人类表皮生长因子受体-2单链抗体抑制高表达人类表皮生长因子受体-2肿瘤生长

Inhibitory effects of human epidermal growth factor receptor-2-single-chain antibody on human epidermal growth factor receptor-2 positive tumors

目的探讨抗人类表皮生长因子受体-2（Her-2）单链抗体（scFv）抑制高表达Her-2的肿瘤细胞生长增殖，探讨其应用于Her-2阳性肿瘤治疗的可行性。方法应用聚合酶链反应（PCR）技术从携带Her-2-scFv片段的质粒pUC57-Her-2-scFv获得目的基因，双酶切法将目的基因Her-2-scFv插入质粒载体pET28a构建表达质粒，转化入Rosetta大肠杆菌诱导表达，获得目的蛋白，并复性、纯化。Western blot证实表达。噻唑蓝（MTT）法检测目的蛋白对肿瘤细胞T6-17增殖的抑制作用。将肿瘤细胞T6-17在裸鼠皮下成瘤，观察抗Her-2单链抗体抑制肿瘤细胞生长。结果经基因测序鉴定，重组质粒pET28a-Her-2-scFv构建成功。转化入Rosetta菌表达并分离纯化，可获得纯度约为90%的目的蛋白。MTT法测定发现，抗Her-2-scFv组对T6-17细胞的的生长抑制率为33.52%，曲妥珠单抗治疗组的生长抑制率为34.79%，两组比较差异无统计学意义（P=0.429），而与阴性对照组比较，差异均有统计学意义（P=0.000）。进而检测Her-2-scFv对裸鼠肿瘤模型生长抑制作用发现，Her-2-scFv各浓度治疗组均表现出抑瘤效应，100、250、500 ng/kg组的抑瘤率分别为33.7%、27.2%、63.3%，与空白对照组比较，差异均有统计学意义（P=0.027、0.035、0.000）。赫赛汀组抑瘤率为70.2%，与500 ng/kg组比较，差异无统计学意义（P=0.643）。结论抗Her-2单链抗体在体内外均可显著抑制Her-2阳性肿瘤细胞T6-17的生长增殖。

Objective To study the inhibitory effect of single-chain antibody （scFv） against human epidermal growth factor receptor-2 （Her-2） on tumor cell T6-17 proliferation. To investigate the potential of Her-2-scFv protein targeted therapy of Her-2 positive tumors.Methods In our study, Her-2-scFv gene fragment was amplified from plasmid pUC57-Her-2-scFv by polymerase chain reaction （PCR）. Then Her-2-scFv gene fragment was cloned into the prokaryotic expression vector pET28a though double enzyme, and get the recombinant plasmid pET28a-Her-2-scFv. Plasmid pET28a-Her-2-scFv was transformed into Rosetta of E. coli. Her-2-scFv protein successful expression was detected by Western blotting. Her-2 positive tumor cell T6-17 proliferation inhibition were tested by methyl thiazol tetrazolium （MTT） assay. The tumor volume was measured by animal model of gastric cancer in vivo test.Results Her-2-scFv gene fragments were received by PCR successfully. Location of Her-2-scFv gene and its sequence in recombinant plasmid pET28a-Her-2-scFv is in a right direction by gene sequencing. MTT assay showed that the growth inhibition ratio of Her-2-positive tumor cell T6-17 in Her-2-scFv group was 33.52%, trastuzumab treatment group was 34.79%, with no statistic significance between them （P=0.429）. Compared with the negative control group （PBS group）, the differences were statistically significant （P=0.000）. In vivo experiments： The inhibiting tumor rates of 100 ng/kg group, 250 ng/kg group, 500 ng/kg group were 33.7%, 27.2%, 63.3%, separately. Compared with the control group, the difference were statistically significant （P=0.027, 0.035, 0.000）. In Hessaitin group, the inhibition rate was 70.2%, compared with 500 ng/kg group, there was no statistically significant difference （P=0.643）.Conclusion Her-2-scFv protein can inhibit the proliferation of T6-17 positive tumor cells in vitro and in vivo.