【作者】 李妍；

【导师】 张春清；

【作者基本信息】 山东大学 ， 内科学， 2015， 博士

【摘要】 研究背景：肿瘤血管新生在肿瘤的生长、侵袭、转移过程中均有及其重要的作用。近年来,抗血管新生靶向治疗已成为肿瘤治疗的新秀方案,获得了广泛的关注。肝细胞癌血供丰富,肿瘤生长快,易发生侵袭和转移；抑制血管新生,有助于减少肝癌血供,延缓肝癌进展,改善疾病的预后。血管内皮生长因子(vascular endothelial growth factor, VEGF)在肿瘤血管新生过程中起着关键性作用。目前临床工作中使用的抗血管新生靶向治疗药物主要针对VEGF靶点,包括：抗VEGF中和抗体、VEGF受体激酶抑制剂、多激酶抑制剂等。然而,人体内VEGF靶器官众多,下游生理机制复杂,VEGF靶向治疗用于肝癌治疗易发生耐药性,且具有较多副作用,限制了其临床应用和发展。因此,我们应着力寻找更为精准的治疗靶点,尽可能减少抗血管新生药物的副作用,提高疗效,以期取得更好的临床效果。孤儿核受体TR3/Nur77(人TR3；小鼠Nur77；大鼠NGFI-B),又称神经生长因子诱导基因B,属于转录因子第四亚家族,与DNA顺式作用元件结合,发挥转录因子功能。哺乳动物细胞中不存在其天然配体,人工合成化合物如cytosporone B、 diindolymethanes类似物等可激活其转录活性,参与调控下游基因的转录起始,进而调节相关生理过程。TR3/Nur77生理作用广泛,可参与调节细胞凋亡、棕色脂肪产热、肿瘤发生与发展、炎症、代谢性疾病、应激、药物成瘾等多个生理过程。我们的前期工作发现,TR3/Nur77是VEGF发挥作用过程中的关键蛋白,介导VEGF诱导的内皮细胞增殖、迁移、通透性增高及体内血管新生,应用TR3反义链可对抗VEGF诱导的内皮细胞增殖、迁移、通透性增高及体内血管新生。此外,TR3/Nur77还可介导组胺、五羟色胺诱导的内皮细胞增殖、迁移、通透性改变和体内血管新生。内皮细胞过表达TR3/Nur77时,内皮细胞增殖、迁移、通透性均明显增高。小鼠荷瘤实验表明,较之野生型小鼠,Nur77基因敲除鼠荷瘤后血管新生较少,且肿瘤生长较慢。因此,我们认为TR3/Nur77是肿瘤抗血管新生靶向治疗的良好靶点。同其它核受体一样,TR3/Nur77有三个主要功能域：转录激活域(transactivation domain, TAD), DNA结合域(DNA binding domain, DBD),以及配体结合域(ligand binding domain, LBD)。其中,TR3/Nur77介导VEGF、组胺、五羟色胺诱导的血管内皮细胞增殖、迁移、通透性增加均有赖于其转录因子活性。然而,TR3/Nur77调控血管新生的分子机制,以及TR3/Nur77诱导血管新生的结构与功能之间的相关关系尚无报道。本研究旨在探寻TR3/Nur77诱导血管新生过程中结构与功能之间的关系,为肿瘤抗血管新生的分子治疗提供理论和实验基础。研究目的：本研究旨在探寻TR3/Nur77诱导血管新生过程中基因结构与功能之间的关系,了解TR3/Nur77促进内皮细胞增殖、迁移、通透性改变、细胞骨架重排的内在分子机制,寻找潜在治疗靶点,为下一步探索肿瘤抗血管新生的分子靶向治疗提供研究基础。研究方法：1、细胞培养：HEK293细胞系培养,以及HUVEC原代细胞培养。2、重组质粒构建：根据突变部位及特点,设计引物,采用重组PCR法扩增包含突变位点在内的核酸片段,并通过酶切、连接获得重组质粒。3、重组腺病毒制备：使用pAd/CMV/V5-DESTTM Gateway(?) Vector Kit将目标质粒重组整合至腺病毒表达载体,转染HEK293细胞获取重组腺病毒,扩增、纯化后分装、冻存备用。4、采用Western Blotting检测重组腺病毒目的蛋白表达情况。5、采用CyQUANT(?) Cell Proliferation Assay Kit检测各重组腺病毒对HUVEC原代细胞增殖能力的影响。6、采用单层细胞划痕实验检测各重组腺病毒对HUVEC原代细胞迁移能力的影响。7、采用单层细胞FITC-dextran(异硫氰酸荧光素标记的右旋糖酐)透过实验检测各重组腺病毒对HUVEC原代细胞通透性的影响。8、采用TRITC-phalloidin(罗丹明标记的鬼笔环肽)染色检测各重组腺病毒对HUVEC原代细胞细胞骨架的影响。9、计算机模拟分析TR3/Nur77转录激活域(TAD)蛋白质空间构象及可能的蛋白结合位点。10、采用SPSS 22.0统计软件分析数据。各组数据均以x±s描述,采用t-test与对照组比较。结果：1) TR3/Nur77可诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成；2)TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成,有赖于其转录因子活性；3) TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成,有赖于其DNA结合域内GRR(344-346)片段存在、Ser351去磷酸化参与；4) TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成,有赖于转录激活域内N端41-60位氨基酸片段参与；5) TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成,有赖于其转录激活域内N端41-61位氨基酸片段的磷酸化参与；6)计算机模拟分析TR3/Nur77转录激活域空间构象,预测该功能域内含三个可能与蛋白结合有关的位点,分别位于27-34位氨基酸片段,56-63位氨基酸片段与69-77位氨基酸片段。结论：我们的研究通过构建一系列TR3/Nur77突变基因用于内皮细胞干预,探索了TR3/Nur77各功能域、常见氨基酸位点在其诱导血管新生过程中的作用,初步定位了TR3/Nur77促血管新生功能的关键位点；并通过计算机模拟实验预测其转录激活域的蛋白质空间构象与可能的蛋白结合位点,得出如下结论：1)HUVEC细胞内TR3/Nur77过表达可诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成；2)TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成,有赖于其转录因子活性；3)TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成有赖于其DNA结合域内GRR(344-346AAS)模序存在、Ser351去磷酸化参与；4)TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成有赖于转录激活域内N端41-60位氨基酸片段参与；5)TR3/Nur77诱导内皮细胞增殖、迁移、通透性增加、细胞骨架重排、应力纤维形成有赖于其转录激活域内N端41-61位氨基酸片段的磷酸化参与：6)计算机模拟分析TR3/Nur77转录激活域空间构象,预测该功能域内含三个可能与蛋白质结合有关的位点,分别位于27-34位氨基酸片段,56-63位氨基酸片段与69-77位氨基酸片段。本研究通过探寻TR3/Nur77诱导血管新生的分子机制,明确其基因结构与促血管新生功能之间的关系,详细阐释了孤儿核受体TR3/Nur77诱导血管新生过程中起到关键作用的功能域与关键氨基酸位点,进一步证实了TR3是抗血管新生治疗的良好靶点,为人工合成特异性激活／抑制TR3/Nur77促血管新生转录活性的配体化合物提供理论依据,为今后肿瘤抗血管新生靶向治疗的研究奠定了理论和实验基础。更多还原

