融合蛋白anti-CD19(Fab)-LDM对B细胞淋巴瘤BJAB细胞杀伤作用及其机制研究

Cytotoxicity of Engineered Fusion Protein Anti-CD19 (Fab)-LDM on B-cell Lymphoma BJAB Cells and Its Mechanism

[目的]探讨融合蛋白anti-CD19(Fab)-LDM对B细胞淋巴瘤细胞株BJAB细胞的杀伤作用及其机制研究。[方法]利用流式细胞分析技术(FACS)和激光共聚焦显微镜技术,检测融合蛋白anti-CD19(Fab)-LDM与BJAB细胞的结合活性。MTT法检测融合蛋白anti-CD19(Fab)-LDM对BJAB细胞的体外杀伤活性。彗星电泳实验检测融合蛋白anti-CD19 (Fab)-LDM对BJAB细胞DNA的损伤。FACS检测不同浓度融合蛋白anti-CD19(Fab)-LDM处理BJAB细胞后细胞周期的变化。[结果] FACS和激光共聚焦显微镜技术实验结果表明,融合蛋白anti-CD19(Fab)-LDM能与BJAB细胞结合。MTT法实验结果表明,融合蛋白anti-CD19(Fab)-LDM对BJAB细胞杀伤活性较单用力达霉素或阿霉素强,IC50值分别为(0.15±0.02)nmol/L、(0.38±0.03)nmol/L和(57.15±2.30)nmol/L。彗星电泳实验结果表明,用5pmol/L的融合蛋白anti-CD19(Fab)-LDM及力达霉素处理BJAB细胞后,可引起细胞不同程度的DNA损伤,由于融合蛋白anti-CD19(Fab)-LDM具有靶向性和细胞内化的特性,对DNA造成的损伤较力达霉素组明显。FACS结果显示,随着融合蛋白anti-CD19(Fab)-LDM浓度的增加,S期的比例从53.78%升高至77.29%,呈剂量依赖性。[结论]融合蛋白anti-CD19 (Fab)-LDM可以靶向杀伤B细胞淋巴瘤细胞株BJAB,引起细胞周期阻滞,在B细胞淋巴瘤生物治疗中具有潜在的应用价值。

[Purpose] To investigate the cytotoxicity of the engineered fusion protein anti-CD19 (Fab)-LDM to B-cell lymphoma BJAB cells and its mechanism.[Methods] The antigen binding activity of anti-CD19 (Fab)-LDM on BJAB cells was observed by FACS and confocal microscopy. The cytotoxicity of the engineered fusion protein anti-CD19 (Fab)-LDM to BJAB cells was tested by MTT assay. Comet assay was used to determine the DNA damage in BJAB cells induced by antiCD19 (Fab)-LDM. Changes of cell cycle of BJAB cells treated with anti-CD19 (Fab)-LDM were detected by FACS.[Results] FACS and confocal microscopy showed that the antibody bound specifically to BJAB cells. MTT assay demonstrated that the engineered fusion protein anti-CD19 (Fab)-LDM strongly enhanced the cytotoxicity to BJAB cells compared with adriamycin or lidamycin (LDM). The comet assay showed that cells treated with anti-CD19 (Fab)-LDM induced more DNA damage than cells treated with LDM. BJAB cells treated with anti-CD19 (Fab)-LDM resulted in cell cycle arrest in a concentration dependence manner.[Conclusion] The fusion protein anti-CD19 (Fab)-LDM can target to kill CD19-positive B lymphoma cells and induce cell cycle arrest. These findings may be useful in clinical practice for B-cell lymphoma treatment.