止血材料开发及其表面功能蛋白研究
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摘要
不可控制的大出血是战争、车祸及其他意外事故死亡的主要原因。开发短时间内止血的紧急救生止血材料,能够有效降低出血导致的死亡。然而大量商业化止血材料都难以满足材料生物相容性好、止血效果快、方便使用等要求。自2002年沸石止血产品Quikclot面世以来,已经在战场上救活了成百上千人,然而Quikclot存在严重的放热效应,降低其放热量会导致止血效果也降低,这大大限制了Quikclot止血材料的应用。因此研究开发止血效果好、副作用低的止血材料是一个迫切需要解决的问题。虽然沸石止血材料有着十几年的应用历史,但是其机理仍然不明。近年来纳米材料-生物体系的相互作用研究表明,当材料进入生物体系时其表面会被生物分子(蛋白质等)覆盖形成“蛋白质环’'(Protein Cronoa),然而“蛋白质环”中功能蛋白与材料生物学效应之间的联系仍然没有阐明,因此研究沸石表面功能蛋白与凝血活性间的关系不仅有利于揭示沸石止血材料的凝血机理,还有利于阐明蛋白质环与材料生物活性之间的关系。
本论文主要围绕紧急救生止血材料的开发、材料止血机理及其表面功能蛋白的活性研究等展开。
论文第二章主要围绕止血效果好、放热效应低的止血材料的开发展开。我们研究了离子交换、基团修饰等对浙江缙云天然沸石体外凝血效果的影响,并考察了天然沸石在家兔致命性股动脉出血实验中的止血效果及伤口愈合情况、细胞毒性、全身毒性和放热效应等。结果表明,天然沸石能够大大降低股动脉出血时间,提高动物存活率,降低负效应,并且有利于伤口愈合。同时天然沸石具有良好的生物相容性、无细胞毒性、无全身毒性和过敏性等；因而天然沸石作为外用止血材料具有广阔的商业化应用前景。另一方面,天然矿物止血材料在我国有着上千年的药用历史,因而我们也考察了天然矿物(蒙脱土、膨润土、鹅管石、花蕊石等)的体外凝血时间和放热效应。与Quikclot相比,这些天然矿物都具有相近的体外凝血效果和较低的放热效应。结果表明天然矿物作为紧急救生止血材料具有潜在的市场应用前景。
论文第三章我们研究了Ca-沸石／血浆复合材料的止血效果及沸石表面功能蛋白。研究发现Ca-沸石进入血液时其表面会被血浆蛋白包裹形成一层具有超高促凝血活性和凝血酶活力的“蛋白质环”,而其他材料及其他离子交换沸石表面蛋白质环促凝血活性很低。免疫分析和生物质谱分析表明,Ca-沸石/蛋白质环中的功能蛋白为凝血酶,凝血酶的活力决定了材料的促凝血活性。当通过EDTA移除沸石中的钙离子,Ca-沸石/蛋白质环中的凝血酶逐渐被抗凝血酶抑制失去活性。稳定性分析表明,Ca-沸石/蛋白质环中的凝血酶能够在室温下稳定保存30个月。通过血友病小鼠(Factor Ⅷ缺乏)止血实验发现蛋白质环包裹的沸石可以有效地用于血友病出血的止血,Ca-沸石/血浆复合材料能够使小鼠在4.5min内止血,而商业化止血产品Quikclot在1h仍不能止血。本章中我们首次从蛋白质水平揭示了沸石止血材料的促凝机理以及蛋白质环中凝血酶与沸石促凝血活性之间的关系,并且发现无机材料(沸石)可以调控其表面蛋白的相互作用。
中药是我国瑰宝,中药的许多矿物药都具有止血效果,然而关于其机理与影响因素仍然不明。因此在本论文第四章我们研究了具有近千年药用历史的花蕊石的止血效果影响因素及止血机理。研究发现,花蕊石的产地与炮制方法对花蕊石止血效果有重要影响,花蕊石中的碳酸钙在炮制过程中生成的乙酸钙是其炮制后能够快速止血的主要原因；并且在一定范围内,随着炮制过程中乙酸钙生成的增加,碳酸钙表面吸附凝血酶的含量和活性也增加。该研究从分子水平揭示了花蕊石的止血机理,有利于中药的应用推广
论文第五章我们研究了二氧化硅表面基团对表面蛋白质环中原位产生凝血酶的吸附及其与抗凝血酶相互作用的调控。研究发现负电基团修饰(-OH,-SH,-COOH)的SBA-15可以促进凝血,正电基团(-NH2)修饰的SBA-15抑制凝血,并且基团修饰对材料表面蛋白质环形成及蛋白质环中凝血酶活性有重要影响；-OH和-SH基团修饰的SBA-15与BSA作用较弱,能够吸附较多的凝血酶,因此蛋白质环形成后具有较高的凝血酶活性。-COOH修饰的SBA-15与BSA作用力较强,材料表面与凝血酶作用力较弱,凝血酶被抗凝血酶结合,因此蛋白质环凝血酶活力低。该研究首次揭示了表面基团对蛋白质环中功能蛋白吸附及活性的影响。
论文第六章我们研究了二氧化钛晶相对表面蛋白质环形成及表面凝血酶活性的影响。研究发现锐钛矿型二氧化钛对血浆蛋白吸附弱,表面蛋白质环中蛋白质少,而金红石相二氧化钛与血浆蛋白作用力强,吸附大量血浆蛋白。进一步研究发现锐钛矿表面吸附凝血酶活性保持不变,金红石表面吸附凝血酶的活性则大量丧失。该研究首次揭示了材料晶相对蛋白质环形成及表面吸附酶活性的影响,对于二氧化钛的生物应用具有重要指导意义。
最后,在论文第七章我们对全文进行了总结,并对止血材料研究及材料-蛋白质环相互作用研究的发展趋势进行了展望。
Uncontrollable hemorrhage is the leading cause of death in battlefields, vehicle accidents, street violence, wilderness accidents etc. The development of a high-efficiency life-saving hemostatic can effectively reduce the mortality due to hemorrhage. However, large numbers of commercial hemostatic can not meet the requirements of high biocompatibility, quick clotting, ease of use etc. Since2002zeolite hemostatic Quikclot has saved hundreds of lives in battlefields, while there is severe exothermic reaction for Quikclot. The hemostatic efficiency will also decrease when reduce its exothermic reaction by pre-hydration, which greatly limited its application. Therefore, it is an urgent problem to develop a high-efficiency hemostatic with lower side effect. On the other hand, the coagulation mechanism of Quikclot is still unknown although it has been used over ten years. Recently, the development in nanomaterials-biological fluid interaction