医用臭氧的体外体液灭菌作用研究

发布时间：2018-05-04 01:21

本文选题：医用臭氧 + 杀菌作用　；参考：《南方医科大学》2009年硕士论文

【摘要】： 臭氧(O_3)是由三个氧原子组成的分子,臭氧气体是一种淡蓝色带有刺激性酸味的气体,在空气中极不稳定,在常温常压空气中易还原为氧气(半衰期40min)。医用臭氧(Medical ozone)为医用纯氧通过臭氧发生器产生的臭氧与医用纯氧的混合气体,臭氧浓度在0.05%～5%之间。与日常生活用和工业用臭氧不同,医用臭氧由医用纯氧而非空气生成,故不含氮氧化合物(NO_2)等有毒气体分子,且要求其浓度能够被精确调控,另外由于其极不稳定性故必须即时制备即时使用(为叙述方便,如无特殊说明下文中的“臭氧”均代指“医用臭氧”)。臭氧具有极强的氧化性,其氧化性仅次于氟和过硫酸盐。已有大量研究表明,体外在生物耐受剂量浓度下(20～80μg/ml)的臭氧就能有效杀灭或灭活水体中多种微生物,包括G~+或G~-菌、HAV、HIV等。我国有慢性肝炎病毒包括乙肝病毒(HBV)和丙肝病毒(HCV)感染者约1.5亿,主要传播途径为血液传播。由于检测技术的限制及病毒感染窗口期的存在,血液制品的生物安全性尚未得到有效解决。降低血液肝炎病毒载量对移植肝再感染也有利。对于慢性乙型肝炎病毒感染者进行肝移植,目前主要预防方法为口服核苷类似物联合静脉用乙肝免疫球蛋白(HBIG),费用高昂,且HBIG国内暂无上市;对于HCV感染者的移植肝再感染目前尚无有效的预防方法。自体输血作为目前最为安全有效的输血方式虽具有节约血源、减少不必要的输血反应和感染性疾病传播等诸多优势,但可能发生细菌污染,输用后将造成严重甚至致命的后果。以上问题的解决均依赖于安全、有效的血液净化方式。腹水是肝硬化最常见的并发症之一。自发性腹膜炎(Spontaneous bacterialperitonitis,SBP)在肝硬化病例中的发生率为10%-25%,在腹水病例中的平均发生率约30%,是肝病严重的标志,病死率很高。SBP主要发生机制为宿主免疫防御功能减弱、肠道细菌移位(BT),感染的病原菌半数是大肠埃希菌,其次是金黄色葡萄球菌、肠球菌等。如能对此类患者腹水进行有效灭菌处理,其腹水浓缩后亦可回输体内,避免蛋白丢失或可提高治疗效果。本研究目的在于利用臭氧体外水介质中能有效杀灭多种微生物的特性,试验臭氧在体外对体液(血浆、腹水等)的杀菌效果,为利用臭氧在体外对体液进行生物净化的可行性进行评价并提供实验参数。第一部分医用臭氧对血浆中细菌杀灭作用的研究 1.1研究方法向5份10ml含等量(0.5麦氏浓度)细菌的生理盐水中分别通入浓度为5μg/ml、10μg/ml、15μg/ml、20μg/ml、90μg/ml的过滤臭氧作用5min。对照组为10ml含等量细菌的生理盐水,以同样方法通入等量过滤医用纯氧。取两组稀释液各1ml接种于细菌培养皿,倾注普通营养琼脂15～20ml并充分混匀,每组做3个培养皿重复,37℃培养24h后计数两组菌落数,计算杀菌率。杀菌率=(对照组平均菌落数-试验组平均菌落数)/对照组平均菌落数×100%。每个实验重复5次。验证臭氧对生理盐水中细菌的杀灭效果。取等浓度的大肠埃希菌和金黄色葡萄球菌菌悬液0.5ml加入10ml血浆中。探索高浓度(90μg/ml)的过滤医用臭氧分别作用5min、10min和20min对其杀菌效果。向无菌生理盐水中添加人新鲜冰冻血浆,按生理盐水与血浆体积比29:1、19:1、9:1、4:1、1:1、0:10混合,得到血浆终浓度分别为3.3%、5.0%、10.0%、20.0%、50.0%、100.0%的系列混合物。实验组于血浆浓度为分别为3.3%、5.0%、10.0%、20.0%、50.0%、100.0%生理盐水-血浆混合物各10ml中加入含菌量为0.5麦氏浓度的菌悬液0.5ml,通入80μg/ml过滤臭氧作用不同时间(5min、10min、20min);对照组为加入等量细菌的同稀释度稀释血浆,以同样流速通入等量过滤医用纯氧。探索血浆对臭氧杀菌的干扰作用。 