两种基因重组免疫毒素导向治疗乳腺癌的细胞毒活性研究

Cytotoxic Comparison between Two Recombinant Immunotoxins of HELβ1-PE38KDEL and HELβ1-PE33 in Selective Therapy for Breast Cancer

目的:探讨HELβ1-PE33、HELβ1-PE38KDEL两种基因重组免疫毒素对乳腺癌细胞的细胞毒活性。方法:构建亚克隆质粒pHELβ1-PE33;诱导生成、纯化HELβ1-PE33蛋白,并用凝胶电泳法检测其与HELβ1-PE38KDEL蛋白纯度;采用MTT法、软琼脂集落形成法检测这两种蛋白对乳腺癌细胞增殖的影响,比较两种蛋白在不同的乳腺癌细胞系中的细胞毒活性。结果:纯化后的两种蛋白均有导向作用,其纯度均超过98%。在免疫毒素敏感乳腺癌细胞内,MTT实验表明HELβ1-PE38KDEL对DY36T2、MDA-MB-453、47T1LZ1、BT474M1、SKBR3的IC50分别为(0.25±0.02)ng/ml、(0.35±0.05)ng/ml、(0.02±0.02)ng/ml、(0.02±0.02)ng/ml、(20.3±0.01)ng/ml,而HELβ1-PE33的IC50分别为(5.90±0.05)ng/ml、(3.91±0.11)ng/ml、(0.09±0.02)ng/ml、(0.06±0.05)ng/ml、(125.00±0.12)ng/ml、(10.80±0.01)ng/ml,两者的细胞毒活性在统计学上有显著意义(P<0.01);软琼脂集落形成实验表明HELβ1-PE38KDEL对MDA-MB-453、BT474M1的IC50分别为(0.05±0.01)、(0.005±0.000)ng/ml,而HELβ1-PE33对上述两种细胞的IC50则为(5.00±0.02)ng/ml和(0.05±0.01)ng/ml,二者的细胞毒活性在统计学上有显著意义(P<0.01)。HELβ1-PE33和HELβ1-PE38KDEL对乳癌细胞抑制作用的时间效?

Objective: This study was designed to compare the cytotoxic activity between two recombinant immunotoxins of HELβ1 PE33,HELβ1 PE38KDEL in the selective therapy for breast cancer. Methods: Plasmid pHELβ1 PE33 was subcloned. The protein expression of HELβ1 PE33 and HELβ1 PE38KDEL was induced and purified. The concentration of these two proteins was detected by PAGE. Cytotoxic comparison of these two proteins was made by MTT assay, soft agar colony formation. Result: Both HELβ1 PE33 and HELβ1 PE38KDEL showed the selective therapy against breast cancer cells. The purification of both immunotoxin was more than 98%. In the immunotoxin sensitive breast cancer cells, the results of MTT showed that IC50 of HELβ1 PE38KDEL against DY36T2, MDA MB 453, 47T1LZ1, BT474M1, SKBR3 was 0.25±0.02, 0.35±0.05, 0.02±0.02, 0.02±0.02, and 20.3±0.01, respectively, while IC50 of HELβ1 PE33 was 5.9±0.05, 3.91±0.11,0.09±0.02, 0.06±0.05,125.00±0.12,and 10.80±0.01 ng/ml, respectively. HELβ1 PE38KDEL was more significantly potent than HELβ1 PE33(P< 0.01). Soft agar colony formation demonstrated that IC50 of HELβ1 PE38KDEL against MDA MB 453, BT474M1 was 0.05±0.01, and 0.005±0.000 ng/ml, respectively. while HELβ1-PE33 was 5±0.02, 0.05±0.01 ng/ml, respectively. HELβ1 PE38KDEL was more significantly potent than HELβ1 PE33 (P< 0.01). The cytotoxic experiment by time course revealed that HELβ1 PE38KDEL was more superior to HELβ1 PE33. Conclusion: KDEL tetrapeptide [LysAspGluLeu]plays a more important role in enhancing cytotoxic effect than molecular size and proteolysis.