基于血栓调节蛋白的抗炎措施研究
发布时间：2018-08-16 19:40
【摘要】： 研究背景:过度炎症与多种疾病密切相关。血栓调节蛋白(thrombomodulin,TM)是一种表达于血管内皮细胞等多种细胞表面的糖蛋白,其具有重要的抗炎功能。因此,使得TM成为防治炎症相关疾病的措施中极具吸引力的一个重要的药物设计新来源。最近一项研究发现,TM是体内天然存在的唯一可以结合并中和晚期(或宽治疗窗口期)关键细胞因子高迁移率族蛋白B1(high mobility group box 1,HMGB1)而产生明显抗炎效应的分子,并初步明确了其参与作用的区域为TM的PD1(N-terminal lectin-like domain)(155aa)结构域。这一新发现为解决我们前期工作的瓶颈带来了契机。在前期的工作中我们曾想开发能拮抗HMGB1而作为潜在的HMGB1的阻断剂的HMGB1 A box结构域,然而,单独表达A box欠稳定性,且目前所报道的A box抗炎作用的文献中均是以融合蛋白形式来表达A box,鉴于此,我们曾思考是否能在解决A box单独表达欠稳定的同时,赋予其更强大的拮抗HMGB1的功能呢?值此,若能将均具有明显抗炎效应的TMPD1与HMGB1 A box融合表达,则有望获得一种具有双重抑制HMGB1的且稳定性更好的新型抗炎衍生分子,从而为相关疾病的治疗提供一种新型候选制剂。 针对基于TM的抗炎措施,除了设计来源于TM的新型抗炎衍生分子外,如能设法上调TM的表达,无疑有助于与炎症相关的多种疾病的防治。法尼酯X受体(farnesoid X receptor, FXR)和肝X受体(Liver X receptor,LXR)均是核受体超家族成员,作为多功能转录因子,除了参与胆固醇、脂类和葡萄糖的代谢调节外,近年发现,抗炎作用是FXR及LXR的一项新功能,但关于其抗炎机制还有待深入阐明。由于FXR及LXR和TM均表达于血管内皮细胞,且均具重要抗炎功能,那么Tm是否是FXR或LXR的一个新靶基因?“通过上调TM而发挥抗炎功能”有无可能是FXR或LXR的一个抗炎新机制呢?弄清该问题,必须在首先证明FXR或LXR确实能影响TM表达,特别是能影响TM作为抗炎蛋白的活性的基础上,才能进行后续研究,从而为进一步深入而全面地阐明FXR或LXR的抗炎机理积累新的理论资料,为探索以FXR或LXR和TM为靶标的抗炎新措施提供科学依据。 研究目的:鉴于TM在抗炎方面的重要作用,制备并筛选由TMPD1与HMGB1 A box组成的能双重抑制HMGB1的融合蛋白;同时,研究FXR、LXR对TM表达的影响,并初步探讨其可能机制。 研究方法: 1.基于TMPD1和HMGB1 A box的新型HMGB1拮抗分子的制备和功能鉴定分别克隆来源于人和小鼠的TMPD1 cDNA,在测序正确的基础上,用基因工程的方法分别将人或小鼠TMPD1 cDNA(分别处于融合分子的N端或C端)与本室已克隆好的人HMGB1 A box(人和小鼠HMGB1 A box氨基酸序列完全一致)直接相连或在二者之间引入一个柔性连接子Gly4Ser,待测序正确后,将各融合分子分别亚克隆于原核表达载体pQE-80L并在大肠杆菌DH5α中进行表达,进而纯化各种蛋白;通过体外抗炎实验,分别观察不同来源的融合蛋白是否具有优于单独TMPD1或HMGB1 A box拮抗HMGB1的功能,同时也可通过比较各融合蛋白在功能强度上的差异而筛选出其中活性最好的分子;进而在体外抗炎实验中分别观察不同来源的最优融合蛋白拮抗HMGB1是否具有剂量依赖性,进一步证实其抗炎活性;为防止小鼠产生免疫反应影响在体抗炎实验效果,我们使用来源于小鼠TMPD1的最优融合蛋白在LPS诱导的小鼠内毒素血症模型(模拟内毒素引起的系统性炎症)上验证其抗炎作用暨增加小鼠存活率的情况。 2.法尼酯X受体、肝X受体可在血管内皮细胞中上调TM的表达选取人血管内皮细胞株Ea.hy926为细胞模型。在证明FXR及LXR在我们所选取的实验体系中是有功能活性的基础上,经FXR激动剂GW4064、CDCA及LXR激动剂GW3965分别处理24h后,RT-PCR和Real-time PCR法检测TM mRNA表达的变化;延长激动剂处理时间至48h,western blot法或流式细胞仪检测TM蛋白表达的差异;与此同时,发色底物法观察在FXR、LXR上调TM的表达水平后,能否增强抗炎效应分子活化蛋白C(activated protein C,APC)的产生能力,进一步证明FXR、LXR对TM活性的影响;在线分析人Tm基因转录起始位点上游(即5'调控区中)有可能存在的FXR或LXR结合位点,以此为线索,克隆人Tm基因启动子区域不同长度的片段,荧光素酶报告基因检测FXR或LXR对Tm基因启动子活性的影响,初步探讨FXR、LXR上调TM表达及活性的机制,为后续研究回答“Tm是否是FXR或LXR的一个新靶基因”奠定基础。 