1.2研究结果 20μg/ml以上臭氧作用5min对10ml生理盐水中大肠埃希菌、金黄色葡萄球菌杀灭率均可达100%,如减小臭氧浓度至5μg/ml对两种细菌的杀菌率分别降至46.72%和38.50%。臭氧对血浆中大肠埃希菌、金黄色葡萄球菌杀灭效果较生理盐水中差,90μg/ml臭氧作用5min对10ml血浆中大肠埃希菌、金黄色葡萄球菌的杀菌率分别只有18.14%和17.86%。延长作用时间可提高杀菌效果,臭氧作用20min对两种细菌的杀菌率可分别提高至50.35%和43.58%。向生理盐水中添加血浆,随血浆浓度的升高,不同血浆浓度(3.3%、5.0%、10.0%、20.0%、50.0%、100.0%)经浓度为80μg/ml的臭氧作用20min对大肠埃希菌及金黄色葡萄球菌的杀菌率从100%降低至50%以下。不同血浆浓度(3.3%、5.0%、10.0%、20.0%、50.0%、100.0%)组间对大肠埃希菌和金黄色葡萄球菌两种细菌的杀菌率均存在显著性差异(F=51420.241,P=0.000和F=75088.996,P=0.000),当血浆浓度在10.0%以上时以80μg/ml臭氧作用20min(相当于臭氧总量19.2mg)即可有细菌存活。不同作用时间组间对大肠埃希菌的杀菌率存在显著性差异(F=30885.690,P=0.000),对金黄色葡萄球菌亦然(F=27441.387,P=0.000),作用时间长者杀菌效果较好。 1.3研究结论 1、臭氧对生理盐水中大肠埃希菌和金黄色葡萄球菌有明显的杀菌效果,20μg/ml以上臭氧作用5min对10ml生理盐水中两种细菌杀灭率即可达100%。 2、臭氧对大肠埃希菌的杀菌效果优于金黄色葡萄球菌。 3、向生理盐水中添加血浆后,臭氧对其杀菌效率迅速降低。血浆浓度在10.0%以上时以80μg/ml臭氧作用20min(相当于臭氧总量19.2mg)即可有细菌存活。使用90μg/ml臭氧作用20min对血浆中的大肠埃希菌和金黄色葡萄球菌的杀灭率仅为50.35%和43.58%。说明血浆中存在抗氧化物质,能干扰臭氧的杀菌作用。进一步延长作用时间或可获得更高杀菌率。第二部分医用臭氧对肝硬化所致腹水中细菌的杀灭作用研究 2.1研究方法收集4例肝硬化顽固性腹水患者腹水标本共6份(其中一名患者于不同时间点取腹水3次)。4名患者的腹水按腹水TP含量由小到大的顺序编号1～4同一患者的不同腹水按腹水TP含量由小到大的顺序编号A～C(4号标本与C标本为同一份标本)。腹水采集前1周内未全身或局部使用抗生素。经检验腹水无菌,加入等量的大肠埃希菌和金黄色葡萄球菌,37℃培养24h后观察不同腹水中培养的菌落数。每份标本做7个培养皿重复,取其平均值。取每份腹水标本各10ml中加入等浓度菌悬液0.5ml,均通入80μg/ml臭氧分别作用5min、10min、20min,含等量细菌的同来源腹水作为对照组以同样方法通入等量过滤医用纯氧。取两组的稀释液接种细菌培养皿,倾注普通营养琼脂后于37℃培养24h,进行活菌菌落计数,计算杀菌率,观察80μg/ml臭氧作用不同时间对4份不同来源腹水的杀菌效果以及同一病人不同腹水的杀菌效果。实验当天同时检测腹水总蛋白(TP)等腹水常规及生化指标,观察臭氧对不同腹水的杀菌效果与该腹水TP含量的关系。 2.2研究结果 1～4号标本的腹水TP值分别为0.6、2.2、16.6、25.7 g/L。等量细菌加入4份腹水培养24小时后菌落数存在差异,1号标本形成菌落数最多,2号大于3号,4号菌落数最少。80μg/ml臭氧对不同来源腹水中大肠埃希菌的杀菌效果存在显著性差异(F=40499.603,P=0.000),对不同腹水中金黄色葡萄球菌杀菌率亦存在显著性差异(F=92525.40,P=0.000),其中80μg/ml臭氧对1号腹水标本(腹水TP含量为0.6g/L)和2号标本(腹水TP含量为2.0g/L)作用10 min(相当于臭氧总量9.6mg)后对两种细菌的杀菌率即可达100%,而对3号和4号标本(腹水TP含量分别为16.6g/L和25.7 g/L)作用10min后对两种细菌杀菌率仍均低于70%。