新型PARP抑制剂的筛选及其活性评价

发布时间：2018-03-13 08:31

本文选题：hPARP1　切入点：PARP抑制剂　出处：《江南大学》2013年硕士论文　论文类型：学位论文

【摘要】：聚ADP核糖聚合酶（Poly（ADP-ribose）polymerase，PARP）是一种参与DNA修复的核酶，在DNA损伤修复中起重要作用，为肿瘤的治疗提供一个新的靶点。因此，PARP抑制剂一直是近年来的研究热点，部分抑制剂已进入临床试验阶段。本论文对PARP抑制剂进行体外筛选并初步评价其药效，这为筛选有自主知识产权的新型PARP抑制剂奠定了基础。本课题通过基因重组技术将hPARP1基因插入到pFastBacTM1载体中，获得质粒pFast-hPARP1；将pFast-hPARP1转化入DH10Bac感受态细胞中，通过位点特异性转座，hPARP1基因可以整合入Bacmid穿梭载体中，获得表达质粒Bacmid-hPARP1；转染Bacmid-hPARP1至Sf9昆虫细胞进行表达，表达产物经3-氨基苯甲酰胺亲和层析柱分离纯化并通过SDS-PAGE、Western blotting和酶活检测法进行鉴定。通过测定已知PARP抑制剂DPQ和菲啶酮PHE对昆虫表达hPARP1的IC_(50)，验证此方法可以用来进行新型PARP抑制剂的筛选。通过克隆形成实验和CCK8细胞增殖-毒性检测实验，筛选对PARP抑制剂PHE敏感的细胞，用于筛选PARP抑制剂的细胞模型。实验结果表明，本论文成功构建了pFast-hPARP1质粒，通过Bac-to-Bac杆状病毒表达系统，实现了hPARP1酶在Sf9昆虫细胞中的表达，产物经3-氨基苯甲酰胺柱亲和层析成功得到纯化，得率为80.8%，纯化倍数为38.72倍，比活达到1.988U/μg。SDS-PAGE和Western blotting结果显示纯化产物在116kDa处有特异性目标条带。PARP抑制剂体外筛选结果显示，在72种化合物中筛选出41个具有生物活性的PARP抑制剂，其中有7个化合物的IC_(50)低于500nmol/L，显示出良好的PARP抑制作用。细胞模型筛选结果显示，中国人源胰腺癌细胞JF305对PARP抑制剂菲啶酮具有一定的敏感性。CCK8结果显示，筛选获得的PARP抑制剂HC-232（A）具有明显抑制JF305细胞的生长作用。本课题通过分子构建、表达、纯化等步骤获得了hPARP1。利用hPARP1，成功筛选出具有一定生物活性的PARP抑制剂。获得了对PARP抑制剂菲啶酮敏感的中国人源胰腺癌细胞JF305，这为筛选出具有自主知识产权的PARP抑制剂奠定了基础，并对后续研究提供了理论依据。
[Abstract]:Poly (ADP) ribosome polymerase (ADP) is a ribozyme involved in DNA repair, which plays an important role in the repair of DNA and provides a new target for tumor treatment. Some of the inhibitors have entered the stage of clinical trial. In this paper, PARP inhibitors were screened in vitro and their efficacy was preliminarily evaluated, which laid a foundation for the screening of new PARP inhibitors with independent intellectual property rights. In this study, the hPARP1 gene was inserted into the pFastBacTM1 vector by gene recombination technique, and the plasmid pFast-hPARP1 was obtained, and the pFast-hPARP1 was transformed into the DH10Bac receptive cells, which could be integrated into the Bacmid shuttle vector by site-specific transposition of hPARP1 gene. The expression plasmid Bacmid-hPARP1 was obtained and transfected into Sf9 insect cells for expression. The expressed product was separated and purified by 3- aminobenzoamide affinity chromatography and identified by SDS-PAGEG Western blotting and enzyme activity assay. It was proved that this method could be used to detect the expression of hPARP1 in insects by detecting the known PARP inhibitors DPQ and phenidinone PHE. Screening of novel PARP inhibitors. Clone formation assay and CCK8 cell proliferation-toxicity test were used. Screening of cells sensitive to PARP inhibitor PHE was used to screen cell models of PARP inhibitors. The results showed that the pFast-hPARP1 plasmid was successfully constructed and the expression of hPARP1 in Sf9 insect cells was realized by Bac-to-Bac baculovirus expression system. The product was purified by 3- aminobenzoamide column affinity chromatography. The specific activity of the purified product was 1.988 U / 渭 g. SDS-PAGE and Western blotting. The results showed that the purified product had a specific target band at 116 kDa. The in vitro screening results showed that 41 bioactive PARP inhibitors were screened out of 72 compounds. In 7 of them, the ICS 50) was lower than 500nmol / L, showing a good inhibitory effect on PARP. The cell model screening showed that JF305, a Chinese pancreatic cancer cell line, was sensitive to the PARP inhibitor phenidronone. CCK8 showed that, The selected PARP inhibitor, HC-232, can significantly inhibit the growth of JF305 cells. This topic is expressed by molecular construction, PARP inhibitors with certain biological activity were successfully screened by using hPARP1.The Chinese pancreatic cancer cell line JF305, which was sensitive to PARP inhibitor phenidinone, was obtained, which was used to screen PARP with independent intellectual property rights. The inhibitor laid the foundation, It also provides the theoretical basis for the follow-up study.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：TQ464.8

【共引文献】