【摘要】：目的以GLP-1受體為靶點(diǎn),建立GLP-1類似物活性檢測(cè)的細(xì)胞模型,為GLP-1類似物以及GLP-1受體激動(dòng)劑的藥物篩選提供一種簡單可靠的評(píng)價(jià)方法。方法首先將人源GLP-1受體基因插入pEGFP-N3,構(gòu)建真核表達(dá)載體pEGFP-GLP-1R,并轉(zhuǎn)染至HEK293A細(xì)胞中,經(jīng)G418壓力篩選后獲得穩(wěn)定表達(dá)GLP-1R-GFP的293A細(xì)胞株,然后通過GFP熒光信號(hào)和Western blot檢測(cè)GLP-1R-GFP融合蛋白在該細(xì)胞中的表達(dá)與分布;最后利用GLP-1類似物利拉魯肽刺激細(xì)胞,均相時(shí)間分辨熒光法檢測(cè)細(xì)胞內(nèi)cAMP含量變化。結(jié)果成功構(gòu)建了GLP-1R-GFP-293A穩(wěn)轉(zhuǎn)細(xì)胞株,GLP-1RGFP蛋白主要分布在細(xì)胞膜表面,該細(xì)胞對(duì)GLP-1類似物利拉魯肽具有高靈敏度和產(chǎn)生cAMP的特異性反應(yīng)。結(jié)論利用所構(gòu)建的細(xì)胞模型,可對(duì)小分子和GLP-1類似物進(jìn)行體外活性分析,為篩選GLP-1受體激動(dòng)劑奠定了模型基礎(chǔ)。
[Abstract]:Objective to establish a cell model for the detection of the activity of GLP-1 analogues using GLP-1 receptor as the target, and to provide a simple and reliable method for the screening of GLP-1 analogues and GLP-1 receptor agonists. Methods Human GLP-1 receptor gene was inserted into pEGFP-N3, to construct eukaryotic expression vector pEGFP-GLP-1R, and transfected into HEK293A cells. After G418 pressure screening, 293A cell line expressing GLP-1R-GFP stably was obtained. Then the expression and distribution of GLP-1R-GFP fusion protein in the cells were detected by GFP fluorescence signal and Western blot. Finally, GLP-1 analogues were used to stimulate the cells, and the changes of cAMP content in the cells were detected by homogeneous time resolved fluorescence method. Results the GLP-1R-GFP-293A stable cell line was successfully constructed. The GLP-1RGFP protein was mainly distributed on the surface of the cell membrane. The cell had a high sensitivity to the GLP-1 analogue and a specific response to the production of cAMP. Conclusion the cell model can be used to analyze the activity of small molecules and GLP-1 analogues in vitro, which lays a foundation for screening GLP-1 receptor agonists.
【作者單位】： 中山大學(xué)藥學(xué)院新藥篩選中心新藥成藥性評(píng)估與評(píng)價(jià)國家地方聯(lián)合工程實(shí)驗(yàn)室;深圳翰宇藥業(yè)股份有限公司;
【基金】：廣東省自然科學(xué)基金資助項(xiàng)目(No2016A030313335)
【分類號(hào)】：R96

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