臭氧不同作用时间(5min、10min、20min)间对大肠埃希菌的杀菌率存在显著性差异(F=28514.907,P=0.000),对金黄色葡萄球菌的杀菌率亦存在显著性差异(F=13066.314,P=0.000),作用时间长者杀菌效果较好,作用20min时对4份腹水中两种细菌的杀菌率均达到70%以上。不同腹水及不同臭氧作用时间对臭氧杀灭大肠埃希菌效率的影响存在交互效应(F=9775.527,P=0.000),对金黄色葡萄球菌的杀菌效果的影响亦存在交互作用(F=3242.915,P=0.00)(表2-3、表2-4)。臭氧作用5 min、10min、20min均显示80μg/ml的臭氧对4份腹水的杀菌效率1号优于2号,其次为3号,对4号标本杀菌效果最差(图2-3),即不同病人腹水其TP含量高者臭氧对其杀灭效果较差。 A～C号标本的腹水TP值分别为14.6、22.1、25.7 g/L。80μg/ml臭氧对同一病人3份不同腹水中大肠埃希菌的杀菌效果存在显著性差异(F=395.821,P=0.000),对不同腹水中金黄色葡萄球菌杀菌率亦存在显著性差异(F=520.915,P=0.000)。不同作用时间组间对大肠埃希菌的杀菌率存在显著性差异(F=38534.299,P=0.000),对金黄色葡萄球菌的杀菌率亦存在显著性差异(F=10800.111,P=0.000),作用时间长者杀菌效果较好。同一病人的不同腹水及不同臭氧作用时间对臭氧杀灭大肠埃希菌效率的影响存在交互效应(F=227.996,P=0.000),对金黄色葡萄球菌的杀菌效果的影响亦存在交互作用(F=140.123,P=0.00)(表2-6、表2-7)。臭氧作用5 min、10min、20min均显示80μg/ml的臭氧对3份腹水的杀菌效率A标本优于B标本,C标本最差(图2-5)。对同一病人的腹水来说,腹水中TP含量高者臭氧对其杀灭效果较差。 2.3研究结论 1、不同腹水环境对细菌生长有影响,腹水TP较高的标本细菌存活数量较少。 2、臭氧对腹水的杀菌效果优于血浆,但对不同腹水杀菌效果存在差异。验证了在蛋白含量较血浆低下的体液如腹水中生物安全剂量范围(不超过80μg/ml)内的臭氧即可发挥有效杀菌效果(即使对TP含量达16.6g/L的腹水以80μg/ml臭氧作用20min其杀菌率亦可达90%以上),腹水TP含量低者臭氧对其杀菌效率较高。 3、延长作用时间可提高臭氧杀菌效率。
[Abstract]:Ozone (O_3) is a molecule consisting of three oxygen atoms. The ozone gas is a light blue and irritating sour gas. It is extremely unstable in the air and is easily reduced to oxygen (half life 40min) in the atmospheric pressure air. Medical ozone (Medical ozone) is a mixture of ozone and medical pure oxygen produced by the ozone generator for medical pure oxygen. The concentration of ozone is between 0.05% and 5%. Unlike daily and industrial ozone, medical ozone is produced from medical pure oxygen instead of air, so it does not contain noxious gas molecules such as nitrogen and oxygen compounds (NO_2) and requires its concentration to be regulated accurately. If there is no special description, ozone is referred to as "medical ozone".