【Abstract】 Background:Angiogenesis plays a vital role in growth, invasion and metastasis of tumors. Recently, it is recognized that anti-angiogenic therapy of tumors is a prospective modality in tumor treatment. Hepatic cell carcinoma has redundant blood supplies and anti-angiogenesis might be beneficial in development as well as prognosis of hepatic cell carcinoma. VEGF is a key cytokine in tumor angiogenesis. Anti-VEGF neutralizing antibody, VEGF kinase inhibitor and multiple kinase inhibitors are now introduced in clinic for treatment of hepatic cell carcinoma. However, the human body is a complex and VEGF has too many target organs within the human body, which result in multiple side effects and intrinsic resistance. Hence it is required to investigate more precise therapeutic target to decrease adverse effect of anti-angiogenic therapy in tumor treatment as possible.Orphan nuclear receptor TR3/Nur77 (human TR3; mouse Nur77; rat NGFI-B), also known as nerve growth factor induced gene B, is a member of nuclear receptor IV subfamily of transcription factors. It has no physiological ligand in mammalian cell lines, although previous study has identified several synthesized agonists, including cytosporone B and a series of methylene-substituted diindolymethanes. TR3/Nur77 is of vital importance in cancer cell biology, brown fat thermosis, inflammation, metabolism diseases, stress and addiction. It currently acts as a transcriptional factor and initiates or blocks transcription of downstream genes, in which manner it regulates downstream physiological processes.Our previous work demonstrated that TR3/Nur77 is the key cytokine mediating VEGF induced proliferation, migration, increased permeability of endothelial cells and angiogenesis in vivo. Application of TR3/Nur77 antisense oligonucleotide will inhibit endothelial cell proliferation, migration and tube formation induced by VEGF. And so it is the same with angiogenic activities of endothelial cells induced by histamine and serotonin in vitro. Up regulation of TR3/Nur77 in endothelial cells directly induced cell proliferation, migration and increased monolayer permeability. Compared to wild type mice, Nur77 knock-out mice has significantly inhibited tumor growth and angiogenesis. Hence we demonstrated that TR3/Nur77 is an excellent target for pro-angiogenesis and anti-angiogenesis therapies.Similar to other members of orphan nuclear receptor superfamily, TR3/Nur77 has three predominant domains:transactivation domain, DNA binding domain, and ligand binding domain. Among them, transactivity is required in mediating VEGF induced endothelial cell proliferation, migration, and increased permeability. However, requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis is still unknown. Our study is aimed to investigate the structure-function relationships and set foundation for the subsequent anti-angiogenic therapy.Aims:The present study is aimed to investigate the structural and functional relationships in TR3/Nur77 induced angiogenesis, and to identify the potential therapeutic target of anti-angiogenic therapy in tumor treatment.Methods:1、Cell culture:HEK293 cells were routinely maintained in DMEM with 10% fetal bovine serum. And HUVECs were routinely maintained in EBM/EGM SingleQuots on 30μg/ml vitrogen coated plates.2、Construction of recombinant plasmid:Recombinant PCR was employed