suggested that the material surface would be covered by a HPC (hard protein corona) after entering into the biological fluid and it was the HPC lead to the main biologic identity of the materials. However, the relationship between the functional proteins in the HPC to the biological impact of the materials is still unclear. The research on the relationships between the functional proteins in the HPC of zeolite surface and the hemostatic efficiency of zeolite not only helps to reveal the underlying procoaglant mechanism of zeolite hemostatic, but also plays critical roles in clarifying the relationship between the HPC and the biological activity of the materials.
This dissertation mainly focused on the development of life-saving hemostatic materials, hemostatic mechanism and functional proteins in the HPC over the material surface.
In chapter2, we focused on developing high-efficiency hemostatic with low side effect. We studied the effect of ion exchange and group modification on the in vitro coagulation efficiency of Jinyun natural zeolite, and evaluated its hemostatic efficiency in the lethal groin injury model of rabbit, cell toxicity, body toxicity and exothermic reaction. The results show that the ion exchange and group modification can not significantly improve the coagulation efficiency. The natural zeolite can significantly reduce the bleeding time, increase the survival of animals, reduce the exothermic effect and favor the wound healing process. Meanwhile, natural zeolite has good biocompatibility, non-cytotoxicity, and non-systemic toxicity, which make natural zeolite as a promising hemostatic. At the same time, we also developed other natural hemostatic with good clotting efficiency and low exothermic effect, such as montmorillonite and bentonite.
In chapter3, we studied the procoagulant activity and thrombin activity of Ca-zeolite/HPC. It was found that there would be formed a HPC with high thrombin activity and high-procoagulant activity on the surface of Ca-zeolite when it was incubated in plasma solution, while limited thrombin activity was observed on other materials or other cations exchange zeolite. Immunoassays and mass spectrometry analysis show that it is the thrombin in the Ca-zeolite/HPC that leads to the high procoagulant activity of Ca-zeolite. The thrombin in the Ca-zeolite/HPC will be inhibited by antithrombin to form a thrombin-antithrombin complex when removing the calcium ions in the zeolite by EDTA. The stability assay of Ca-zeolite/HPC shows that the thrombin activity is stable over a30months storage at room temperature. Finally, we studied the hemostatic efficiency of Ca-zeolite/HPC in a tail bleeding model of hemophilia mouse (Factor VIII deficiency). The results shows that the mice can effectively stop bleeding within4.5min after the application of Ca-zeolite/HPC, but it will still bleed after1h for the Quikclot group. In this chapter we first revealed the underlying procoagulant mechanism of zeolite and clarified the relationship between the procoagulant activity of zeolite and HPC.
