肿瘤抑素相关肽T42肽的纯化及抗肝癌细胞Hepg2活性的测定

Determination of anti-Hepg2 cells activity and purification of tumor chalone T42 peptide

目的:利用已成功构建的肿瘤抑素相关活性肽T42肽高效表达基因工程重组菌pTYB2-T42,提取纯化T42肽,检测其抗肿瘤活性,为研究T42肽抗肿瘤作用机制及新型抗肿瘤药物研发提供实验基础。方法:将pTYB2-T42大肠杆菌进行培养,IPTG诱导其产生融合蛋白,经几丁质柱亲和层析得到目的蛋白,葡聚糖凝胶G15去除DTT干扰,TricineSDS-PAGE鉴定T42肽,紫外分光光度法检测其浓度。MTT检测T42肽对细胞的生长增殖抑制作用,AO/EB荧光染色检测细胞凋亡形态学变化。结果:提取的T42肽其相对分子质量在5000左右,与理论值4600一致。MTT实验结果显示:T42肽作用下Hepg2细胞生长增殖受到抑制,且随着T42肽浓度的增加,Hepg2细胞存活率逐渐下降,其半数抑制浓度(IC50)为30mmol.L-1;AO/EB荧光染色结果表明:经过30mmol.L-1T42肽作用24h后的Hepg2细胞表现出细胞皱缩呈圆形或卵圆形,核皱缩变形且胞膜受损等一系列细胞凋亡特征性的形态变化。结论:T42肽具有显著的抗肿瘤细胞活性,可明显抑制人肝癌细胞Hepg2的生长增殖,并促进其凋亡。

Objective To extract and purify anti-tumor peptide of tumor chalone T42 peptide from gene engineering bacteria pTYB2-T42 and identify its biological activity, and provide the experimental basis for the research of its function mechanism and the clinical treatment of tumors. Methods The pTYB2-T42 in E. coli was cultivated, and fusion protein was produced by IPTG, T42 peptide was extracted by the method of affinity chromatograph, DTT was removed by molecular sieve G15 and then T42 peptide was identified by Trincine SDS-PAGE. The influence of T42 peptide on the cell activity was detected by MTT. The apoptosis of T42 peptide was detected by AO/EB. Results The recombinant expression vector coding tumstatin T42 peptide was correct in size, its relative molecular mass was about 5 000. The result of MTT showed that the proliferation of growth of Hepg2 cells was inhibited by T42 peptide, and the survival rate of Hepg2 cells was decreased with the increase of T42 peptide dose, and IC50 was 30 mmol · L^-1. The result of AO/EB indicated that the Hepg2 cells appeared typical characteristics of apoptosis after treated with T42 peptide for 24 h. Conclusion T42 peptide evidently inhibit the proliferation of growth of Hepg2 cells in a dose-dependent manner, and it can promote the apoptosis of Hepg2 cells.