to prepare the mutants, with corresponding oligonucleotides as primers.3、Preparation of recombinant adenovirus:pAd/CMV/V5-DESTTM Gateway(?) Vector Kit was employed to prepare adenovirus vectors expressing TR3 and its mutants. Transfect HEK293 cells with adenovirus vectors expressing TR3 and its mutants and purify the virus. Store in -80 ℃ and thaw it on ice before each use.4、Western Blot was employed to screen the protein expression of HUVECs infected with different adenovirus.5、CyQUANT(?) Cell Proliferation Assay was employed to determine proliferation of HUVECS infected with different adenovirus.6、Monolayer migration assay was employed to determine migration of HUVECS infected with different adenovirus.7、Monolayer permeability assay was employed to determine migration of HUVECS infected with different adenovirus.8、Rhodamine phalloidin staining was employed to observe cytoskeleton changes of HUVECS infected with different adenovirus.9、Computational protein structure modeling and binding site potential analysis10、SPSS17.0 software was employed for statistical analysis and results are presented as mean± SD. Student’s t-test was employed to determine statistical significance.Results:1、TR3/Nur77 promotes proliferation, migration, permeability and cytoskeleton rearrangement.2、TR3/Nur77 transcriptional activity is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.3、De-phosphorylation of Ser351 and amino acid residues GRR in the DNA-binding domain are required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton.4、Amino acid 41-60 segment of TR3/Nur77 in the transactivation domain is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.5、Phosphorylation of Thr 48,55 and 61, Ser 52 and 54, and Tyr 60 is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.6、Protein structure and binding site prediction for TR3 N-terminal transactivation domain reveals there are 3 potential protein binding sites:site 1 (T27-T34), site 2 (F56-E63) and site 3 (Y69-P77).Conclusions:The main findings of the present studies are 1)TR3/Nur77 promotes proliferation, migration, permeability and cytoskeleton rearrangement.2)TR3/Nur77 transcriptional activity is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.3)De-phosphorylation of Ser351 and amino acid residues GRR in the DNA-binding domain are required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton.4)Amino acid 41-60 segment of TR3/Nur77 in the transactivation domain is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.5) Phosphorylation of Thr 48,55 and 61, Ser 52 and 54, and Tyr 60 is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.7)Protein structure and binding site prediction for TR3 N-terminal transactivation domain reveals there are 3 potential protein binding sites: site 1 (T27-T34), site 2 (F56-E63) and site 3 (Y69-P77).Our previous studies demonstrated that TR3 is an excellent and specific target for therapies of pro-angiogenesis and anti-angiogenesis. These studies further elucidate the structure and functional relationship of TR3 in angiogenesis by identification of novel amino acid residues that are critical for the function of TR3 in endothelial cells. Our current studies set the foundation for identification of compound(s) to inhibit or promote TR3 transcriptional activity for anti-angiogenic and pro-angiogenic therapies.更多还原