Traditional Chinese medicine (TCM) is a great treasure of our country. Many TCM also have procoagulant effect, however the coagulation mechanism and influencing factors are still unknown. Therefore, we studied the influence factors on the hemostatic efficiency and hemostatic mechanism of ophicalcite in chapter4. The results show that the producing area and processing methods play critical roles in the hemostatic efficiency of ophicalcite. The calcium acetate formed in the processing process contributed to the acceleration of the blood coagulation. In certain range, the thrombin activity and thrombin adsorption on the surface of calcite increase as the increase in the formation of calcium acetate. The study reveals the underlying procoagulant mechanism of ophicalcite and favors the application of TCM.
In chapter5, we studied the surface group mediated thrombin generation and adsorption in the HPC of SBA-15. It was found that the negatively charged groups (-OH,-SH,-COOH) could promote the blood clotting of SBA-15, while positively charged group (-NH2) inhibit the blood coagulation. More important, the surface groups play critical roles in the HPC formation and thrombin activity in the HPC:-OH and-SH group modified SBA-15show moderate adsorption to BSA and leads to more thrombin absorbed in the HPC, thus the HPC show high thrombin activity.-COOH modified SBA-15show strong adsorption to BSA and leads to the weak interaction between thrombin, thus most of the thrombin in the HPC was inhibited by anti-thrombin. The research reveals the effects of the surface groups on the adsorption of the functional protein in the HPC.
In Chapter6, we studied the effect of crystal phase of titanium dioxide on the HPC formation and the activity of the adsorbed thrombin. The results show that anatase has weak affinity to the plasma proteins and leads to less protein in the HPC, while rutile has strong affinity to the plasma proteins and forms a HPC with much more proteins. The results of thrombin activity on the titanium dioxide surface reveal that there is a sharp decrease in thrombin activity after adsorption to rutile surface. On the other side, the thrombin activity does not show significant change after adsorption to anatase surface. The research clarified the effect of crystal phase on the HPC formation and the thrombin activity on the surface of titanium dioxide, and played a critical role for the biological applications titanium dioxide.
Finally, we summarized the dissertation, and made an outlook for the development trend of hemostatic materials and material-HPC interactions in Chapter
本论文主要围绕紧急救生止血材料的开发、材料止血机理及其表面功能蛋白的活性研究等展开。
论文第二章主要围绕止血效果好、放热效应低的止血材料的开发展开。我们研究了离子交换、基团修饰等对浙江缙云天然沸石体外凝血效果的影响,并考察了天然沸石在家兔致命性股动脉出血实验中的止血效果及伤口愈合情况、细胞毒性、全身毒性和放热效应等。结果表明,天然沸石能够大大降低股动脉出血时间,提高动物存活率,降低负效应,并且有利于伤口愈合。同时天然沸石具有良好的生物相容性、无细胞毒性、无全身毒性和过敏性等；因而天然沸石作为外用止血材料具有广阔的商业化应用前景。另一方面,天然矿物止血材料在我国有着上千年的药用历史,因而我们也考察了天然矿物(蒙脱土、膨润土、鹅管石、花蕊石等)的体外凝血时间和放热效应。与Quikclot相比,这些天然矿物都具有相近的体外凝血效果和较低的放热效应。结果表明天然矿物作为紧急救生止血材料具有潜在的市场应用前景。
