CAJ | 학술논문

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。
목적구건종류세포/당지류복합물연합TLR9배체적종류역묘，병탐토상술종류역묘대소서결장암적치료효과。방법운용만병독전염GFP-CD1d지MC38종류세포구건CD1d-MC38세포계，병가재α-Galcer（α-galactosylceramide ，α-GC）지상술세포계제비CD1d-MC38/α-Galcer복합물。이용RT-PCR、류식세포술검측MC38세포계CD1d적표체정황이급대우α-Galcer결합효솔。최후건립소서MC38결장암피하종류모형，관찰종류세포/당지류복합물연합TLR9배체적종류역묘（ CD1d-MC38/α-GC＋CpG1826）대우소서종류적치료작용，면역조화방법검측종류조직중CD4＋T화CD8＋T세포침윤정황。결과성공구건료표체CD1d분자적CD1d-MC38세포계，CD1d양성적CD1d-MC38세포비례위（98．10±2．53）％；차해세포계능유효결합α-Galcer，수착α-Galcer농도승고화가재시간적연장，기결합효솔축점상승；CD1d-MC38/α-GC＋CpG1826종류역묘능유효연완소서피하종류생장（여기여각조비교，P＜0．01），연장하류소서생존시간（여CD1d-MC38/α-GC조비교，P＜0．05；여기여각조비교，P＜0．01）；종류조직면역조화현시，실험조종류조직중유경다적CD4＋T세포화CD8＋T세포침윤，여CD1d-MC38/α-GC조비교，차이무통계학의의（P＞0．05），여기여각조비교，차이유통계학의의（P＜0．01）。결론종류세포/당지류복합물연합TLR9배체적종류역묘능억제소서종류생장，연장하류소서생존기，발휘항종류작용유뢰우CD4＋T세포화CD8＋T세포。
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.