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目的:研究Flt-3配体对树突状细胞结直肠癌细胞融合疫苗的制备和抗肿瘤免疫效应的影响。方法:①酪氨酸激酶受体3配体(FL)与粒细胞巨噬细胞集落刺激因子(GMCSF)、白细胞介素4(IL4)、肿瘤坏死因子(TNF)联用,刺激外周血单个核细胞(PBMC)中的树突状细胞(DC)前体细胞成熟,应用聚乙烯二醇(PEG)融合技术融合DC和Lovo细胞,制备融合疫苗。②通过细胞生长曲线、细胞表面表达和细胞形态来观察细胞的生物学特性。③裸鼠体内融合疫苗的成瘤活性的检测。④MTT比色法检测肿瘤细胞体外杀伤细胞毒活性。结果:在FL的参与下,成熟DC的细胞数目及细胞形态无显著改变;CD80、CD83、CD86等在细胞表面的表达较未加FL组显著丰富;融合疫苗呈悬浮生长,具有两种亲代细胞的细胞形态结构特点,融合细胞数量较稳定,不出现明显的细胞倍增;融合疫苗接种裸鼠体内后观察30d,未见肿瘤组织生长;FL组融合疫苗对肿瘤细胞具有明显的抑制作用。结论:Flt3L可显著促进DC的成熟;FL组融合细胞的分子表达较非FL组有很大提高,融合细胞具有作为抗肿瘤疫苗应具备的低增殖能力和不成瘤的安全性;DC与结直肠癌细胞融合后作为肿瘤疫苗在体外具有高效而特异的抗肿瘤效应,说明FL对增强融合疫苗抗结直肠癌特异性免疫效应有明显的作用。
Objective: To investigate the effect of Flt-3 ligand on the preparation of dendritic cell colorectal cancer cell fusion vaccine and anti-tumor immune effect. Methods: ① The combination of tyrosine kinase receptor 3 ligand (FL) and granulocyte macrophage colony stimulating factor (GMCSF), interleukin 4 (IL4) and tumor necrosis factor (TNF) stimulated peripheral blood mononuclear cells Dendritic cells (DCs) precursor cells in cells (PBMCs) were matured and fused with DCs and Lovo cells using polyethylene glycol (PEG) fusion technology to prepare fusion vaccines. ② through the cell growth curve, cell surface expression and cell morphology to observe the biological characteristics of cells. ③ nude mice in vivo fusion vaccine test tumor activity. ④ MTT colorimetric assay of cytotoxic activity of tumor cells in vitro. RESULTS: With the help of FL, there was no significant change in the numbers and morphology of mature DCs. The expression of CD80, CD83 and CD86 on the cell surface was significantly higher than that of the untreated FL group. The fusion vaccine grew in suspension with two parental cells The number of fusion cells was stable and no obvious cell doubling was observed. After the fusion vaccine was inoculated in nude mice for 30 days, no tumor growth was observed. The fusion vaccine of FL group had obvious inhibitory effect on tumor cells. Conclusion: Flt3L can significantly promote the maturation of DC. The expression of fusion cells in FL group is much higher than that in non-FL group. The fused cells have low proliferative capacity and no tumor-forming safety as anti-tumor vaccine. DC and colorectal Cancer cells as a tumor vaccine after fusion with highly efficient and specific antitumor effect in vitro, indicating that FL enhance the fusion vaccine against colorectal cancer-specific immune effects have a significant role.