摘要目的 探讨人类表皮生长因子受体2(Her-2)靶向重组型caspase-6融合蛋白对骨肉瘤的促凋亡作用.方法 将抗Her-2单链抗体基因e23sFv、绿脓杆菌外毒素PE的转膜结构域基因(PEⅡ)和重组型caspage-6基因连接,构建成Immunocasp-6(e23sFv-PE Ⅱ-casp-6)融合蛋白,将其亚克隆入真核表达载体pCMV中,构建pCMV-e23sfv-PE Ⅱ-caspase-6载体(简称pCMV-6).将荷瘤裸鼠随机分为治疗组和对照组,采用阳离子脂质体包裹法,将pCMV-6和pCMV分别肌肉注射入荷瘤裸鼠身上,观察肿瘤体积和裸鼠体重的变化,并称取肿瘤重量.采用HE染色法观察肿瘤组织形态学变化,采用末端脱氧核苷酸转移酶标记技术(TUNEL)检测肿瘤组织的凋亡形态,采用免疫组织化学染色检测目的基因的表达.结果 对照组的肿瘤体积、肿瘤重量和裸鼠体重分别为(975.09±49.76)mm3、(1.08±0.16)g和(4.07±0.49)g,治疗组分别为(376.01±265.18)mm2、(0.64±0.18)g和(6.20±1.14)g.HE染色和TUNEL法检测显示,治疗组裸鼠肿瘤组织呈现出典型的凋亡特性,而对照组裸鼠肿瘤组织结构正常.免疫组织化学染色显示,caspase-6在治疗组裸鼠肿瘤组织中呈阳性,而在对照组肿瘤组织和肌肉组织中呈阴性.结论 人重组型caspase-6融合蛋白能靶向性识别、结合Her-2阳性的骨肉瘤细胞,并诱导其发生凋亡,为骨肉瘤的临床治疗提供了一定的实验基础.

abstractsObjective To investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells. Methods Recombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv) , a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i. e. pCMV-e23sfv-PE Ⅱ -caspase-6 ( abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i. m. injections of liposotme encapsulated pCMV-6 or pCMV alone . The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene exression was analyzed by immunohistochemical staining. Results The tumor growth of the mice in the treatment group was significantly slower than that of the control group ( P =0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0. 0002). The tumor weight of the mice in the treatment group was signicificantly lower than that of the control group (P = 0. 0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry. Conclusion Immunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.