Ozone is highly oxidizable, and its oxidizability is second only to fluorine and persulfate. A large number of studies have shown that in vitro, ozone can effectively kill or inactivate a variety of microorganisms in the water body, including G~+ or G~- bacteria, HAV, HIV, and so on, under the biological tolerance dose concentration (20~80 Mu g/ml).
There are about 150 million of chronic hepatitis viruses, including hepatitis B virus (HBV) and hepatitis C virus (HCV) infection in our country. The main route of transmission is blood transmission. The biological safety of blood products has not been effectively solved because of the restriction of detection technology and the presence of the virus infection window period. The lower blood hepatitis virus load is also beneficial to the reinfection of the transplanted liver. For patients with chronic hepatitis B virus infection, the main prevention method is the combination of oral nucleoside analogues and intravenous hepatitis B immunoglobulin (HBIG). The cost is high, and HBIG is not available at home. There is no effective prevention method for reinfection of liver in HCV infected people. Autologous blood transfusion is the most safe and safe at present. Although effective blood transfusion has many advantages such as saving blood source, reducing unnecessary transfusion reaction and infectious disease transmission, bacterial contamination may result in serious or even fatal consequences. The solutions of the above problems are all dependent on safety and effective blood purification.
Ascites is one of the most common complications of liver cirrhosis. The incidence of Spontaneous bacterialperitonitis (SBP) in the cases of liver cirrhosis is 10%-25%, the average incidence in the cases of ascites is about 30%. It is a sign of serious liver disease, and the high mortality rate of.SBP is that the host immune defense function is weakened and the intestinal bacteria are weakened. BT, half of the infected pathogens are Escherichia coli, followed by Staphylococcus aureus, Enterococcus and so on. If it can effectively sterilize the ascites of such patients, the ascites can also be returned to the body after concentration, to avoid protein loss or to improve the therapeutic effect.
The purpose of this study is to test the bactericidal effect of ozone in body fluid (plasma, ascites, etc.) in vitro by using ozone in water medium, and to evaluate the feasibility of using ozone to purify the body fluid in vitro and provide experimental parameters.
The first part is about the killing effect of medical ozone on bacteria in plasma.
1.1 research methods
The concentration of 5 10ml containing equal (0.5 MF) bacteria was injected into the physiological saline of 5 mu g/ml, 10 mu g/ml, 15 mu g/ml, 20 mu g/ml, 90 u g/ml, 5min. control group was the normal saline containing the equal amount of bacteria in 10ml, and the same method was used to filter the medical pure oxygen in the same amount. The two groups of diluents were inoculated in the bacterial culture dish and poured out. Common nutrient agar was 15 ~ 20ml and well mixed, 3 cultures were repeated in each group. Two groups of colonies were counted and bactericidal rate was counted at 37 C for 24h. Bactericidal rate = (average colony number of the control group - average colony number in test group) / the average colony number of control group was repeated 5 times per experiment. The effect of ozone on bacteria in normal saline was verified.
