论文部分内容阅读
目的:本研究旨在通过纯化分泌型簇集素(secretory clusterin,sCLU)蛋白,并对其生物学功能进行初步探索。方法:以人肺癌NCI-H460细胞cDNA为模板,构建真核表达质粒pRAG5-ag-sCLU,并转染HEK-293F细胞表达目的蛋白;采用亲和层析柱纯化目的蛋白后,分别采用Flag抗体及clusterin抗体行蛋白质印迹法检测鉴定纯化的sCLU蛋白。采用不同浓度纯化的sCLU蛋白处理NCI-H460细胞,观察其对细胞形态的影响。采用Boyden小室法鉴定sCLU蛋白对肺癌LETP-a-2细胞迁移能力的影响。结果:成功纯化获得具有二级结构的sCLU蛋白;sCLU蛋白浓度为5.4μmol/L时可使NCI-H460细胞的形态发生改变,并促进LETP-a-2细胞的迁移(P=0.0021)。结论:sCLU蛋白可能通过上皮间质转化促进肺癌细胞的迁移,sCLU蛋白的纯化将为其进一步应用于临床研究奠定了良好的实验基础。
OBJECTIVE: The aim of this study was to purify the secretory clusterin (sCLU) protein and to explore its biological function. Methods: The eukaryotic expression plasmid pRAG5-ag-sCLU was constructed by using human lung cancer NCI-H460 cell cDNA as a template, and transfected into HEK-293F cells to express the target protein. The target protein was purified by affinity chromatography, And clusterin antibody was used to detect the purified sCLU protein by Western blotting. NCI-H460 cells were treated with different concentrations of sCLU protein and their effects on cell morphology were observed. The Boyden chamber method was used to identify the effect of sCLU protein on the migration of lung cancer LETP-a-2 cells. Results: The sCLU protein with secondary structure was successfully purified. When the concentration of sCLU protein was 5.4μmol / L, the morphology of NCI-H460 cells was changed and the migration of LETP-a-2 cells was promoted (P = 0.0021). Conclusion: sCLU protein may promote the migration of lung cancer cells through epithelial-mesenchymal transition. The purification of sCLU protein will lay a good experimental foundation for its further application in clinical research.