肿瘤细胞／糖脂类分子联合 TLR 9配体治疗小鼠结肠癌的效果研究

Alpha-galactosylceramide loaded tumor cells in combination with TLR9 agonists induce potent anti-tumor responses in a murine model of colon cancer

目的构建肿瘤细胞/糖脂类复合物联合TLR9配体的肿瘤疫苗，并探讨上述肿瘤疫苗对小鼠结肠癌的治疗效果。方法运用慢病毒转染GFP-CD1d至MC38肿瘤细胞构建CD1d-MC38细胞系，并加载α-Galcer（α-galactosylceramide ，α-GC）至上述细胞系制备CD1d-MC38/α-Galcer复合物。利用RT-PCR、流式细胞术检测MC38细胞系CD1d的表达情况以及对于α-Galcer结合效率。最后建立小鼠MC38结肠癌皮下肿瘤模型，观察肿瘤细胞/糖脂类复合物联合TLR9配体的肿瘤疫苗（ CD1d-MC38/α-GC＋CpG1826）对于小鼠肿瘤的治疗作用，免疫组化方法检测肿瘤组织中CD4＋T和CD8＋T细胞浸润情况。结果成功构建了表达CD1d分子的CD1d-MC38细胞系，CD1d阳性的CD1d-MC38细胞比例为（98．10±2．53）％；且该细胞系能有效结合α-Galcer，随着α-Galcer浓度升高和加载时间的延长，其结合效率逐渐上升；CD1d-MC38/α-GC＋CpG1826肿瘤疫苗能有效延缓小鼠皮下肿瘤生长（与其余各组比较，P＜0．01），延长荷瘤小鼠生存时间（与CD1d-MC38/α-GC组比较，P＜0．05；与其余各组比较，P＜0．01）；肿瘤组织免疫组化显示，实验组肿瘤组织中有更多的CD4＋T细胞和CD8＋T细胞浸润，与CD1d-MC38/α-GC组比较，差异无统计学意义（P＞0．05），与其余各组比较，差异有统计学意义（P＜0．01）。结论肿瘤细胞/糖脂类复合物联合TLR9配体的肿瘤疫苗能抑制小鼠肿瘤生长，延长荷瘤小鼠生存期，发挥抗肿瘤作用有赖于CD4＋T细胞和CD8＋T细胞。

Objective To design a new cancer vaccine by using alpha-galactosylceramide （α-Galcer,α-GC） loaded tumor cells in combination with TLR 9 ligand and to evaluate its therapeutic effects on colon canc-er in mice.Methods MC38 cells were transfected with lentivirus （GFP-CD1d） to prepare CD1d-MC38 cells. The expression of CD1d molecules in CD1d-MC38 cells was detected by fluorescence microscopy , RT-PCR and flow cytometry.The sorted CD1d-MC38 cells were loaded with α-Galcer to prepare CD1d-MC38/α-GC complex. Flow cytometry was performed to evaluate the efficiency of combination .A mouse model of colon cancer was es-tablished to investigate the therapeutic effects of α-Galcer loaded tumor cells in combination with TLR 9 ligand （ CD1d-MC38/α-GC＋CpG1826） on colon cancer in mice by analyzing tumor growth and mice survival time .Im-munohistochemical staining was used to detect CD 4＋T and CD8＋T infiltrating lymphocytes in tumor tissues .Re-sults The MC38 cancer cells that expressed CD 1d and GFP were successfully constructed , among which 98.10%±2.53%were positive for CD1d.Moreover, the CD1d-MC38 cells could combine with α-Galcer effec-tively in a dose and time dependent manner .Compared with PBS treated group ,α-GC treated group and TLR9 ligand treated group , the experimental vaccine strategy was sufficient to inhibit the growth of established tumors and prolong survival of tumor-bearing mice （P〈0.01）.Immunohistochemistry analysis revealed that levels of CD4＋T cells and CD8＋T cells in experiment group were significantly higher than those in groups treated with PBS,α-GC and TLR9 ligand （P〈0.01）.Conclusion CD1d-MC38/α-GC in combination with CpG1826 could efficiently inhibit the growth of established tumors and prolong survival of tumor-bearing mice .Immunohisto-chemistry analysis revealed that CD 4＋T cells and CD8＋T cells played important roles in anti-tumor immunity.