The concentration of Escherichia coli and Staphylococcus aureus suspension 0.5ml were added to the 10ml plasma. The bactericidal effect of 5min, 10min and 20min on the high concentration (90 u g/ml) of filtered medical ozone was investigated respectively.
A mixture of fresh frozen plasma was added to aseptic saline, and a series of 3.3%, 5%, 10%, 20%, 50%, 100% mixture of 3.3%, 5%, 20%, 50%, 100%, respectively. The plasma concentration of the experimental group was 3.3%, 5%, 10%, 20%, 50%, 100% saline plasma mixture, respectively. Each 10ml was added to the bacterial suspension containing 0.5 malt concentration of bacteria 0.5ml, and the ozone action was filtered through 80 g/ml (5min, 10min, 20min). The control group was diluted with the same dilution of the same amount of bacteria and filtered the medical pure oxygen at the same flow rate, and the interference effect of plasma on ozone sterilization was explored.
1.2 research results
The killing rate of 5min for Escherichia coli and Staphylococcus aureus in 10ml normal saline up to 20 g/ml was 100%, such as reducing the ozone concentration to 5 u g/ml to 46.72% and 38.50%. ozone against Escherichia coli in plasma, the killing effect of Staphylococcus aureus was worse than that in saline, 90 mu g/ml stinky. The bactericidal rate of 5min in 10ml plasma of Escherichia coli and Staphylococcus aureus, respectively, only 18.14% and 17.86%. prolongation time can improve the germicidal efficacy. The bactericidal rate of ozone action 20min on two kinds of bacteria can be increased to 50.35% and 43.58%. to the physiological saline, with the increase of plasma concentration and different plasma concentration. 3.3%, 5%, 10%, 20%, 50%, 100%) the bactericidal rate of 20min to Escherichia coli and Staphylococcus aureus from 100% to 50% below the concentration of 80 g/ml ozone. The bactericidal rates of Escherichia coli and Staphylococcus aureus among the groups of different plasma concentrations (3.3%, 5%, 10%, 20%, 50%, etc.) were all significant The difference (F=51420.241, P=0.000 and F=75088.996, P=0.000), when the plasma concentration is above 10%, the bacteria can survive with the action of 80 mu g/ml (equivalent to the total amount of ozone 19.2mg). The bactericidal rate of Escherichia coli between different time groups is significantly different (F= 30885.690, P=0.000), and to Staphylococcus aureus (F=27441.38). 7, P=0.000), the effect of germicidal effect is better.
1.3 research conclusions
1, ozone has obvious bactericidal effect on Escherichia coli and Staphylococcus aureus in normal saline, and the killing rate of two kinds of bacteria in 10ml normal saline by ozone over 20 g/ml is 100%.
2, ozone has a better germicidal efficacy against Escherichia coli than Staphylococcus aureus.
3, after the plasma was added to the saline, the bactericidal efficiency of ozone decreased rapidly. When the plasma concentration was above 10%, the bacteria could survive with 80 mu g/ml ozone action 20min (equivalent to the total ozone amount 19.2mg). The killing rate of Escherichia coli and Staphylococcus aureus in plasma was only 50.35% and 43.58% with 90 g/ml ozone action 20min. It indicates that there are antioxidants in the plasma, which can interfere with the germicidal efficacy of ozone. Further prolonging the time of action may lead to a higher bactericidal rate.
The second part is about the killing effect of medical ozone on bacteria in ascites caused by cirrhosis.
2.1 research methods
A total of 6 specimens of ascites in 4 patients with cirrhosis of the refractory ascites were collected (one of the patients took ascites at different time points for 3 times). The ascites of the.4 patients were numbered in the order of the TP content of ascites from small to large, and the different ascites of the same patient were numbered A to C in the order of TP in the ascites. (specimens of No. 4 and C specimens were the same specimen). Antibiotics were not used whole-body or local within 1 weeks before water collection. After testing ascitic asepsis, adding the equal amount of Escherichia coli and Staphylococcus aureus, the number of colonies in different ascites was observed after culture of 24h at 37 C. The average value of each specimen was repeated in 7 Petri dishes.