论文第三章我们研究了Ca-沸石／血浆复合材料的止血效果及沸石表面功能蛋白。研究发现Ca-沸石进入血液时其表面会被血浆蛋白包裹形成一层具有超高促凝血活性和凝血酶活力的“蛋白质环”,而其他材料及其他离子交换沸石表面蛋白质环促凝血活性很低。免疫分析和生物质谱分析表明,Ca-沸石/蛋白质环中的功能蛋白为凝血酶,凝血酶的活力决定了材料的促凝血活性。当通过EDTA移除沸石中的钙离子,Ca-沸石/蛋白质环中的凝血酶逐渐被抗凝血酶抑制失去活性。稳定性分析表明,Ca-沸石/蛋白质环中的凝血酶能够在室温下稳定保存30个月。通过血友病小鼠(Factor Ⅷ缺乏)止血实验发现蛋白质环包裹的沸石可以有效地用于血友病出血的止血,Ca-沸石/血浆复合材料能够使小鼠在4.5min内止血,而商业化止血产品Quikclot在1h仍不能止血。本章中我们首次从蛋白质水平揭示了沸石止血材料的促凝机理以及蛋白质环中凝血酶与沸石促凝血活性之间的关系,并且发现无机材料(沸石)可以调控其表面蛋白的相互作用。
中药是我国瑰宝,中药的许多矿物药都具有止血效果,然而关于其机理与影响因素仍然不明。因此在本论文第四章我们研究了具有近千年药用历史的花蕊石的止血效果影响因素及止血机理。研究发现,花蕊石的产地与炮制方法对花蕊石止血效果有重要影响,花蕊石中的碳酸钙在炮制过程中生成的乙酸钙是其炮制后能够快速止血的主要原因；并且在一定范围内,随着炮制过程中乙酸钙生成的增加,碳酸钙表面吸附凝血酶的含量和活性也增加。该研究从分子水平揭示了花蕊石的止血机理,有利于中药的应用推广
论文第五章我们研究了二氧化硅表面基团对表面蛋白质环中原位产生凝血酶的吸附及其与抗凝血酶相互作用的调控。研究发现负电基团修饰(-OH,-SH,-COOH)的SBA-15可以促进凝血,正电基团(-NH2)修饰的SBA-15抑制凝血,并且基团修饰对材料表面蛋白质环形成及蛋白质环中凝血酶活性有重要影响；-OH和-SH基团修饰的SBA-15与BSA作用较弱,能够吸附较多的凝血酶,因此蛋白质环形成后具有较高的凝血酶活性。-COOH修饰的SBA-15与BSA作用力较强,材料表面与凝血酶作用力较弱,凝血酶被抗凝血酶结合,因此蛋白质环凝血酶活力低。该研究首次揭示了表面基团对蛋白质环中功能蛋白吸附及活性的影响。
论文第六章我们研究了二氧化钛晶相对表面蛋白质环形成及表面凝血酶活性的影响。研究发现锐钛矿型二氧化钛对血浆蛋白吸附弱,表面蛋白质环中蛋白质少,而金红石相二氧化钛与血浆蛋白作用力强,吸附大量血浆蛋白。进一步研究发现锐钛矿表面吸附凝血酶活性保持不变,金红石表面吸附凝血酶的活性则大量丧失。该研究首次揭示了材料晶相对蛋白质环形成及表面吸附酶活性的影响,对于二氧化钛的生物应用具有重要指导意义。
最后,在论文第七章我们对全文进行了总结,并对止血材料研究及材料-蛋白质环相互作用研究的发展趋势进行了展望。
Uncontrollable hemorrhage is the leading cause of death in battlefields, vehicle accidents, street violence, wilderness accidents etc. The development of a high-efficiency life-saving hemostatic can effectively reduce the mortality due to hemorrhage. However, large numbers of commercial hemostatic can not meet the requirements of high biocompatibility, quick clotting, ease of use etc. Since2002zeolite hemostatic Quikclot has saved hundreds of lives in battlefields, while there is severe exothermic reaction for Quikclot. The hemostatic efficiency will also decrease when reduce its exothermic reaction by pre-hydration, which greatly limited its application. Therefore, it is an urgent problem to develop a high-efficiency hemostatic with lower side effect. On the other hand, the coagulation mechanism of Quikclot is still unknown although it has been used over ten years. Recently, the development in nanomaterials-biological fluid interaction suggested that the material surface would be covered by a HPC (hard protein corona) after entering into the biological fluid and it was the HPC lead to the main biologic identity of the materials. However, the relationship between the functional proteins in the HPC to the biological impact of the materials is still unclear. The research on the relationships between the functional proteins in the HPC of zeolite surface and the hemostatic efficiency of zeolite not only helps to reveal the underlying procoaglant mechanism of zeolite hemostatic, but also plays critical roles in clarifying the relationship between the HPC and the biological activity of the materials.