研究结果:(1)成功制备并纯化hTMPD1、A/ hTMPD1、A/linker/hTMPD1、hTMPD1/ A、hTMPD1/linker/A、mTMPD1、A/mTMPD1、A/linker/mTMPD1、mTMPD1/A、mTMPD1/linker /A共10个重组蛋白,其中8个为融合蛋白;(2)体外抗炎实验表明,hTMPD1/A与A/mTMPD1分别是来源于人TMPD1组和小鼠TMPD1组中拮抗HMGB1功能的最优分子;(3)hTMPD1/A与A/mTMPD1拮抗HMGB1均具有剂量依赖性;(4)A/mTMPD1在LPS诱导的小鼠内毒素血症模型中能明显增加小鼠存活率;(5)FXR呈配体剂量依赖性的上调TM mRNA和蛋白表达;(6)FXR激动剂GW4064上调TM活化APC的能力;(7)荧光素酶报告基因检测初步提示FXR极有可能在Tm基因启动子区域-646～-481存在结合位点IR8(AGGTCCtcccaaagTGCCCT -503～-484);(8)LXR呈配体剂量依赖性的上调TM mRNA和蛋白表达;(9)LXR激动剂GW3965上调TM活化APC的能力;(10)LXR激动剂不影响我们所克隆到的Tm基因启动子区域-2494～+160的活性。 结论:(1)所制备并筛选的TMPD1与HMGB1 A box融合蛋白是能双重抑制HMGB1且稳定性更好的新型抗炎衍生分子;(2)将TMPD1作为与A box组成融合蛋白的伴侣分子是既能增加其稳定性又能赋予其更强大的拮抗HMGB1功能的有效方法,从而为感染和炎症等相关疾病的治疗提供一种新型候选制剂制备策略;(3)融合蛋白的不同融合形式对融合蛋白的功能发挥影响很大,不同的融合形式适用于不同融合蛋白;(4)FXR极有可能结合Tm基因启动子区域的IR8位点直接上调TM表达;(5)LXR上调TM表达,但具体机制尚需进一步研究;(6)被FXR和LXR上调的TM是具有功能活性的。
[Abstract]:Background: Hyperinflammation is closely related to a variety of diseases. Thrombomodulin (TM) is a glycoprotein expressed on the surface of vascular endothelial cells and other cells. It has important anti-inflammatory functions. Therefore, TM has become an attractive drug design for the prevention and treatment of inflammatory-related diseases. Source. A recent study found that TM is the only molecule naturally present in the body that can bind to and produce significant anti-inflammatory effects on high mobility group box 1 (HMGB1), a key cytokine in the intermediate and late (or wide therapeutic window period), and preliminarily identified TM's PD1 (N-terminal lectin-like) region. The discovery of the domain (155aa) domain provides an opportunity to address the bottleneck of our previous work. In our previous work, we wanted to develop the HMGB1 A box domain, which can antagonize HMGB1 and act as a potential HMGB1 blocker. However, the single expression of the A box is unstable, and the reported anti-inflammatory effects of A box are based on In view of this, we have considered whether to confer stronger antagonistic effect on HMGB1 while resolving the unstable expression of box alone. Therefore, if TMPD1 and HMGB1 box with obvious anti-inflammatory effect can be fused and expressed, it is hopeful to obtain a dual inhibition of HMGB1 and more stable. Good new anti-inflammatory derived molecules, thus providing a new candidate for the treatment of related diseases.
