膜型MHC-Ⅰ类分子高表达的GBM U251细胞疫苗的研制及其体外抗瘤作用

摘要

ABSTRACT:OBJECTIVE To investigate the manufacture of human glioblastoma multiforme ( GBM ) U251 cell vaccine with membrane-enriched majorhistocompatibility complex class I( MHC classI) molecules and the mechanism of its antitumor in vitro rudimentally.METHODS The human GMB U251 cells were incubated with varying doses of IFN-γranging between 0 and 2000U· mL-1 for 48 hours;Flow cytometry was applied to analyze the expression rates of MHC class Imolecules on the membrane of U251 cells which were had been incubated with vary doses of IFN-γfor 48 hours.U251 cell vaccine were manufactutred by optimum inducement schemes which induced membrane-en-riched MHC class Imolecule.PBMCs of healthy donator were coincubated with the U251 cell vaccine.then collected as effector cells.The competence of specific killing of the three effector cells is determined by MTT assay;At the same time,the interdiction test that mouse anti-human HLA-A,B,C monoclonal antibody was applied in the specific killing test were carried out;Flow cytometry was applied to analyzed the changing of the proportions of CD4 +and CD8 +T lymphocytes;the secretion of IFN-γand IL-2 of the effector cells.RESULTS The expression rate of MHC class Imolecules on the membrane of U251 cells was 89.9%and topmost among the experimented groups when U251 cells was treated by IFN-γat 500U· mL-1 for 48 hours in vitro.CTLs stimulated by U251 cell vaccine which were the membrane-enriched MHC class I molecule had stronger competence specifically killing wild type U251 cells than untreated control group(P<0.05).The proportion of CD4 +and CD8 +T lymphocyte of PBMCs coincubated with it had increased dramatically(P<0.05),and could secrete a greatest deal of IFN-γand IL-2(P<0.05).CONCLU-SION Treament namely IFN-γat 500U· mL-1 for 48 hours is optimum scheme which induces the expression of membranemolecule MHC class I of U251 cells.The effector cells stimulated by the membrane-enriched MHC class Imolecules U251 cell vaccine own higher specific killing competence.which maybe relate to MHC class Imolecules.%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。