CAJ | 학술논문

目的观察2E8(自制鼠抗人CD19新单克降抗体)-4',5',7-三羟基异黄酮(Genistein,Gen)免疫毒素(2E8-Gen)体外靶向杀伤Nalm-6细胞作用及其机制,为靶向治疗B细胞系自血病、淋巴瘤奠定基础.方法通过细胞形态观察、台盼蓝拒染法、细胞生长曲线比较、MTT比色法和流式细胞术.结果作用24 h,2E8-Gen对Nalm-6细胞具有明显的杀伤作用,浓度在20～100 nmol/L 9个观测点的细胞存活率[从20 nmol/L时的(71.8±7.9)%逐步降到100 nmol/L时的(16.6±12.9)%,n=3]与对照组[(100±13.9)%]相比差异均有统计学意义(P<0.05),浓度在80 nmol/L以上杀伤作用显著加强;100 nmol/L 2E8-Gen在24、48、72 h对Nalm-6细胞生长抑制率分别为82%、84%、94%,呈时间依赖关系.100 nmo/L浓度2E8-Gen组的Nalm-6细胞生长曲线与空白组、PBS组和相同浓度的纯2E8组的Nalm-6细胞生长曲线相比,差异有统计学意义(F=152.15,P=2.15×10-7),但后3组间差异无统计学意义(F=1.51,P=0.29);而100 nmol/L 2E8-Gen对CD19阴性的Molt-3细胞的生长曲线与空白组相比无明显影响(F=0.34,P=0.59).24 h 100 nmol/L 2E8-Gen组早期凋亡Nalm-6细胞阳性率[(33.45±8.77)%]明显高于对照组[(10.44±1.28)%,t=-4.39,P=0.001].结论 2E8-Gen对有CD19抗原表达的Nalm-6细胞具有良好的选择性杀伤作用,并呈时间依赖关系,浓度在80 nmol/L以上杀伤作用显著加强,凋亡机制参与了该杀伤作用.
목적 관찰2E8(자제서항인CD19신단극강항체)-4',5',7-삼간기이황동(Genistein,Gen)면역독소(2E8-Gen)체외파향살상Nalm-6세포작용급기궤제,위파향치료B세포계자혈병、림파류전정기출.방법 통과세포형태관찰、태반람거염법、세포생장곡선비교、MTT비색법화류식세포술.결과작용24 h,2E8-Gen대Nalm-6세포구유명현적살상작용,농도재20～100 nmol/L 9개관측점적세포존활솔[종20 nmol/L시적(71.8±7.9)%축보강도100 nmol/L시적(16.6±12.9)%,n=3]여대조조[(100±13.9)%]상비차이균유통계학의의(P<0.05),농도재80 nmol/L이상살상작용현저가강;100 nmol/L 2E8-Gen재24、48、72 h대Nalm-6세포생장억제솔분별위82%、84%、94%,정시간의뢰관계.100 nmo/L농도2E8-Gen조적Nalm-6세포생장곡선여공백조、PBS조화상동농도적순2E8조적Nalm-6세포생장곡선상비,차이유통계학의의(F=152.15,P=2.15×10-7),단후3조간차이무통계학의의(F=1.51,P=0.29);이100 nmol/L 2E8-Gen대CD19음성적Molt-3세포적생장곡선여공백조상비무명현영향(F=0.34,P=0.59).24 h 100 nmol/L 2E8-Gen조조기조망Nalm-6세포양성솔[(33.45±8.77)%]명현고우대조조[(10.44±1.28)%,t=-4.39,P=0.001].결론 2E8-Gen대유CD19항원표체적Nalm-6세포구유량호적선택성살상작용,병정시간의뢰관계,농도재80 nmol/L이상살상작용현저가강,조망궤제삼여료해살상작용.
Objective Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma. Methods 2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing. Results After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8±7.9)% down to (16.6±12.9)%, respectively (n=3), which were all significantly lower than that of control group(100±13.9)% (P<0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups(F=152.15, P=2.15×10<'-7>), but there was no significant difference among the three control groups (F=1.51, P= 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen ( 00 nmol/L) and with negative control of blank did not show any significant difference (F=0.34, P= 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45±8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44%±1.28%, t=-4.39, P=0.001) at 24 hours of culture. Conclusion 2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.