In addition to designing novel anti-inflammatory derivatives derived from TM, the up-regulation of TM expression will undoubtedly contribute to the prevention and treatment of many inflammatory diseases. Farnesoid X receptor (FXR) and liver X receptor (LXR) are members of the nuclear receptor superfamily as multifunctional transcription. In addition to its involvement in the regulation of cholesterol, lipids and glucose metabolism, anti-inflammatory effects have been found to be a new function of FXR and LXR in recent years, but the anti-inflammatory mechanism remains to be elucidated. Is it possible that up-regulation of TM may be a new anti-inflammatory mechanism of FXR or LXR? To clarify this problem, we must first prove that FXR or LXR can indeed affect TM expression, especially TM as an anti-inflammatory protein activity, before we can carry out further studies, so as to further clarify the FXR or LXR in a comprehensive way. New theoretical data are accumulated on anti-inflammatory mechanism, which provides scientific basis for exploring new anti-inflammatory measures targeting FXR or LXR and TM.
OBJECTIVE: In view of the important role of TM in anti-inflammatory, to prepare and screen the fusion protein of TMPD1 and HMGB1 A box which can double inhibit HMGB1, and to study the effect of FXR and LXR on the expression of TM, and to explore the possible mechanism.
Research methods:
1. Based on the preparation and functional characterization of novel HMGB1 antagonist molecule from TMPD1 and HMGB1 A box, the TMPD1 cDNA from human and mouse was cloned. On the basis of correct sequencing, the human or mouse TMPD1 cDNA (located at the N-terminal or C-terminal of the fusion molecule respectively) and the human HMGB1 A box (human and mouse HMGB1 A box) were cloned by genetic engineering. The fusion proteins were subcloned into prokaryotic expression vector pQE-80L and expressed in E. Whether the fusion protein has the function of antagonizing HMGB1 better than TMPD1 or HMGB1 A box alone, and the most active molecule can be screened by comparing the functional strength of each fusion protein; then, in vitro anti-inflammatory experiment, we observed whether the optimal fusion protein from different sources antagonized HMGB1 in a dose-dependent manner. In order to prevent the immune response of mice and influence the anti-inflammatory effect in vivo, we used the optimal fusion protein from mouse TMPD1 to verify its anti-inflammatory effect and increase the survival rate of mice in LPS-induced endotoxemia model.
2. Farnesoid X receptor and hepatic X receptor can up-regulate the expression of TM in vascular endothelial cells. Human vascular endothelial cell line Ea. hy926 was selected as the cell model. On the basis of the functional activity of FXR and LXR in our selected experimental system, after being treated with FXR agonists GW4064, CDCA and LXR agonists GW3965 for 24 hours, RT-PCR and Real-time PC were performed. The expression of TM mRNA was detected by R assay, and the expression of TM protein was detected by Western blot or flow cytometry after prolonging the treatment time of agonists to 48 hours. At the same time, the ability of anti-inflammatory molecule activated protein C (APC) production was further confirmed by chromogenic substrate assay after FXR and LXR up-regulated the expression of TM. To investigate the effect of FXR and LXR on the activity of TM; to analyze the possible FXR or LXR binding sites upstream of human Tm gene transcription initiation site (i.e. 5'regulatory region), and to clone fragments of different length of human Tm gene promoter region, luciferase reporter gene to detect the effect of FXR or LXR on the activity of Tm gene promoter, and to preliminarily explore the FXR, LXR binding sites. The mechanism of up-regulation of TM expression and activity by XR lays a foundation for further research to answer whether Tm is a new target gene for FXR or LXR.
Results: (1) Ten recombinant proteins were successfully prepared and purified, including hTMPD1, A/hTMPD1, A/linker/hTMPD1, hTMPD1/A, hTMPD1/linker/A, mTMPD1, A/mTMPD1, A/linker/mTMPD1, mTMPD1/A, mTMPD1/A, and mTMPD1/linker/A, eight of which were fusion proteins; (2) In vitro anti-inflammatory tests showed that hTMPD1/A and A/mTMPD1 were antagonistic to human and mouse PD1, respectively. (3) hTMPD1/A and A/mTMPD1 antagonized HMGB1 in a dose-dependent manner; (4) A/mTMPD1 significantly increased the survival rate of mice in LPS-induced endotoxemia; (5) FXR up-regulated TM mRNA and protein expression in a dose-dependent manner; (6) FXR agonist GW4064 up-regulated TM-activated APC; (7) fluorescein Enzyme reporter gene detection preliminarily suggested that FXR might have binding sites IR8 (AGGTCCtcccaaag TGCCCT-503-484) in the promoter region of Tm gene; (8) LXR up-regulated TM mRNA and protein expression in a dose-dependent manner; (9) LXR agonist GW3965 up-regulated TMAPC activity; (10) LXR agonist did not affect the cloned Tm gene. Gene promoter region -2494 ~ +160 activity.
