H22 hepatocelluler carcinoma cells. Flow cytometry was used to detect the expression of CD4 and CD25 molecules of the T cells which came from the tumor-bearing mice. The Foxp3 gene expression was detected by RT-PCR and flow cytometry. CD4+ CD25+ T cells and CD4+ CD25- T cells were separated and purified by immuno-magnetic beads. The proliferation and suppressive function of the CD4+ CD25+ T cells coming from tumor-bearing mice was measured by 3H]-thymidines incorporation experiment in vitro, and then effect of CD4+ CD25+ T cells originated from hepatocellular carcinoma-bearing mice on tumor growth was observed in vivo. RESULTS: (1) Compared with mice of the control group, the percentage of CD4+ CD25+ T cells of CD4+ T cells in tumor-bearing mice is not only higher in draining lymph nodes (18.80% < or = 0.06%) vs. (9.50% +/- 0.03%), (P < 0.01), but also higher in non-draining lymph nodes (LN) and spleen (SP), LN: (16.28% +/- 0.02%) vs. (9.50% +/- 0.03%), P < 0.01; SP: (17.28% +/- 0.06%) vs. (11.08% +/- 0.04%), (P < 0.05). The expression of regulatory T cell specific marker Foxp3 gene was also increased. In the same tumor-bearing mice, the number of CD4+ CD25+ T cells in draining lymph node was relatively higher than the contralateral nondraining lymph node, but the difference was statistically not significant (18.8% +/- 0.06%) vs. (16.28% +/- 0.02%), (P > 0.05). (2) The CD4+ CD25+ T cells purified from tumor-bearing mice--like naturally occurring regulatory T cells--were anergic to anti-CD3 monoclonal antibody stimulation in vitro, but it could suppress CD4+ CD25- T cells proliferation. (3) The percentage of CD4+ CD25+ T cells was positively related to tumor size. It could also suppress the anti-tumor effect of CD4+ CD25- T cells in vivo. Conclusion The growth of hepatocellular carcinoma in mice can boost the amount of regulatory T cells. The amount of regulatory T cells is positively related to tumor size, indicating that attack on regulatory T cells could be used as one of modalities in cancer treatment in the future.