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目的 在体外用抗CD3单抗、抗CD2 8单抗和白细胞介素 2 (IL 2 )扩增肿瘤特异性CTL ,为肿瘤过继治疗提供足够数量的、具有高度杀瘤活性的效应细胞。方法 采用两种方案培养肿瘤细胞免疫的小鼠脾细胞。 (1)抗CD3单抗刺激 48h后 ,加入抗CD3单抗和 2 0U mlrIL 2 (抗 CD3+IL 2组 ) ;(2 )抗CD3单抗和抗CD2 8单抗同时刺激 48h后 ,加入抗CD3单抗、抗CD2 8单抗和 2 0U mlrIL 2 (抗 CD3+抗 CD2 8+IL 2组 )。分别检测 2组效应细胞的增殖水平、杀瘤活性及表型。结果 抗 CD3+IL 2组细胞的3H TdR掺入量在第 6天、12天、2 0天分别为 2 2 12 6、5 42 6、2 0 72 ,抗 CD3+抗 CD2 8+IL 2组细胞的3H TdR掺入量在第 6天、12天、2 0天分别为 32 16 8、12 92 2、32 6 5 ,后者明显高于前者。在培养 12d时 ,抗 CD3+IL 2组细胞对FBL 3的最大杀伤活性为 6 6 .4% ,抗 CD3+抗 CD2 8+IL 2组细胞对FBL 3的最大杀伤活性为 77.8%。细胞表型FACS分析结果表明 ,抗 CD3+抗 CD2 8+IL 2组培养 12d后的细胞 90 %以上为Thy1.2 + 细胞 ,且CD2 5 + 细胞在抗 CD3+抗 CD2 8+IL 2组、抗 CD3+IL 2组分别为 2 3.0 0 %、8.15 %。结论 抗CD3单抗、抗CD2 8单抗和低剂量IL 2同时非特异性刺激 ,可获得大量扩增的、具有高度杀瘤活性的肿瘤特异性CTL。
Objective To amplify tumor-specific CTLs by anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, and interleukin 2 (IL 2) in vitro, and to provide a sufficient number of effector cells with high killing activity for tumor adoptive treatment. Methods Two protocols were used to culture mouse spleen cells immunized with tumor cells. (1) 48 hours after anti-CD3 monoclonal antibody stimulation, anti-CD3 monoclonal antibody and 20 uMlLIL2 (anti-CD3+IL2 group) were added; (2) anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody were stimulated for 48 hours and then added anti-CD3 monoclonal antibody. CD3 mAb, anti-CD28 mAb, and 20 uMrIL2 (anti-CD3+ anti-CD28+ IL2 group). The proliferation, killing activity, and phenotype of the two groups of effector cells were examined. Results 3H TdR incorporation in anti-CD3+IL 2 cells was 2 2 12 6 , 5 42 6 and 20 72 on days 6, 12 and 20, respectively. Anti-CD3+ anti-CD28+ IL 2 cells The 3H TdR incorporation amounted to 32 16 8, 12 92 2, 32 6 5 on the 6th, 12th, and 20th days, respectively, and the latter was significantly higher than the former. At the 12th day of culture, the anti-CD3+IL2 group had the highest cytotoxic activity against FBL3 of 66.4%, and the anti-CD3+ anti-CD28+IL2 group had the highest cytotoxic activity against FBL3 of 77.8%. The cell phenotype FACS analysis showed that more than 90% of the cells in the anti-CD3+anti-CD28+IL2 group cultured for 12 days were Thy1.2+ cells, and the CD25+ cells were in the anti-CD3+anti-CD28+IL2 group, anti-CD3 +IL 2 groups were 2 3.0 0 % and 8.15 %, respectively. Conclusion Anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, and low-dose IL 2 are simultaneously stimulated non-specifically, and a large number of tumor-specific CTLs with high tumorigenicity can be obtained.