CONCLUSIONS: (1) The fusion protein of TMPD1 and HMGB1 box is a novel anti-inflammatory derivative that can inhibit both HMGB1 and HMGB1 with better stability; (2) TMPD1 as a chaperone molecule of fusion protein with A box is an effective way to increase its stability and give it stronger antagonistic effect on HMGB1, so as to prevent infection and inflammation. The treatment of symptoms and other related diseases provides a new candidate preparation strategy; (3) different fusion forms of fusion proteins have a great impact on the function of fusion proteins, and different fusion forms are suitable for different fusion proteins; (4) FXR is most likely to bind to the IR8 site of Tm gene promoter region directly up-regulate TM expression; (5) LXR up-regulates TM expression. However, specific mechanisms still need further study. (6) TM, which is upregulated by FXR and LXR, has functional activity.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R341
本文编号：2186982
[Abstract]:Background: Hyperinflammation is closely related to a variety of diseases. Thrombomodulin (TM) is a glycoprotein expressed on the surface of vascular endothelial cells and other cells. It has important anti-inflammatory functions. Therefore, TM has become an attractive drug design for the prevention and treatment of inflammatory-related diseases. Source. A recent study found that TM is the only molecule naturally present in the body that can bind to and produce significant anti-inflammatory effects on high mobility group box 1 (HMGB1), a key cytokine in the intermediate and late (or wide therapeutic window period), and preliminarily identified TM's PD1 (N-terminal lectin-like) region. The discovery of the domain (155aa) domain provides an opportunity to address the bottleneck of our previous work. In our previous work, we wanted to develop the HMGB1 A box domain, which can antagonize HMGB1 and act as a potential HMGB1 blocker. However, the single expression of the A box is unstable, and the reported anti-inflammatory effects of A box are based on In view of this, we have considered whether to confer stronger antagonistic effect on HMGB1 while resolving the unstable expression of box alone. Therefore, if TMPD1 and HMGB1 box with obvious anti-inflammatory effect can be fused and expressed, it is hopeful to obtain a dual inhibition of HMGB1 and more stable. Good new anti-inflammatory derived molecules, thus providing a new candidate for the treatment of related diseases.
In addition to designing novel anti-inflammatory derivatives derived from TM, the up-regulation of TM expression will undoubtedly contribute to the prevention and treatment of many inflammatory diseases. Farnesoid X receptor (FXR) and liver X receptor (LXR) are members of the nuclear receptor superfamily as multifunctional transcription. In addition to its involvement in the regulation of cholesterol, lipids and glucose metabolism, anti-inflammatory effects have been found to be a new function of FXR and LXR in recent years, but the anti-inflammatory mechanism remains to be elucidated. Is it possible that up-regulation of TM may be a new anti-inflammatory mechanism of FXR or LXR? To clarify this problem, we must first prove that FXR or LXR can indeed affect TM expression, especially TM as an anti-inflammatory protein activity, before we can carry out further studies, so as to further clarify the FXR or LXR in a comprehensive way. New theoretical data are accumulated on anti-inflammatory mechanism, which provides scientific basis for exploring new anti-inflammatory measures targeting FXR or LXR and TM.
OBJECTIVE: In view of the important role of TM in anti-inflammatory, to prepare and screen the fusion protein of TMPD1 and HMGB1 A box which can double inhibit HMGB1, and to study the effect of FXR and LXR on the expression of TM, and to explore the possible mechanism.
