内皮抑素30肽对HepG2细胞黏附、侵袭能力影响的机制研究

Endostatin 30 peptide inhibit the cell adhesion and invasion of HepG2 cells

摘要:

目的  探讨RGD修饰的人内皮抑素30肽对HepG2细胞黏附、侵袭的影响及作用机制。方法  人工合成人内皮抑素1～30位氨基酸（30肽，序列25～31由RGIRGAD改为RGDRGD）所对应的核苷酸序列，连接到质粒pTYB2中，再转化至大肠埃希菌BL21（DE3）中表达，同法合成人内皮抑素1～27位氨基酸构成的27肽作为对照。细胞黏附实验及Transwell实验分别检测30肽和27肽对HepG2细胞黏附能力和侵袭能力的影响，同时筛选与侵袭作用相关的整合素；免疫荧光检测30肽和27肽对αvβ3整合素的聚集作用，流式细胞仪检测30肽和27肽对HepG2细胞表面相应整合素表达的影响；逆转录-聚合酶链反应（RT-PCR）及Western blot检测30肽和27肽对HepG2细胞TIMP1和TIMP2 mRNA和蛋白质表达量的影响。结果  30肽能明显抑制HepG2细胞黏附、侵袭，对侵袭作用的影响与整合素αvβ3相关，同时，30肽能上调HepG2细胞中TIMP1和TIMP2 mRNA及蛋白质的表达，30肽加αvβ3抗体后，上述作用明显增强。结论  30肽可能是通过整合素αvβ3发挥其抗肿瘤侵袭转移作用，可以成为临床上肝癌的辅助化疗药物发挥作用。

关键词:

内皮抑素;RGD序列;侵袭;TIMP

Abstract:

Objective To study the influence of human endostatin 30 peptide modified RGD on the cell adhesion and invasion of HepG2 cells and the mechanism. Methods The nucleotide sequence encoding amino acids 1-30 of endostatin (peptide 30, with amino acids 25-31 mutated from RGIRGAD to RGDRGD) was artificially synthesized and cloned into the plasmid pTYB2 and expressed in E. coli (DE3), Similarly, peptide 27, corresponding to amino acids 1-27 of endostatin, was produced as a control. The cell adhesion assay and transwell was used to inspect the influence of cell adhesion and invasion activity of HepG2 caused by 30 peptide and 27 peptide, respectively. Meanwhile, integrin associate with invasion was screened out; The aggregation of integrin αvβ3 was detected using by immunofluorescence The expression of integrin on the HepG2 cell surface was measured by flow cytometry in order to test the effect of 30 peptide and 27 peptide. The expression of mRNA and protein was tested by RT-PCR and Western blot, including TIMP1 and TIMP2. Results The cell adhesion and invasion of HepG2 was inhibited obviously by 30 peptide, and it's influence of invasive effect on HepG2 associate with integrin αvβ3. At the same time, the mRNA and protein expression of TIMP1 and TIMP2 were down-regulated by 30 peptide. Above function enhanced obviously after αvβ3 antibody joined to 30 peptide. Conclusions 30 peptide can give full play to its anti-transferance for tumor through integrin αvβ3, 30 peptide may help chemotherapeutics to play their function of anti-hepatoma in the clinical treatment.

Key words:

Endostatin; RGD sequence; invasion; TIMP