人重组型caspase-6融合蛋白对骨肉瘤E10细胞的靶向促凋亡作用

The targeted pro-apoptotic effect of human recombinant caspase-6 fusion protein on osteosarcoma E10 cells

目的:探讨Her2靶向重组人caspase-6融合蛋白对骨肉瘤E10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)和重构型caspase-6基因连接,构建成immunocaspase-6(e23sFv-PEⅡ-caspase-6)基因,将其克隆入质粒pCMV中,阳离子脂质体包裹法转染E10细胞;HE染色、电子显微镜观测转染后细胞形态学变化;免疫细胞化学染色检测目的基因表达;MTT法检测目的基因转染后细胞的生长状况。结果:目的基因转染后,HE染色、电子显微镜观察到细胞呈现典型的凋亡特征,免疫细胞化学染色检测出caspase-6的表达,MTT法检测发现细胞的增殖被明显抑制(P≤0.01)。结论:Her2靶向重组的人caspase-6融合蛋白能识别、结合Her2+骨肉瘤E10细胞,并促使其凋亡。

Objective To investigate the pro-apoptotic effect of Her2 targeted recombinant caspase-6 fusion protein on osteosarcoma E10 cells. Methods Recombinant immunocaspase-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her2 antibody （e23sFv）, a PEA translocation domain （PEA aa253-364）, and an recombinant caspase-6. The pCMV-immunocaspase-6 was produced by cloning immunocaspase-6 gene into pCMV plasmid, and then the pCMV-immunocaspase-6 mixed cationic liposome was transfected into E10 cells. The effects of target gene expression on morphology and growth status of E10 cells were observed by HE staining and electron microscopy and analyzed by immunocytochemical staining and MTr assay. Results The fusion protein was detected in the cytoplasm of the transfected E10 cells. These cells presented the typical characteristics of apoptosis as detected by HE staining and electron microscopy. MTT assay revealed that the proliferation of immunocaspase-6 gene-transfected E10 cells was much lower than that of non- or mock-transfected cells （P≤0.01）. Conclusion Her2 targeted human recombinant caspase-6 fusion protein can induce apoptosis of Her2-overexpressing E10 cells.