基于血栓調(diào)節(jié)蛋白的抗炎措施研究
發(fā)布時間：2018-08-16 19:40
【摘要】： 研究背景:過度炎癥與多種疾病密切相關(guān)。血栓調(diào)節(jié)蛋白(thrombomodulin,TM)是一種表達(dá)于血管內(nèi)皮細(xì)胞等多種細(xì)胞表面的糖蛋白,其具有重要的抗炎功能。因此,使得TM成為防治炎癥相關(guān)疾病的措施中極具吸引力的一個重要的藥物設(shè)計新來源。最近一項研究發(fā)現(xiàn),TM是體內(nèi)天然存在的唯一可以結(jié)合并中和晚期(或?qū)捴委煷翱谄?關(guān)鍵細(xì)胞因子高遷移率族蛋白B1(high mobility group box 1,HMGB1)而產(chǎn)生明顯抗炎效應(yīng)的分子,并初步明確了其參與作用的區(qū)域為TM的PD1(N-terminal lectin-like domain)(155aa)結(jié)構(gòu)域。這一新發(fā)現(xiàn)為解決我們前期工作的瓶頸帶來了契機。在前期的工作中我們曾想開發(fā)能拮抗HMGB1而作為潛在的HMGB1的阻斷劑的HMGB1 A box結(jié)構(gòu)域,然而,單獨表達(dá)A box欠穩(wěn)定性,且目前所報道的A box抗炎作用的文獻(xiàn)中均是以融合蛋白形式來表達(dá)A box,鑒于此,我們曾思考是否能在解決A box單獨表達(dá)欠穩(wěn)定的同時,賦予其更強大的拮抗HMGB1的功能呢?值此,若能將均具有明顯抗炎效應(yīng)的TMPD1與HMGB1 A box融合表達(dá),則有望獲得一種具有雙重抑制HMGB1的且穩(wěn)定性更好的新型抗炎衍生分子,從而為相關(guān)疾病的治療提供一種新型候選制劑。 針對基于TM的抗炎措施,除了設(shè)計來源于TM的新型抗炎衍生分子外,如能設(shè)法上調(diào)TM的表達(dá),無疑有助于與炎癥相關(guān)的多種疾病的防治。法尼酯X受體(farnesoid X receptor, FXR)和肝X受體(Liver X receptor,LXR)均是核受體超家族成員,作為多功能轉(zhuǎn)錄因子,除了參與膽固醇、脂類和葡萄糖的代謝調(diào)節(jié)外,近年發(fā)現(xiàn),抗炎作用是FXR及LXR的一項新功能,但關(guān)于其抗炎機制還有待深入闡明。由于FXR及LXR和TM均表達(dá)于血管內(nèi)皮細(xì)胞,且均具重要抗炎功能,那么Tm是否是FXR或LXR的一個新靶基因?“通過上調(diào)TM而發(fā)揮抗炎功能”有無可能是FXR或LXR的一個抗炎新機制呢?弄清該問題,必須在首先證明FXR或LXR確實能影響TM表達(dá),特別是能影響TM作為抗炎蛋白的活性的基礎(chǔ)上,才能進(jìn)行后續(xù)研究,從而為進(jìn)一步深入而全面地闡明FXR或LXR的抗炎機理積累新的理論資料,為探索以FXR或LXR和TM為靶標(biāo)的抗炎新措施提供科學(xué)依據(jù)。 研究目的:鑒于TM在抗炎方面的重要作用,制備并篩選由TMPD1與HMGB1 A box組成的能雙重抑制HMGB1的融合蛋白;同時,研究FXR、LXR對TM表達(dá)的影響,并初步探討其可能機制。 研究方法: 1.基于TMPD1和HMGB1 A box的新型HMGB1拮抗分子的制備和功能鑒定分別克隆來源于人和小鼠的TMPD1 cDNA,在測序正確的基礎(chǔ)上,用基因工程的方法分別將人或小鼠TMPD1 cDNA(分別處于融合分子的N端或C端)與本室已克隆好的人HMGB1 A box(人和小鼠HMGB1 A box氨基酸序列完全一致)直接相連或在二者之間引入一個柔性連接子Gly4Ser,待測序正確后,將各融合分子分別亞克隆于原核表達(dá)載體pQE-80L并在大腸桿菌DH5α中進(jìn)行表達(dá),進(jìn)而純化各種蛋白;通過體外抗炎實驗,分別觀察不同來源的融合蛋白是否具有優(yōu)于單獨TMPD1或HMGB1 A box拮抗HMGB1的功能,同時也可通過比較各融合蛋白在功能強度上的差異而篩選出其中活性最好的分子;進(jìn)而在體外抗炎實驗中分別觀察不同來源的最優(yōu)融合蛋白拮抗HMGB1是否具有劑量依賴性,進(jìn)一步證實其抗炎活性;為防止小鼠產(chǎn)生免疫反應(yīng)影響在體抗炎實驗效果,我們使用來源于小鼠TMPD1的最優(yōu)融合蛋白在LPS誘導(dǎo)的小鼠內(nèi)毒素血癥模型(模擬內(nèi)毒素引起的系統(tǒng)性炎癥)上驗證其抗炎作用暨增加小鼠存活率的情況。 