CN105267223B - A kind of plasma promotees blood coagulation adjuvant - Google Patents
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Abstract
本发明涉及一种等离子体促凝血辅助剂，其活性成分为高铁血红素。将配制的促凝血辅助剂滴加在血液中，在低温等离子体处理下，可以使血液中纤维蛋白原聚合，从而使血液迅速凝固。利用该发明进行低温等离子体凝血具有易操作、成本低、耗时短、凝血效果好等优势，因而有极好的潜在应用前景。
The invention relates to a plasma coagulation-promoting auxiliary agent, the active ingredient of which is heme. The prepared coagulation-promoting auxiliary agent is added dropwise to the blood, and under the low-temperature plasma treatment, the fibrinogen in the blood can be polymerized, so that the blood can coagulate rapidly. Using the invention to carry out low-temperature plasma coagulation has the advantages of easy operation, low cost, short time consumption, good coagulation effect, etc., and thus has excellent potential application prospects.
Description
技术领域technical field
本发明涉及一种等离子体促凝血辅助剂。The invention relates to a plasma coagulation promoting auxiliary agent.
背景技术Background technique
凝血是指血液凝固，由流体状态变为不能流动凝胶状态的过程。一般凝血机制包括产生凝血酶以稳定血小板血栓，以及形成纤维蛋白凝块。血液凝固过程大致分为凝血酶原激活物形成、凝血酶形成、纤维蛋白形成三个阶段。纤维蛋白形成一般是凝血酶形成后可催化血浆中可溶性纤维蛋白原(Fibrinogen，Fg)转变为纤维蛋白，形成不可溶性的纤维蛋白多聚体(血纤维)，并网罗血细胞形成凝胶状的血凝块。显然，对于凝血机制的研究有助于医学上止血处理。医学上为达到更好的止血效果，往往还使用辅助止血剂进行配合使用，比如较普遍的止血敷料有藻酸盐、明胶海绵/粉末、胶原海绵/粉末/纤维/薄片、氧化纤维素、凝血酶等，现在多用胶原复合物，包括结合有抑肽酶、凝血酶、纤维蛋白封闭剂等止血剂的胶原，还有结合抑肽酶的明胶、纤维蛋白胶、壳聚糖和沸石等，其中，胶原、壳聚糖和淀粉类止血材料应用越来越普遍。Coagulation is the process by which blood coagulates and changes from a fluid state to an immobile gel state. The general coagulation mechanism involves the generation of thrombin to stabilize platelet thrombi, and the formation of fibrin clots. The blood coagulation process is roughly divided into three stages: prothrombin activator formation, thrombin formation, and fibrin formation. Fibrin formation is generally after the formation of thrombin, which can catalyze the conversion of soluble fibrinogen (Fibrinogen, Fg) in plasma into fibrin, form insoluble fibrin polymers (blood fibers), and recruit blood cells to form a gelatinous blood clot. Obviously, the research on the mechanism of blood coagulation is helpful for medical hemostasis treatment. In order to achieve a better hemostatic effect in medicine, auxiliary hemostatic agents are often used in combination. For example, the more common hemostatic dressings include alginate, gelatin sponge/powder, collagen sponge/powder/fiber/sheet, oxidized cellulose, coagulation Enzymes, etc., now use collagen complexes, including collagen combined with hemostatic agents such as aprotinin, thrombin, and fibrin sealant, as well as gelatin, fibrin glue, chitosan, and zeolite combined with aprotinin, among which , Collagen, chitosan and starch-based hemostatic materials are becoming more and more common.
同时，人们还在发明和发展一些新的凝血技术，如等离子体凝血技术。等离体子具有物理、化学复合作用的优势，已经用于医疗器械的除垢消毒、皮肤病的治疗、牙齿根管治疗和手术伤口消毒等。最近，研究人员发现等离子体可以应用于凝血处理，但低温等离子体凝血效率还有待提高。但据了解，目前在利用低温等离子体技术凝血中加入高铁血红素作为促凝血辅助剂却未有报道。At the same time, people are still inventing and developing some new blood coagulation techniques, such as plasma coagulation technology. Plasma has the advantages of physical and chemical compound effects, and has been used in descaling and disinfection of medical equipment, treatment of skin diseases, root canal treatment of teeth, and disinfection of surgical wounds. Recently, researchers have found that plasma can be applied to blood coagulation treatment, but the coagulation efficiency of low-temperature plasma has yet to be improved. However, as far as we know, there is no report on the addition of hemoglobin as a coagulation-promoting auxiliary agent in blood coagulation using low-temperature plasma technology.
