猪食欲肽受体2激动剂细胞筛选模型的建立及对天然中药提取物受体激动剂的筛选

Establishment of Agonist Screening Model Based on Pig Orexin-2 Receptor and Screening of Receptor Agonists for Traditional Chinese Medicine

本研究旨在构建猪食欲肽受体2(pOX2R)的真核表达载体,探究其对天然中药提取物的药理学活性。将成功构建的pcDNA3.1(+)-myc/pOX2R和报告质粒PGL4.29[luc2p/CRE/Hygro]分别瞬时转染HEK293T细胞,利用萤火虫荧光素酶报告基因检测法测定阳性细胞株在不同浓度激动剂Orexin A和Orexin B以及颉颃剂TCS作用下胞内cAMP水平。将目的质粒与报告质粒稳定共转染CHO-K1细胞,利用G418和潮霉素B进行抗性筛选,获得6株阳性细胞株和5株阴性细胞株。对具有促进食欲作用的18种天然中药材的36种提取物进行筛选,并用瞬时共转染了pcDNA3.1(+)-myc/pOX2R和PGL4.29[luc2p/CRE/Hygro]的HEK293T细胞作为研究对象,对阳性提取物进行剂量效应分析。结果显示,Orexin A与Orexin B单独作用细胞时,可以使萤火虫荧光素酶的活性提高,呈明显的剂量效应关系,当与颉颃剂TCS联合作用时,TCS可以颉颃激动剂对受体的激活作用。通过优化药物刺激时间和二甲亚砜(DMSO)用量后,建立了以pOX2R为靶点的稳定、可靠的激动剂细胞筛选模型。经过初筛和复筛,得到了甘草水提物、白莲子和山药醇提物3种可以使阳性细胞荧光素酶表达量升高的提取物。提取物在一定浓度范围内可以刺激胞内荧光素酶的表达,呈剂量效应关系。以上结果为筛选出可以提高猪生产效率的先导化合物及相关药物研究提供参考依据。

This study was aimed to construct a eukaryotic expression vector for porcine Orexin 2 receptor(pOX2R)and explore its pharmacological function when treated with traditional Chinese medicine.The pcDNA3.1(+)-myc/pOX2R and PGL4.29[luc2p/CRE/Hygro]were transiently transfected into HEK293T cells.The intracellular cAMP level of the positive cell lines under the action of Orexin A and Orexin B and antagonist TCS was measured by luciferase reporter gene assay.6 positive cells and 5 negative cells were obtained by stably transfecting target plasmid and reporter plasmid into CHO-K1 cells,G418 and hygromycin B were used for resistance screening.36 kinds of extracts from 18 kinds of traditional Chinese medicine were screened,HEK293T cells transfected with pcDNA3.1(+)-myc/pOX2R and PGL4.29[luc2p/CRE/Hygro]were used to analyze the dose effect on the positive extracts.The results showed that Orexin A and Orexin B could increase the activity of firefly luciferase,and TCS could antagonize the activation of receptor.A stable and reliable agonist screening model was constructed based on the expression level of firefly luciferase by optimizing drug stimulation time and DMSO dosage.Licorice extracted by water,lotus seed and Chinese yam extracted by alcohol could increase the expression of firefly luciferase.The extract could stimulate the expression of intracellular luciferase in a certain concentration range,which was dose-effect relationship.These results would provide reference for screening compounds and related drugs that could improve pig production efficiency.