DC荷载α-GalCer联合肿瘤特异性CTL对小鼠Heps肝癌移植瘤生长的影响

Effects of DC load α- GalCer combined with tumor specific cytotoxic T lymphocyte on the growth of transplanted Heps hepatoma in mice

目的:探讨树突状细胞(DC)荷载α-半乳糖神经酰胺(α-GalCer)联合肿瘤特异性细胞毒T淋巴细胞(CTL)对小鼠Heps肝癌移植瘤生长的抑制作用。方法:诱导扩增小鼠骨髓来源的DC细胞和T淋巴细胞,培养成为具有肿瘤特异性的CTL,DC细胞体外荷载α-GalCer。建立Heps肝癌移植瘤模型,将36只模型鼠随机分为4组(n=9),分别尾静脉给予生理盐水(对照组)、CTL(CTL组)、DC荷载α-GalCer(DC荷载α-GalCer组)、DC细胞荷载α-GalCer+CTL(联合治疗组)输注。每隔两天测量移植瘤体积,观察体积变化。于治疗后第14d,处死小鼠,取眼球血及瘤体组织。流式细胞术(FCM)检测外周血CD4^+、CD8^+T细胞比例。免疫组织化学染色观察肿瘤组织中Bcl-2/Bax凋亡蛋白的表达。结果:与对照组相比,各治疗组均可抑制肿瘤的生长,差异具有统计学意义(P<0.01);联合治疗组较DC荷载α-GalCer组及CTL组肿瘤体积明显减小(P<0.05)。联合治疗组小鼠外周血CD4^+、CD8^+T细胞数量较另外三组显著升高(P<0.05);对照组、CTL组、DC荷载α-GalCer组小鼠外周血中CD4^+、CD8^+T细胞含量无明显差异(P>0.05)。与对照组相比,各治疗组移植瘤内Bax阳性细胞数量明显增加(P<0.05),Bcl-2阳性细胞数量明显减少(P<0.05);与CTL组和α-GalCer组相比,联合治疗组移植瘤内Bax阳性细胞明显增加(P<0.05),Bcl-2阳性细胞明显下降(P<0.05)。结论:DC荷载α-GalCer与肿瘤特异性CTL联合应用能够对小鼠Heps肝癌移植瘤具有协同杀伤作用。

Objective： To investigate the inhibitory effects of dendritic cells（DC） load alpha - Galactosylceramide （ α- GalCer） combined with tumor specific cytotoxic T lymphocyte（CTL） on the growth of transplanted Heps hepatoma in mice. Methods：Induce the DC cells and T lymphocyte that expanded the mice bone marrow, making it become a tumor specific CTL. DC cells load α- GalCer. First establish Heps liver cancer xenograft model, then randomly divide the 36 model mice into 4 groups （n = 9 ） , and respectively intravenous inject physiological saline （control group） , eytotoxie T lymphocyte（ CTL group）, DC load α -GalCer（ DC load α -GalCer group） and DC load α -GalCer combined with cytotoxic T lymphocyte （combined treatment group）. Measuring the volume of tumor at two days, observing the change of volume. In the fourteenth days after treatment, randomly selected from each group of 9 mices were sacrificed to obtain the ocular blood, spleen and tumor tissue in mice ： Using flow cytometry detect the proportion of peripheral blood CD4＋ , CD8 ＋ T cells. Immunohistochemical staining was performed in the sections for Bcl -2 and Bax to count Bcl - 2 positive cells and Bax positive cells in the local tumor. Results ： The tumor average volume and weight in the control group was higher than in the CTL group, DC loading α - GalCer group and the combined treatment group （ P 〈 0.01 ）. The average volume of the tumors in the combined treatment groups was significantly reduced than in the CTL group and DC loading α -GalCer group（P 〈0.05）. On day 14 after treatment,the CD4＋ ,CD8 ＋ T lymphocyte in the combined treatment group was significantly increased than in the control group, CTL group and DC loading α - GalCer group（P 〈0.05 ）. And the CD4＋ , CD8＋ T lymphocyte was no obvious difference in the control group, CTL group and the DC loading α - GalCer group（ P 〉 0.05 ）. The number of BAX positive cells in transplanted tumor were significantly increased （P 〈 0.05 ）, the expression of Bax protein was significantly increased （P 〈 0.01 ）. The number of Bcl-2 positive cells was significantly reduced（P 〈0.05 ）. The expression of Bcl- 2 protein was significantly decreased （ P 〈 0.01 ）. Compared with the data of CTL group and DC load α- GalCer group, the combined treatment group showed the number and expression of Bax positive cells was significantly increased（ P 〈 0.05 ）. While the number and expression of Bcl - 2 positive cells was significantly decreased（ P 〈 0.05 ）. Conclusion： It has synergistic killing effect on transplanted Heps hepatoma in mice using DC load c~ - GalCer combined with the tumor specific CTL.