发明内容Contents of the invention
本发明要提供一种等离子体促凝血辅助剂，以克服现有低温等离子体凝血技术凝血效果不理想的问题。The present invention provides a plasma coagulation-promoting auxiliary agent to overcome the problem of unsatisfactory coagulation effect of the existing low-temperature plasma coagulation technology.
本发明所采取的技术方案为：The technical scheme that the present invention takes is:
一种等离子体促凝血辅助剂，其活性成分为高铁血红素。A plasma procoagulant auxiliary agent, the active ingredient of which is heme.
优选的，所述促凝血辅助剂用以下步骤配制而成：将高铁血红素放于超纯水，用氢氧化钠溶液调节pH至12使高铁血红素充分溶解；然后，用磷酸钠缓冲液配制成pH值为7.4、高铁血红素浓度为0.01mol/L高铁血红素母液。Preferably, the coagulation-promoting adjuvant is prepared by the following steps: put hemin in ultrapure water, adjust the pH to 12 with sodium hydroxide solution to fully dissolve the hemin; then, prepare it with sodium phosphate buffer The pH value is 7.4, and the hematin concentration is 0.01mol/L hematin mother liquor.
优选的，在用低温等离子体处理血液时，滴加所述高铁血红素母液至血液中，血液中高铁血红素浓度在10-5～10-3mol/L。Preferably, when the blood is treated with low-temperature plasma, the mother liquid of hematin is added dropwise into the blood, and the hematin concentration in the blood is 10 -5 -10 -3 mol/L.
优选的，所述所述氢氧化钠溶液浓度为0.1mol/L。Preferably, the concentration of the sodium hydroxide solution is 0.1mol/L.
本发明还提供了一种上述等离子体促凝血辅助剂的制备方法，包括下列步骤：将高铁血红素放于超纯水，用氢氧化钠溶液调节pH至12，待高铁血红素充分溶解后，用磷酸钠缓冲液配制pH＝7.4、高铁血红素浓度为0.01mol/L高铁血红素母液。The present invention also provides a preparation method of the above-mentioned plasma coagulation-promoting auxiliary agent, comprising the following steps: putting hemin in ultrapure water, adjusting the pH to 12 with sodium hydroxide solution, and after the hemin is fully dissolved, A sodium phosphate buffer solution was used to prepare a mother liquor of pH = 7.4 and a hematin concentration of 0.01 mol/L.
与现有技术相比本发明所取得的有益效果为：本发明首次提出在等离子体凝血处理中加入促凝血辅助剂的设想，并将高铁血红素作为促凝血辅助剂的活性成分配制成促凝血辅助剂，在低温等离子体作用下进行模拟凝血实验，结果表明，添加促凝血辅助剂的血液在30秒内，可以使血液中纤维蛋白原聚合，从而使血液迅速凝固，与不加该促凝血辅助剂的血液样品相比较，凝血效果等到显著提高。由此可见，利用该发明进行低温等离子体凝血具有易操作、成本低、耗时短、凝血效果好等优势，因而有极好的潜在应用前景。Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention proposes for the first time the idea of adding a coagulation-promoting auxiliary agent in the plasma coagulation treatment, and uses hemoglobin as the active ingredient of the anti-coagulation auxiliary agent to formulate Blood coagulation adjuvant, simulated blood coagulation experiment under the action of low-temperature plasma, the results show that adding the blood coagulation adjuvant can polymerize the fibrinogen in the blood within 30 seconds, so that the blood coagulates rapidly Compared with blood samples of coagulation aids, the coagulation effect was significantly improved. It can be seen that using the invention to carry out low-temperature plasma coagulation has the advantages of easy operation, low cost, short time consumption, and good coagulation effect, so it has excellent potential application prospects.
