【作者】 李莹；

【导师】 邱阳；

【作者基本信息】 中国医科大学 ， 内科学， 2018， 硕士

【摘要】 目的:动脉粥样硬化（atherosclerosis,AS）发病率正在逐渐增高,研究AS及其相关疾病的发病机制和防治方法成为热点。二氢睾酮（dihydrotestosterone,DHT）是5α-还原酶还原睾酮双键形成的甾醇,是最具活性的雄激素。既往研究发现,生理水平的DHT可以显著减少巨噬细胞中血凝素样氧化型低密度脂蛋白受体1（lectin-like oxidized low density lipoprotein receptor 1,LOX-1）的mRNA的表达,而且至少部分是通过雄激素受体（androgen receptor,AR）发挥作用的。为进一步探讨雄激素及其受体对动脉粥样硬化发生发展的作用,本课题小组前期通过酵母双杂交筛选出在男性外周血单核细胞内与AR相互作用的蛋白,得到核糖体蛋白L10（RPL10）,前B细胞白血病同源框蛋白作用蛋白1（PBXIP1）,但通过酵母双杂交并不能充分证实蛋白与蛋白间的作用,存在假阳性的可能。为此,本实验旨在利用pull down技术在体外对AR与上述蛋白的相互作用进行进一步验证,进而探讨在男性单核细胞中的蛋白与AR的相互作用的信号传导通路,可能作为辅助调节因子与AR之间存在相互作用,为动脉粥样硬化致病机制及治疗方向提供理论依据和靶点。研究方法:本研究采用基因克隆的技术,将pGADT₇-RPL10、pGADT₇-PBXIP1作为模板,设计特异性引物,通过聚合酶链式反应（PCR）扩增目的片段。将RPL10、PBXIP1基因片段,克隆到利用Eco R I和Bam H I两种限制性内切酶双酶切后的含有GST标签的pGEX₄T₂载体上,构建重组质粒pGEX₄T₂-RPL10与pGEX₄T₂-PBXIP1,进行测序并分析。利用诱导剂IPTG诱导大肠杆菌表达含有GST标签的蛋白RPL10与PBXIP1,经聚丙烯酰胺凝胶电泳分离蛋白,考马斯亮蓝染胶,脱色观察蛋白表达情况。利用pull down技术,将pGBKT₇-AR重组质粒进行体外转录翻译,作为捕获蛋白;提取在大肠杆菌中表达的RPL10与PBXIP1蛋白,作为诱饵蛋白,将其固相化到MagneGST^TMM Particles上,将两种蛋白共同孵育,洗脱,纯化,利用Western blot技术分析相互作用的蛋白复合物,化学发光法鉴定结果并分析。结果:通过PCR技术获得目的基因片段,并重组质粒GST-RPL10、GST-PBXIP1构建成功。IPTG诱导RPL10、PBXIP1融合蛋白成功表达后,用pull down和Western blot技术证实,GST-RPL10融合蛋白大小约在50KD左右,GST-PBXIP1融合蛋白大小约在52KD左右,空白对照GST蛋白大小约在26KD左右,AR-LBD融合蛋白大小约在37KD左右;两种蛋白共同孵育后分别用不同抗体检测可见GST-RPL10/PBXIP1融合蛋白在体外可以特异性的将c-myc-AR-LBD融合蛋白pull down下来,空白对照GST蛋白不能将c-myc-AR融合蛋白pull down下来。结论:通过Pull down技术进一步证实AR编码蛋白分别与RPL10、PBXIP1在体外发生相互作用。更多还原

【Abstract】 Objective:The incidence of atherosclerosis（AS）is gradually increasing,and the pathogenesis,prevention and treatment of AS and its related diseases have become hot spots.Dihydrotestosterone（DHT）is the sterol formed by the 5α-reductase reducing testosterone double bond and is the most active androgen.Previous studies have found that physiological levels of DHT can significantly reduce m RNA expression of lectin-like oxidized low-density lipoprotein receptor 1（LOX-1）in macrophages,and at least In part,it acts through the androgen receptor（AR）.To further explore the role of androgen and its receptors in the development of atherosclerosis,our group previously screened proteins interacting with AR in male peripheral blood mononuclear cells through yeast two-hybrid screening to obtain ribosomal protein L10（RPL10）,pre-B cell leukemia homeobox protein 1（PBXIP1）,but yeast two-hybridization does not fully confirm the role of protein and protein,and there may be false positives.Therefore,this experiment aims to use pull down technology to further verify the interaction between AR and the above proteins in vitro,and then to explore the signal transduction pathway of the interaction between protein and AR in male monocytes,possibly as an auxiliary regulatory factor.The interaction with AR provides a theoretical basis and target for atherosclerosis pathogenesis and treatment.Research methods:This study uses gene cloning technology,pGADT₇-RPL10,pGADT₇-PBXIP1 as templates,to design specificprimers for amplification ofRPL10and PBXIP1 gene fragments,polymerase chain reaction（PCR）method was used to amplify the destination segment（RPL10,PBXIP1）.The RPL10 and PBXIP1 gene fragments were cloned into the pGEX₄T₂ vector containing GST tags after restriction enzyme digestion with Eco R I and Bam H I and the recombinant plasmids pGEX₄T₂-RPL10 and pGEX4T2-PBXIP1 were constructed,sequenced and analyzed.The inducer IPTG was used to induce E.coli expressed protein RPL10,PBXIP1.The protein was separated by polyacrylamide gel electrophoresis and was stained with Coomassie Blue.Protein expression was observed after decolorization.Using pull down system,pGBKT₇-AR recombinant plasmid as a capture protein for in vitro transcription;the RPL10,PBXIP1protein expressed in E.coli was extracted,and immobilized as a bait protein on MagneGST^?Particles.Then the two proteins were co-incubated,eluted and purified.Western blot was used to analysis the interaction of protein complexes,chemiluminescence method was used to identify the results and analysis.Result:The target gene fragment was obtained by PCR and the recombinant plasmids GST-RPL10 and GST-PBXIP1 were successfully constructed.After IPTG-induced RPL10 and PBXIP1 fusion proteins were successfully expressed,they were confirmed by pull down and Western blot techniques.The size of the GST-RPL10fusion protein was approximately 50 KD,the size of the GST-PBXIP1 fusion protein is about 52KD,the blank control GST protein size is about 26KD,and the AR-LBD fusion protein size is about 37KD;the two proteins are incubated together and detected with different antibodies to detect GST-RPL10/The PBXIP1 fusion protein can specifically pull down the c-myc-AR fusion protein in vitro.The blank control GST protein cannot pull down the c-myc-AR fusion protein.Conclusion:It was further confirmed AR protein can interact with RPL10,PBXIP1respectively in vitro by the pull down technology.更多还原