發(fā)布時間：2018-06-20 00:07

  本文選題：GPR119 + β-arrestin��； 參考：《昆明理工大學》2014年碩士論文

【摘要】：GPR119屬于G蛋白偶聯(lián)受體A型家族,主要在腸道內(nèi)分泌L細胞和胰腺β細胞表面表達,當GPR119被激活后能夠直接促進L細胞分泌GLP-1,并引發(fā)GLP-1的全部生理功能,同時也能夠直接促進β細胞分泌胰島素。因此,GPR119成為近年來研究糖尿病治療的新靶點。而以GPR119分子藥理學及其生物學特征為基礎,建立相應的藥物篩選模型并尋找GPR119小分子調(diào)節(jié)劑是抗糖尿病研發(fā)的新途徑。本研究將以GPR119為靶點,通過直接或間接標記該靶點構建熒光標記的穩(wěn)定細胞株,為下一步開展天然產(chǎn)物的藥物篩選(尤其是小分子)奠定基礎。本研究首先構建了GPR119綠色熒光蛋白(GFP)標記的真核表達載體,并先后轉(zhuǎn)染人骨肉瘤U20S細胞和HEK293細胞,初步獲得熒光標記的GPR119轉(zhuǎn)染細胞,并利用蛋白質(zhì)印跡技術驗證了瞬時轉(zhuǎn)染成功和GPR119蛋白的表達。但轉(zhuǎn)染細胞在未加激動劑刺激情況下,也能發(fā)生自聚集反應。在后續(xù)實驗中,經(jīng)抗性篩選后轉(zhuǎn)染細胞均全部死亡,難以形成穩(wěn)定的細胞系,針對可能的原因,設計了多種解決方案,包括更換細胞類型、排除血清干擾、加入鈣離子抑制劑、降低轉(zhuǎn)染時的質(zhì)粒濃度等,效果均不明顯,仍在繼續(xù)尋找合理的原因。考慮到GFP分子量較大,直接標記GPR119可能影響其空間結構,造成建立熒光標記GRP119細胞株的困難。而β-arrestin在G蛋白偶聯(lián)受體信號轉(zhuǎn)導中發(fā)揮著重要作用,它能特異性地與被配體激活的受體復合物結合,因此通過熒光標記的β-arrestin也能檢測GPR119的聚合。因此,利用本實驗室已構建的熒光標記β-arrestin的細胞株,嘗試引入表達天然構象的GPR119建立相應的篩藥細胞株。實驗選用整合型質(zhì)粒pCMV6-A-Hygro,插入GPR119序列,構建了pCMV6-A-Hygro-GPR119質(zhì)粒并轉(zhuǎn)染了熒光標記β-arrestin細胞株,加入激動劑后,已觀察到熒光聚集,經(jīng)過分選有望獲得穩(wěn)定細胞株。本研究通過直接標記和間接標記技術構建穩(wěn)定的GPR119細胞株,兩種方法均已得到瞬時表達的重組細胞,其中間接標記細胞能與激動劑發(fā)生反應。本研究為今后篩選小分子GPR119激動劑和其它調(diào)節(jié)劑提供了基礎。
[Abstract]:GPR119 belongs to the G protein-coupled receptor A family, mainly expressed on the surface of endocrine L cells and pancreatic 尾 cells. When GPR119 is activated, it can directly promote the secretion of GLP-1 by L cells and trigger the whole physiological function of GLP-1. At the same time, it can also directly promote the secretion of insulin by 尾-cells. Therefore, GPR119 has become a new target for the treatment of diabetes in recent years. Based on the molecular pharmacology and biological characteristics of GPR119, it is a new way to develop anti-diabetic drugs to establish the corresponding drug screening model and find the small molecular regulator of GPR119. In this study, GPR119 was used as the target to construct the fluorescent labeled stable cell line by directly or indirectly labeling the target, which laid the foundation for drug screening of natural products (especially small molecules) in the next step. In this study, a GPR119 green fluorescent protein (GFP) labeled eukaryotic expression vector was constructed and transfected into human osteosarcoma U20S cells and HEK293 cells, and fluorescent labeled GPR119 transfected cells were obtained. The transient transfection and the expression of GPR119 protein were verified by Western blot. However, the transfection cells also showed a self-aggregation reaction without agonist stimulation. In the subsequent experiments, all the transfected cells died after the resistance screening, and it was difficult to form a stable cell line. According to the possible reasons, a variety of solutions were designed, including changing cell types, eliminating serum interference, and adding calcium ion inhibitors. The effect of reducing plasmid concentration during transfection was not obvious. Considering the high molecular weight of GFP, the direct labeling of GPR119 may affect its spatial structure and make it difficult to establish a fluorescent labeled GRP119 cell line. 尾 -arrestin plays an important role in the signal transduction of G-protein-coupled receptor, and it can specifically bind to ligand-activated receptor complex. Therefore, 尾 -arrestin can also detect the polymerization of GPR119 by fluorescent labeled 尾 -arrestin. Therefore, using the fluorescent labeled 尾 -arrestin cell line constructed in our laboratory, we try to introduce the natural conformation of GPR119 to establish the corresponding screening cell line. The plasmid pCMV6-A-Hygro119 was inserted into the GPR119 sequence. The plasmid pCMV6-A-Hygro-GPR119 was constructed and transfected into the fluorescent labeled 尾 -arrestin cell line. After the addition of agonist, fluorescence aggregation was observed, and the stable cell line was expected to be obtained by sorting out the pCMV6-A-Hygro-GPR119. In this study, a stable GPR119 cell line was constructed by direct and indirect labeling techniques. Both methods have obtained transient expression of recombinant cells, in which indirect labeled cells can react with agonists. This study provides a basis for screening small molecule GPR119 agonists and other regulators in the future.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96

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