附图说明Description of drawings
图1为等离子体射流装置和电源部分原理示意图。Figure 1 is a schematic diagram of the principle of the plasma jet device and the power supply.
图2为模拟等离子体促凝血辅助剂作用下的凝血实验效果比较图。Fig. 2 is a comparison diagram of coagulation experiment effect under the action of simulated plasma procoagulant adjuvant.
图3等离子体促凝血辅助剂可促使纤维蛋白原聚合的荧光法验证结果。Fig. 3 The results of the fluorescence method verification that the plasma procoagulant adjuvant can promote the polymerization of fibrinogen.
具体实施方式Detailed ways
下面结合具体实施例对本发明的技术方案作进一步具体说明。The technical solutions of the present invention will be further specifically described below in conjunction with specific embodiments.
一种等离子体促凝血辅助剂，其活性成分为高铁血红素，主要通过以下步骤配制：称取适量高铁血红素放于超纯水中，用一定浓度的NaOH溶液调节pH至12，最好用0.1mol/L的NaOH溶液调节pH，高铁血红素在碱性溶液中可以快速溶解。待高铁血红素充分溶解，用磷酸钠缓冲液配制制成pH值为7.4、高铁血红素浓度为0.01mol/L高铁血红素母液。在用低温等离子体处理血液时，滴加上述高铁血红素母液至血液中，血液中高铁血红素浓度在10-5～10-3mol/L。A plasma coagulation-promoting auxiliary agent, the active ingredient of which is hemoglobin, which is mainly prepared by the following steps: weigh an appropriate amount of hemoglobin and put it in ultrapure water, adjust the pH to 12 with a certain concentration of NaOH solution, preferably with 0.1mol/L NaOH solution adjusts the pH, and heme can dissolve quickly in alkaline solution. After the hemin is fully dissolved, a sodium phosphate buffer solution is used to prepare a mother solution of hematin with a pH value of 7.4 and a hematin concentration of 0.01 mol/L. When the blood is treated with low-temperature plasma, the above-mentioned hematin mother solution is added dropwise to the blood, and the hematin concentration in the blood is 10 -5 ~ 10 -3 mol/L.
凝血实验的主要装置为等离子体子射流装置。图1为等离体子射流装置和电源部分原理示意图：一根长为15cm的不锈钢电极，它放在一根直径为5mm的石英管中，电极和石英管固定在绝缘外套中，绝缘外套上方有进气口和电源接线端，绝缘外套下方有出气口，放电时在出口处产生等离子体射流。放电电极下端被石英管封闭，上面则连接电源，电源输出电压为8～10万伏，放电频率约为8～10kHZ。放电射流的流速流量可以通过气阀控制，放电气体为He气，流量大约为1升/分钟。等离子体辐照样品时，射流出口和样品溶液之间的保持距离约1～5毫米，处理时间几秒到几分钟，样品初始温度为室温，样品处理后温度基本不变。The main device of coagulation experiment is plasma sub-jet device. Figure 1 is a schematic diagram of the principle of the plasma jet device and the power supply: a stainless steel electrode with a length of 15 cm is placed in a quartz tube with a diameter of 5 mm, and the electrode and the quartz tube are fixed in the insulating jacket, above the insulating jacket There are air inlets and power terminals, and there is an air outlet under the insulating jacket, and plasma jets are generated at the outlet when discharging. The lower end of the discharge electrode is sealed by a quartz tube, and the upper end is connected to a power supply. The output voltage of the power supply is 80,000 to 100,000 volts, and the discharge frequency is about 8 to 10kHZ. The flow rate and flow rate of the discharge jet can be controlled by an air valve, the discharge gas is He gas, and the flow rate is about 1 liter/minute. When the plasma irradiates the sample, the distance between the jet outlet and the sample solution is about 1-5 mm, the processing time is several seconds to several minutes, the initial temperature of the sample is room temperature, and the temperature of the sample remains basically unchanged after processing.
