可溶性重组hTRAIL蛋白抑制U251细胞生长

Inhibitory Effect of Soluble Recombinant hTRAIL on Human Brain Glioblastoma U251 Cells

肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)是TNF超家族中的成员,能够广泛诱导肿瘤细胞凋亡,对正常细胞无明显毒副作用.TRAIL已成为肿瘤治疗领域的研究热点.人脑胶质瘤是神经系统肿瘤中最常见类型,占颅内肿瘤50%～60%,5年存活率为20%～30%.本研究探讨可溶性TRAIL蛋白对人脑胶质瘤细胞(U251)的抑制作用.由大肠杆菌表达系统表达的TRAIL多为包涵体,为获得可溶性的蛋白,将hTRAIL95～281功能区基因片段插入到pHisSUMO表达载体,经IPTG低温诱导表达,Ni-NTA Agarose纯化后获得可溶性SUMO-hTRAIL,经SUMO ProteaseⅠ切去SUMO融合标签后获得成熟可溶hTRAIL蛋白.以U251细胞为靶细胞,通过MTT法检测TRAIL对肿瘤细胞的抑制作用.结果证明,TRAIL对U251细胞的抑制呈剂量依赖关系,最大抑制率为53.9%.流式细胞仪检测TRAIL诱导U251细胞凋亡实验中,对照组细胞存活率为92.2±0.8%,实验组细胞存活率为35.5±1.2%,证明重组蛋白具有生物学活性,并在体外能明显诱导U251肿瘤细胞发生死亡.本研究结果为TRAIL蛋白在临床上应用于肿瘤治疗奠定了基础.

TNF-related apoptosis inducing ligand（TRAIL） is a member of TNF super family.TRAIL could specifically induce apoptosis in broad kinds of tumor cells,but has little side and toxic effects on normal cells.TRAIL has become a hotspot in tumor therapy.Human brain glioblastoma is a common kind of tumor in nervous system accounting for 50%～60% of intracranial tumors;the five-year survival rate is only 20%～30%.In this study,we investigated the inhibitory effects of recombinant human TRAIL on human brain glioblastoma U251 cell line.TRAIL expressed by E.coli system was usually in elementary body form.The functional fragment of human TRAIL（95～281 amino acids） were cloned into the prokaryotic expressing vector pHisSUMO to acquire soluble TRAIL protein.SUMO-hTRAIL fusion protein was induced by IPTG at low temperature and purified by Ni-NTA Agarose.The SUMO tag was removed by SUMO ProteaseⅠdigestion to obtain the mature hTRAIL protein.The MTT assay indicated that the mature hTRAIL protein could significantly inhibit the growth of U251 cell line.The induction was dose-depended and the highest inhibitory rate was 53.9%.Flow cytometry assay showed that the survival rate of control group was 92.2 ± 0.8% and the survival rate of medication administration group was 35.5±1.2%.We concluded that the protein is biologically active and could significantly induce apoptosis in U251 tumor cells.This study provided a platform for TRAIL protein to use in cancer therapy.