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目的探索膜型MHC-Ⅰ类分子高表达人多形性胶质母细胞瘤(GBM)U251细胞疫苗的制备方法及其体外诱导的抗瘤作用机制。方法U251胶质瘤细胞加入0~2000U·mL~(-1)不同浓度梯度的重组人IFN-γ干预48h;流式细胞仪(FCM)检测不同浓度梯度的重组人IFN-γ诱导后的U251细胞胞膜MHC-Ⅰ类分子表达变化情况;取诱导MHC-Ⅰ类分子高表达最高的IFN-γ干预方案作为疫苗的制备方法;体外刺激健康捐献者PBMCs作为效应细胞,MTT比色法检测其对野生型及IFN-γ诱导48h后靶细胞的特异性杀伤活性,同时以抗人HLA-A,B,C行特异性杀伤试验的阻断试验;ELISA法检测效应细胞攻击靶细胞后的IFN-γ、IL-2的分泌情况;FCM检测疫苗刺激前后PBMCs CD4~+、CD8~+T淋巴细胞比例变化。结果 500U·mL~(-1)IFN-γ诱导48h U251细胞胞膜的MHC-Ⅰ类分子表达率最高,达89.9%;高表达膜型MHC-Ⅰ类分子U251细胞疫苗9在体外具有明显特异性的杀瘤活性的诱导能力(P<0.05);体外可分泌大量的IFN-γ和L-2(P<0.05);CD4+、CD8+明显增高(P<0.05)。结论500U·mL~(-1) IFN-γ诱导48h是U251细胞胞膜MHC-Ⅰ类分子体外诱导表达的最佳方案;膜型MHC-Ⅰ类分子高表达的U251细胞疫苗休外具有特异性的抗瘤作用,其杀瘤活性与膜型MHC-Ⅰ类分子表达上调有关。
Objective To explore the preparation of membrane-type MHC-Ⅰ high-expressing human glioblastoma multiforme (GBM) U251 cell vaccine and its anti-tumor mechanism in vitro. Methods U251 glioma cells were treated with different concentrations of 0 ~ 2000U · mL -1 recombinant human interferon-γ for 48h. Flow cytometry (FCM) was used to detect U251 The expression of MHC class I molecules in the cell membrane was studied. The IFN-γ intervention program which induced the highest expression of MHC class I molecules was used as the vaccine preparation. The PBMCs of healthy donors were stimulated in vitro as effector cells. MTT colorimetric assay The specific killing activity of wild type and IFN-γ induced target cells 48h after the specific anti-human HLA-A, B, C line specific killing test blocking test; ELISA method to detect effector cells after the target cell IFN -γ and IL-2 secretion. The percentage of CD4 ~ + and CD8 ~ + T lymphocytes in PBMCs before and after vaccination were detected by FCM. Results The MHC-I expression of U251 cell membrane was the highest at 48 hours after being treated with 500 U · mL -1 IFN-γ, which was 89.9%. High expression of MHC class I molecule U251 cell vaccine 9 was significantly different in vitro (P <0.05). A large amount of IFN-γ and L-2 were secreted in vitro (P <0.05), CD4 + and CD8 + were significantly increased (P <0.05). CONCLUSION: The optimal cytotoxicity of MHC-I molecules induced by 500 U · mL -1 IFN-γ for 48 h in U251 cells is unique. The antitumor activity of the tumor-killing activity is related to the up-regulation of membrane type MHC-I molecules.