利用上述等离子体装置对模拟血液样品进行凝血实验。模拟血液样品的pH值范围为7.35～7.45，纤维蛋白原在在血浆中浓度范围为2～4克/升。取4份模拟血液样品，每个样品溶液200μL，暴露表面积为1cm2，同时做四个平行凝血实验：(a)为对照样品：未添加高铁血红素、未进行等离子体处理样品；(b)为未添加高铁血红素、等离子体处理30秒的样品；(c)为添加高铁血红素浓度为10-4mol/L、等离子体处理30秒的样品；(d)为添加高铁血红素浓度为10-3mol/L、等离子体处理30秒的样品。实验结果如图2所示，由图可以清楚看到，等离子体处理可以促凝血，加入高铁血红素，则凝血效果显著提高，并且随着浓度升高，凝血效果进一步增强。Coagulation experiments were carried out on simulated blood samples using the above-mentioned plasma device. The pH range of the simulated blood sample is 7.35-7.45, and the concentration range of fibrinogen in plasma is 2-4 g/L. Take 4 simulated blood samples, each sample solution is 200μL, and the exposed surface area is 1cm2, and four parallel coagulation experiments are performed at the same time: (a) is a control sample: no hemin is added, and no plasma treatment is performed; (b) It is the sample without adding hematin and treated with plasma for 30 seconds; (c) is the sample with added hematin concentration of 10 -4 mol/L and treated with plasma for 30 seconds; (d) is the sample with added hematin concentration of 10 -3 mol/L, plasma treated samples for 30 seconds. The experimental results are shown in Figure 2. It can be clearly seen from the figure that plasma treatment can promote blood coagulation, and the addition of hemoglobin can significantly improve the coagulation effect, and the coagulation effect is further enhanced as the concentration increases.
为了进一步验证以上实验效果，我们用含有纤维蛋白原的样品进行测试，即将含有纤维蛋白原的溶液(浓度为2～4克/升)进行等离子体辐照处理，并与加入含高铁血红素10-5M的等离子体促凝血辅助剂的纤维蛋白原的样品进行比较，结果如3所示，由图可知，等离子体处理前加入促凝血辅助剂处理后的样品蛋白质荧光增强幅度最大(图3中的曲线4)，说明添加促凝血辅助剂可以促使纤维蛋白原聚合形成纤维蛋白，而且进一步说明添加促凝血辅助剂的样品中纤维蛋白原转化为纤维蛋白的效率更高，即凝血效果更好。In order to further verify the above experimental results, we used samples containing fibrinogen to test, that is, the solution containing fibrinogen (concentration: 2-4 g/L) was subjected to plasma irradiation treatment, and mixed with the addition of hematin 10 The fibrinogen sample of -5 M plasma procoagulant adjuvant was compared, and the results are shown in 3. It can be seen from the figure that the protein fluorescence enhancement range of the sample treated with procoagulant adjuvant before plasma treatment was the largest (Fig. 3 Curve 4) in the figure shows that the addition of procoagulant adjuvant can promote the polymerization of fibrinogen to form fibrin, and further shows that the conversion efficiency of fibrinogen into fibrin in the sample added with procoagulant adjuvant is higher, that is, the coagulation effect is better .
除此以外，由于考虑到一般血液中还含有其它盐分，在实验中还比较了促凝血辅助剂在氯化钠存在下的凝血效果，结果表明，样品溶液中NaCl(0.9％)的存在对纤维蛋白原聚合影响不大，由此也证明了我们专利方案的实用性和普适性。In addition, due to the consideration that general blood also contains other salts, the coagulation effect of procoagulant adjuvants in the presence of sodium chloride was also compared in the experiment. The results showed that the presence of NaCl (0.9%) in the sample solution had an effect on Proprotein polymerization has little effect, which also proves the practicability and universality of our patented scheme.
最后所应说明的是，以上具体实施方式仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the present invention can be Modifications or equivalent replacements of the technical solutions without departing from the spirit and scope of the technical solutions of the present invention shall fall within the scope of the claims of the present invention.
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