2.法尼酯X受體、肝X受體可在血管內(nèi)皮細(xì)胞中上調(diào)TM的表達(dá)選取人血管內(nèi)皮細(xì)胞株Ea.hy926為細(xì)胞模型。在證明FXR及LXR在我們所選取的實驗體系中是有功能活性的基礎(chǔ)上,經(jīng)FXR激動劑GW4064、CDCA及LXR激動劑GW3965分別處理24h后,RT-PCR和Real-time PCR法檢測TM mRNA表達(dá)的變化;延長激動劑處理時間至48h,western blot法或流式細(xì)胞儀檢測TM蛋白表達(dá)的差異;與此同時,發(fā)色底物法觀察在FXR、LXR上調(diào)TM的表達(dá)水平后,能否增強抗炎效應(yīng)分子活化蛋白C(activated protein C,APC)的產(chǎn)生能力,進(jìn)一步證明FXR、LXR對TM活性的影響;在線分析人Tm基因轉(zhuǎn)錄起始位點上游(即5'調(diào)控區(qū)中)有可能存在的FXR或LXR結(jié)合位點,以此為線索,克隆人Tm基因啟動子區(qū)域不同長度的片段,熒光素酶報告基因檢測FXR或LXR對Tm基因啟動子活性的影響,初步探討FXR、LXR上調(diào)TM表達(dá)及活性的機制,為后續(xù)研究回答“Tm是否是FXR或LXR的一個新靶基因”奠定基礎(chǔ)。 研究結(jié)果:(1)成功制備并純化hTMPD1、A/ hTMPD1、A/linker/hTMPD1、hTMPD1/ A、hTMPD1/linker/A、mTMPD1、A/mTMPD1、A/linker/mTMPD1、mTMPD1/A、mTMPD1/linker /A共10個重組蛋白,其中8個為融合蛋白;(2)體外抗炎實驗表明,hTMPD1/A與A/mTMPD1分別是來源于人TMPD1組和小鼠TMPD1組中拮抗HMGB1功能的最優(yōu)分子;(3)hTMPD1/A與A/mTMPD1拮抗HMGB1均具有劑量依賴性;(4)A/mTMPD1在LPS誘導(dǎo)的小鼠內(nèi)毒素血癥模型中能明顯增加小鼠存活率;(5)FXR呈配體劑量依賴性的上調(diào)TM mRNA和蛋白表達(dá);(6)FXR激動劑GW4064上調(diào)TM活化APC的能力;(7)熒光素酶報告基因檢測初步提示FXR極有可能在Tm基因啟動子區(qū)域-646～-481存在結(jié)合位點IR8(AGGTCCtcccaaagTGCCCT -503～-484);(8)LXR呈配體劑量依賴性的上調(diào)TM mRNA和蛋白表達(dá);(9)LXR激動劑GW3965上調(diào)TM活化APC的能力;(10)LXR激動劑不影響我們所克隆到的Tm基因啟動子區(qū)域-2494～+160的活性。 結(jié)論:(1)所制備并篩選的TMPD1與HMGB1 A box融合蛋白是能雙重抑制HMGB1且穩(wěn)定性更好的新型抗炎衍生分子;(2)將TMPD1作為與A box組成融合蛋白的伴侶分子是既能增加其穩(wěn)定性又能賦予其更強大的拮抗HMGB1功能的有效方法,從而為感染和炎癥等相關(guān)疾病的治療提供一種新型候選制劑制備策略;(3)融合蛋白的不同融合形式對融合蛋白的功能發(fā)揮影響很大,不同的融合形式適用于不同融合蛋白;(4)FXR極有可能結(jié)合Tm基因啟動子區(qū)域的IR8位點直接上調(diào)TM表達(dá);(5)LXR上調(diào)TM表達(dá),但具體機制尚需進(jìn)一步研究;(6)被FXR和LXR上調(diào)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R341
本文編號：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R341
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 彭軍;陳槐卿;劉肖珩;李汝恒;鄭曉紅;;流體剪應(yīng)力對內(nèi)皮細(xì)胞蛋白C的活化以及蛋白C受體和血栓調(diào)節(jié)蛋白表達(dá)的影響(英文)[J];生物醫(yī)學(xué)工程學(xué)雜志;2009年02期
，本文編號：2186982
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2186982.html
最近更新
教材專著
