摘要
为了研究改造后的肿瘤抑素2个抗肿瘤活性肽的作用机制,明确其不同的抗肿瘤活性,采用基因工程技术原理,人工合成肿瘤抑素中185~203位氨基酸所对应的19肽和T7肽(74~98位氨基酸)基础上改造的21肽碱基序列,将其与融合蛋白表达载体pTYB2重组后转化到大肠杆菌BL21(DE3)中进行诱导表达,用几丁质亲和层析柱一步纯化,直接获得19肽和21肽,利用MTT法、细胞生长曲线、TUNEL法、流式细胞仪早期细胞凋亡检测和细胞周期检测,小鼠H22腹水型转移型肝癌实体瘤抑瘤实验并结合组织病理学切片,来研究19肽和21肽单独应用或联合应用对肿瘤细胞和内皮细胞生长和凋亡的影响以及对体内肿瘤的抑制情况.体内外实验表明:获得的19肽抗肿瘤活性以直接作用肿瘤细胞为主,也有抑制新生血管生成的作用.基因重组21肽抗肿瘤作用是通过抑制肿瘤组织新生血管生成实现的.19肽、21肽联合应用对肿瘤细胞、内皮细胞生长抑制和促凋亡作用明显增强,抗肿瘤活性大大提高.联合用药弥补了单独用药不足,产生协同抗肿瘤作用,可能会成为今后肿瘤治疗的一个主要方向.
To study biological activity and mechanism of two modified anti-tumor peptide of tumstatin, the nucleotide sequences of 19peptide (amino acid 185 ~203) and 2 l peptide based on modified T7 peptide (amio acid 75~95) of tumstatin were designed, artificially synthesized and inserted into the fusion protein vector pTYB2. After transformed and expressed in E. coil BL-21 (DE3) by means of fusion protein, the soluble 19peptide and 21peptide were obtained from one step chitin affinity chromatograph. By such experiment as MTT assay, cell growth curve, TUNEL assay, flow cytometry, the effect of the H22 ascitic fluid transfevent hepatoma of mice and histopathological slice, the biological activity of 19peptide and21peptide were studied. Experiments in vitro and in vivo identified that 19peptide could inhibit tumor cell proliferation, promote tumor cell apoptosis and stop tumor cell in G0/G1 cycle. It also could inhibit human umbilical vein endothelial cell proliferation to some extend. The anti-tumor activity of 19peptide mainly relied on affecting tumor cell directly and partly on inhibiting vascularization.2 lpeptide inhibited proliferation of human umbilical vein endothelial cell much better than that of tumor cell. It almost could not promote endothelial cell and tumor cell apoptosis.21peptide mainly exhibited indirect anti-tumor activity by anti-angiogenesis. The combination of 19peptide and 2 lpeptide obviously inhibited endothelial cell and tumor cell proliferation and promoted them apoptosis. A combination application could markedly enhance anti-tumor activity, make up the defect of 19peptide, 21peptide alone and bring about synergism. It would be an efficient method for tumor therapy in future, which will lay foundation on its mechanism of action research and clinical tumor therapy.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2007年第11期1152-1161,共10页
Progress In Biochemistry and Biophysics
基金
国家自然基金资助项目(30472035)
黑龙江省自然科学基金资助项目(TD2005-21)
黑龙江省教育厅自然科学基金资助项目(11511104).~~
关键词
肿瘤抑素
蛋白质表达
细胞增殖
细胞凋亡
肿瘤治疗
tumstatin, protein expression, cell proliferation, cell apoptosis, tumor therapy