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背景与目的:蛇毒去整合素是一类从多种蛇毒和水蛭中分离到的含RGD(Arg-Gly-Asp)或KGD(Lys-Gly-Asp)模体的小分子蛋白质,其由于能竞争性地结合细胞表面的整合蛋白受体,阻断肿瘤细胞与细胞外基质的黏附,从而发挥抗肿瘤转移的作用。rAdinbitor是我实验室采用基因克隆方法从大连产白眉蝮蛇(Gloydiusblomhoffibrevicaudus)的毒腺中获得的一种含有RGD模体的去整合素。本实验的目的是探讨rAdinbitor对人肝癌细胞系SMMC-7721黏附、增殖的抑制作用,为rAdinbitor抗肿瘤转移的作用机制提供实验依据。方法:以细胞黏附实验观察纤连蛋白(FN)基质对SMMC-7721细胞黏附的影响。分别采用结晶紫染色法和MTT比色法测定重组蛋白rAd-inbitor对SMMC-7721向FN黏附的影响,及对已黏附于FN上的SMMC-7721的影响。以MTT比色法检测不同浓度的rAdinbitor对SMMC-7721细胞增殖的影响;HE染色观察200μg/mlAdinbitor诱导SMMC-7721细胞36h凋亡的形态变化;采用流式细胞仪检测rAdinbitor对细胞凋亡的诱导作用。结果:①rAdinbitor对SMMC-7721细胞在FN基质上的黏附有一定的抑制作用,并呈浓度依赖关系,25、50、100、200μg/ml浓度组rAdinbitor的黏附抑制率分别为4.90%、11.73%、22.84%、37.28%,各组差异有显著性(P<0.05)。②rAdinbitor作用后,已黏附的SMMC-7721细胞变圆、脱落,各浓度组黏附细胞的存活率的平均值分别为98.89%、90.01%、63.37%、35.13%,细胞的增殖受到明显抑制(P<0.05)。③rAdinbitor对SMMC-7721细胞的增殖具有较强的抑制作用,且随着药物浓度的增加,其抑制作用增强(P<0.05)。rAdinbitor对SMMC-7721细胞作用48h的IC50值为177.83μg/ml。④200μg/mlrAd-inbitor作用SMMC-7721细胞36h呈现早期细胞凋亡的形态改变,凋亡率达20.68%,明显高于未经处理的对照组的2.38%(P<0.05)。结论:重组白眉蝮蛇去整合素rAdinbitor能够剂量依赖性地抑制SMMC-7721细胞在FN上的黏附,并促使已黏附的SMMC-7721细胞脱落,剂量依赖性抑制SMMC-7721细胞增殖并诱导其凋亡。
BACKGROUND & AIM: Snake venom disintegrins are a class of small molecule proteins with RGD (Arg-Gly-Asp) or KGD (Lys-Gly-Asp) motifs isolated from various venom and leeches. Sexually binding integrin receptors on the cell surface, blocking the adhesion of tumor cells to the extracellular matrix, thereby exerting anti-tumor metastasis. rAdinbitor is an integrin containing RGD motif obtained from the gland of Gloydius bomhoffibrevicaudus by gene cloning method in our laboratory. The purpose of this experiment is to investigate the inhibitory effect of rAdinbitor on the adhesion and proliferation of human hepatocellular carcinoma cell line SMMC-7721 and provide the experimental evidence for the mechanism of rAdinbitor’s anti-tumor metastasis. Methods: The effect of fibronectin (FN) matrix on the adhesion of SMMC-7721 cells was observed by cell adhesion assay. The effect of recombinant protein rAd-inbitor on the adhesion of SMMC-7721 to FN and the adhesion to FN on SMMC-7721 were determined by crystal violet staining and MTT colorimetric assay respectively. The effect of rAdinbitor on the proliferation of SMMC-7721 cells was detected by MTT assay. The morphological changes of SMMC-7721 cells induced by 200 μg / ml Adinbitor were observed by HE staining. The apoptosis induced by rAdinbitor was detected by flow cytometry effect. Results: ① Adinbitor inhibited the adhesion of SMMC-7721 cells to FN matrix in a concentration-dependent manner. The inhibition rates of rAdinbitor at concentration of 25, 50, 100, 200μg / ml were 4.90%, 11.73% 22.84%, 37.28% respectively. There was significant difference between the groups (P <0.05). (2) After treated with rAdinbitor, the adherent SMMC-7721 cells became round and detached, and the average survival rates of adherent cells in each concentration group were 98.89%, 90.01%, 63.37% and 35.13%, respectively. The proliferation of cells was significantly inhibited (P < 0.05). ③RAdinbitor had a strong inhibitory effect on the proliferation of SMMC-7721 cells, and its inhibitory effect was enhanced with the increase of drug concentration (P <0.05). The IC50 value of rAdinbitor on SMMC-7721 cells for 48h was 177.83μg / ml. ④200μg / ml rAd-inbitor SMMC-7721 cells showed morphological changes of early apoptosis, the apoptosis rate was 20.68%, significantly higher than the untreated control group 2.38% (P <0.05). CONCLUSION: The recombinant integrin rAdinbitor inhibits the adhesion of SMMC-7721 cells to FN in a dose-dependent manner and promotes the detachment of adherent SMMC-7721 cells in a dose-dependent manner and induces the proliferation of SMMC-7721 cells Death.