荧光标记GPR119细胞系的构建

发布时间：2018-06-20 00:07

本文选题：GPR119 + β-arrestin　；参考：《昆明理工大学》2014年硕士论文

【摘要】：GPR119属于G蛋白偶联受体A型家族,主要在肠道内分泌L细胞和胰腺β细胞表面表达,当GPR119被激活后能够直接促进L细胞分泌GLP-1,并引发GLP-1的全部生理功能,同时也能够直接促进β细胞分泌胰岛素。因此,GPR119成为近年来研究糖尿病治疗的新靶点。而以GPR119分子药理学及其生物学特征为基础,建立相应的药物筛选模型并寻找GPR119小分子调节剂是抗糖尿病研发的新途径。本研究将以GPR119为靶点,通过直接或间接标记该靶点构建荧光标记的稳定细胞株,为下一步开展天然产物的药物筛选(尤其是小分子)奠定基础。本研究首先构建了GPR119绿色荧光蛋白(GFP)标记的真核表达载体,并先后转染人骨肉瘤U20S细胞和HEK293细胞,初步获得荧光标记的GPR119转染细胞,并利用蛋白质印迹技术验证了瞬时转染成功和GPR119蛋白的表达。但转染细胞在未加激动剂刺激情况下,也能发生自聚集反应。在后续实验中,经抗性筛选后转染细胞均全部死亡,难以形成稳定的细胞系,针对可能的原因,设计了多种解决方案,包括更换细胞类型、排除血清干扰、加入钙离子抑制剂、降低转染时的质粒浓度等,效果均不明显,仍在继续寻找合理的原因。考虑到GFP分子量较大,直接标记GPR119可能影响其空间结构,造成建立荧光标记GRP119细胞株的困难。而β-arrestin在G蛋白偶联受体信号转导中发挥着重要作用,它能特异性地与被配体激活的受体复合物结合,因此通过荧光标记的β-arrestin也能检测GPR119的聚合。因此,利用本实验室已构建的荧光标记β-arrestin的细胞株,尝试引入表达天然构象的GPR119建立相应的筛药细胞株。实验选用整合型质粒pCMV6-A-Hygro,插入GPR119序列,构建了pCMV6-A-Hygro-GPR119质粒并转染了荧光标记β-arrestin细胞株,加入激动剂后,已观察到荧光聚集,经过分选有望获得稳定细胞株。本研究通过直接标记和间接标记技术构建稳定的GPR119细胞株,两种方法均已得到瞬时表达的重组细胞,其中间接标记细胞能与激动剂发生反应。本研究为今后筛选小分子GPR119激动剂和其它调节剂提供了基础。
[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.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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