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目的:探讨Her2靶向重组的caspase-6融合蛋白对骨肉瘤SOSP-9607细胞的促凋亡作用。方法:将抗Her2单链抗体基因e23sFv与绿脓杆菌外毒素PE的转膜结构域基因(PEⅡ)和活性caspase-6基因连接,构建成Immunocasp-6(e23sFv-PEⅡ-casp-6)基因,将其克隆入真核表达载体pCMV中,转染SOSP-9607细胞,间接免疫荧光法检测目的基因表达和细胞形态学变化,通过AnnexinV染色、流式细胞术、MTT法检测来观察其促肿瘤细胞凋亡的作用。结果:转染SOSP-9607细胞后,间接免疫荧光染色检测出caspase-6的表达,SOSP-9607细胞出现明显的核浓缩、碎裂;电镜观察到细胞质浓缩,胞内空泡,核染色质浓集等典型的凋亡特征;MTT法检测发现细胞的增殖明显被抑制;AnnexinV染色流式细胞术检测可见明显的凋亡细胞,凋亡率为31.4%,较对照组细胞(凋亡率为6.0%)有非常显著的增加(P<0.01)。结论:重组Immunocasp-6基因可以在转染的SOSP-9607细胞中表达,并诱导细胞发生凋亡。
AIM: To investigate the apoptosis-promoting effect of Her2-targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells. Methods: The e23sFv anti-Her2 single chain antibody gene was linked with the transmembrane domain gene (PEⅡ) and active caspase-6 gene of Pseudomonas exotoxin PE to construct Immunocasp-6 (e23sFv-PEⅡ-casp-6) The recombinant plasmids were cloned into the eukaryotic expression vector pCMV and transfected into SOSP-9607 cells. The expression of the target gene and the morphological changes of the cells were detected by indirect immunofluorescence. Annexin V staining, flow cytometry and MTT assay were used to observe the growth of tumor cells The role of apoptosis. Results: After transfection with SOSP-9607 cells, the expression of caspase-6 was detected by indirect immunofluorescence staining. SOSP-9607 cells showed obvious nuclear condensation and fragmentation. The cytoplasm concentration, intracellular vacuolization and nuclear chromatin concentration The apoptotic rate was 31.4%, which was significantly higher than that of the control group (the apoptotic rate was 6.0 %) Had a very significant increase (P <0.01). Conclusion: The recombinant Immunocasp-6 gene can be expressed in transfected SOSP-9607 cells and induce cell apoptosis.