Research methods:
1. Based on the preparation and functional characterization of novel HMGB1 antagonist molecule from TMPD1 and HMGB1 A box, the TMPD1 cDNA from human and mouse was cloned. On the basis of correct sequencing, the human or mouse TMPD1 cDNA (located at the N-terminal or C-terminal of the fusion molecule respectively) and the human HMGB1 A box (human and mouse HMGB1 A box) were cloned by genetic engineering. The fusion proteins were subcloned into prokaryotic expression vector pQE-80L and expressed in E. Whether the fusion protein has the function of antagonizing HMGB1 better than TMPD1 or HMGB1 A box alone, and the most active molecule can be screened by comparing the functional strength of each fusion protein; then, in vitro anti-inflammatory experiment, we observed whether the optimal fusion protein from different sources antagonized HMGB1 in a dose-dependent manner. In order to prevent the immune response of mice and influence the anti-inflammatory effect in vivo, we used the optimal fusion protein from mouse TMPD1 to verify its anti-inflammatory effect and increase the survival rate of mice in LPS-induced endotoxemia model.
2. Farnesoid X receptor and hepatic X receptor can up-regulate the expression of TM in vascular endothelial cells. Human vascular endothelial cell line Ea. hy926 was selected as the cell model. On the basis of the functional activity of FXR and LXR in our selected experimental system, after being treated with FXR agonists GW4064, CDCA and LXR agonists GW3965 for 24 hours, RT-PCR and Real-time PC were performed. The expression of TM mRNA was detected by R assay, and the expression of TM protein was detected by Western blot or flow cytometry after prolonging the treatment time of agonists to 48 hours. At the same time, the ability of anti-inflammatory molecule activated protein C (APC) production was further confirmed by chromogenic substrate assay after FXR and LXR up-regulated the expression of TM. To investigate the effect of FXR and LXR on the activity of TM; to analyze the possible FXR or LXR binding sites upstream of human Tm gene transcription initiation site (i.e. 5'regulatory region), and to clone fragments of different length of human Tm gene promoter region, luciferase reporter gene to detect the effect of FXR or LXR on the activity of Tm gene promoter, and to preliminarily explore the FXR, LXR binding sites. The mechanism of up-regulation of TM expression and activity by XR lays a foundation for further research to answer whether Tm is a new target gene for FXR or LXR.
Results: (1) Ten recombinant proteins were successfully prepared and purified, including hTMPD1, A/hTMPD1, A/linker/hTMPD1, hTMPD1/A, hTMPD1/linker/A, mTMPD1, A/mTMPD1, A/linker/mTMPD1, mTMPD1/A, mTMPD1/A, and mTMPD1/linker/A, eight of which were fusion proteins; (2) In vitro anti-inflammatory tests showed that hTMPD1/A and A/mTMPD1 were antagonistic to human and mouse PD1, respectively. (3) hTMPD1/A and A/mTMPD1 antagonized HMGB1 in a dose-dependent manner; (4) A/mTMPD1 significantly increased the survival rate of mice in LPS-induced endotoxemia; (5) FXR up-regulated TM mRNA and protein expression in a dose-dependent manner; (6) FXR agonist GW4064 up-regulated TM-activated APC; (7) fluorescein Enzyme reporter gene detection preliminarily suggested that FXR might have binding sites IR8 (AGGTCCtcccaaag TGCCCT-503-484) in the promoter region of Tm gene; (8) LXR up-regulated TM mRNA and protein expression in a dose-dependent manner; (9) LXR agonist GW3965 up-regulated TMAPC activity; (10) LXR agonist did not affect the cloned Tm gene. Gene promoter region -2494 ~ +160 activity.
CONCLUSIONS: (1) The fusion protein of TMPD1 and HMGB1 box is a novel anti-inflammatory derivative that can inhibit both HMGB1 and HMGB1 with better stability; (2) TMPD1 as a chaperone molecule of fusion protein with A box is an effective way to increase its stability and give it stronger antagonistic effect on HMGB1, so as to prevent infection and inflammation. The treatment of symptoms and other related diseases provides a new candidate preparation strategy; (3) different fusion forms of fusion proteins have a great impact on the function of fusion proteins, and different fusion forms are suitable for different fusion proteins; (4) FXR is most likely to bind to the IR8 site of Tm gene promoter region directly up-regulate TM expression; (5) LXR up-regulates TM expression. However, specific mechanisms still need further study. (6) TM, which is upregulated by FXR and LXR, has functional activity.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R341
【参考文献】
相关期刊论文 前1条
1 彭军;陈槐卿;刘肖珩;李汝恒;郑晓红;;流体剪应力对内皮细胞蛋白C的活化以及蛋白C受体和血栓调节蛋白表达的影响(英文)[J];生物医学工程学杂志;2009年02期
，本文编号：2186982
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