This dissertation mainly focused on the development of life-saving hemostatic materials, hemostatic mechanism and functional proteins in the HPC over the material surface.
In chapter2, we focused on developing high-efficiency hemostatic with low side effect. We studied the effect of ion exchange and group modification on the in vitro coagulation efficiency of Jinyun natural zeolite, and evaluated its hemostatic efficiency in the lethal groin injury model of rabbit, cell toxicity, body toxicity and exothermic reaction. The results show that the ion exchange and group modification can not significantly improve the coagulation efficiency. The natural zeolite can significantly reduce the bleeding time, increase the survival of animals, reduce the exothermic effect and favor the wound healing process. Meanwhile, natural zeolite has good biocompatibility, non-cytotoxicity, and non-systemic toxicity, which make natural zeolite as a promising hemostatic. At the same time, we also developed other natural hemostatic with good clotting efficiency and low exothermic effect, such as montmorillonite and bentonite.
In chapter3, we studied the procoagulant activity and thrombin activity of Ca-zeolite/HPC. It was found that there would be formed a HPC with high thrombin activity and high-procoagulant activity on the surface of Ca-zeolite when it was incubated in plasma solution, while limited thrombin activity was observed on other materials or other cations exchange zeolite. Immunoassays and mass spectrometry analysis show that it is the thrombin in the Ca-zeolite/HPC that leads to the high procoagulant activity of Ca-zeolite. The thrombin in the Ca-zeolite/HPC will be inhibited by antithrombin to form a thrombin-antithrombin complex when removing the calcium ions in the zeolite by EDTA. The stability assay of Ca-zeolite/HPC shows that the thrombin activity is stable over a30months storage at room temperature. Finally, we studied the hemostatic efficiency of Ca-zeolite/HPC in a tail bleeding model of hemophilia mouse (Factor VIII deficiency). The results shows that the mice can effectively stop bleeding within4.5min after the application of Ca-zeolite/HPC, but it will still bleed after1h for the Quikclot group. In this chapter we first revealed the underlying procoagulant mechanism of zeolite and clarified the relationship between the procoagulant activity of zeolite and HPC.
Traditional Chinese medicine (TCM) is a great treasure of our country. Many TCM also have procoagulant effect, however the coagulation mechanism and influencing factors are still unknown. Therefore, we studied the influence factors on the hemostatic efficiency and hemostatic mechanism of ophicalcite in chapter4. The results show that the producing area and processing methods play critical roles in the hemostatic efficiency of ophicalcite. The calcium acetate formed in the processing process contributed to the acceleration of the blood coagulation. In certain range, the thrombin activity and thrombin adsorption on the surface of calcite increase as the increase in the formation of calcium acetate. The study reveals the underlying procoagulant mechanism of ophicalcite and favors the application of TCM.
In chapter5, we studied the surface group mediated thrombin generation and adsorption in the HPC of SBA-15. It was found that the negatively charged groups (-OH,-SH,-COOH) could promote the blood clotting of SBA-15, while positively charged group (-NH2) inhibit the blood coagulation. More important, the surface groups play critical roles in the HPC formation and thrombin activity in the HPC:-OH and-SH group modified SBA-15show moderate adsorption to BSA and leads to more thrombin absorbed in the HPC, thus the HPC show high thrombin activity.-COOH modified SBA-15show strong adsorption to BSA and leads to the weak interaction between thrombin, thus most of the thrombin in the HPC was inhibited by anti-thrombin. The research reveals the effects of the surface groups on the adsorption of the functional protein in the HPC.
In Chapter6, we studied the effect of crystal phase of titanium dioxide on the HPC formation and the activity of the adsorbed thrombin. The results show that anatase has weak affinity to the plasma proteins and leads to less protein in the HPC, while rutile has strong affinity to the plasma proteins and forms a HPC with much more proteins. The results of thrombin activity on the titanium dioxide surface reveal that there is a sharp decrease in thrombin activity after adsorption to rutile surface. On the other side, the thrombin activity does not show significant change after adsorption to anatase surface. The research clarified the effect of crystal phase on the HPC formation and the thrombin activity on the surface of titanium dioxide, and played a critical role for the biological applications titanium dioxide.
Finally, we summarized the dissertation, and made an outlook for the development trend of hemostatic materials and material-HPC interactions in Chapter
引文
1. http://www.who.int/mediacentre/news/releases/2007/pr17/en/index.html.
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