The same concentration bacteria suspension 0.5ml was added to the 10ml of each ascitic specimen. The same amount of ascitic water containing the same amount of bacteria was injected into the same amount of 80 u g/ml ozone to filter medical pure oxygen in the same amount as the control group. Two groups of diluents were inoculated to the bacterial culture dish, and then the common nutrient agar was poured into 24h at 37. The bacteria colony count was counted and the bactericidal rate was calculated. The bactericidal effect of 80 g/ml ozone action on 4 different sources of ascites and the bactericidal effect of different ascites in the same patient were observed.
On the same day, routine and biochemical indexes of ascites, such as ascites total protein (TP), were detected simultaneously. The relationship between the effect of ozone and the TP content of ascites was observed.
2.2 research results
The TP value of the ascites of 1~4 specimens was 0.6,2.2,16.6,25.7 g/L. equal amount of bacteria added to 4 ascites for 24 hours, and the number of colony number was different, the number of colony 1 was the most, 2 greater than 3, and the number of colony number 4 was least.80 mu g/ml ozone on the bactericidal effect of Escherichia coli in the ascites of different sources (F=40499.603, P=0.) 000), the bactericidal rate of Staphylococcus aureus in different ascites was also significantly different (F=92525.40, P=0.000). The bactericidal rate of 80 g/ml ozone on 1 ascites (ascites TP content 0.6g/L) and 2 specimens (TP content of ascites 2.0g/L) was 10 min (equivalent to ozone total 9.6mg), and the bactericidal rate of two bacteria was 100%, and 3 and 4. The bactericidal rate of two bacteria was still lower than that of 70%. ozone (5min, 10min, 20min), and the bactericidal rate of Escherichia coli was significantly different (F=28514.907, P=0.000), and the bactericidal rate of Staphylococcus aureus was also significantly different (F=13066.314, P=). The bactericidal rate of the two bacteria was still lower than that of 70%. ozone (5min, 10min, 20min). The bactericidal rate of the specimens (ascites TP and 25.7 g/L respectively) 0), the germicidal effect of the action time elders was better, the bactericidal rate of two bacteria in the 4 ascites was above 70% when the effect of 20min. The effect of different ascites and different ozone time on the efficiency of ozone killing Escherichia coli (F=9775.527, P=0.000), and the effect on the bactericidal effect of Staphylococcus aureus also existed. Interaction (F=3242.915, P=0.00) (table 2-3, table 2-4). Ozone action 5 min, 10min, 20min showed that 80 micron of ozone bactericidal efficiency for 4 ascites was better than No. 2, followed by No. 3, and the worst bactericidal effect on specimen 4 (Figure 2-3), that is, the ozone of different patients with high TP content in the ascites was less effective.
The TP value of the ascites of A ~ C was 14.6,22.1,25.7 g/L.80 micron g/ml, respectively, for the bactericidal effect of the 3 different ascites of the same patient (F=395.821, P=0.000). The bactericidal rate of Staphylococcus aureus in different ascites was also significantly different (F=520.915, P=0.000). The bactericidal rate of Escherichia coli has significant difference (F=38534.299, P=0.000), the bactericidal rate of Staphylococcus aureus also has significant difference (F=10800.111, P=0.000), and the effect of germicidal effect is better. The influence of different ascites and different ozone action on the efficiency of ozone killing Escherichia coli in the same patient Effect (F=227.996, P=0.000), the effect on the bactericidal effect of Staphylococcus aureus also had interaction (F=140.123, P=0.00) (table 2-6, table 2-7). Ozone action 5 min, 10min, 20min showed that 80 mu of ozone on 3 ascites bactericidal efficiency A specimens were superior to B standard, C specimens (Figure 2-5). For the ascites of the same patient, TP in the ascites. The ozone with high content was less effective in killing it.
2.3 research conclusions
1, different ascites environment had an effect on bacterial growth, and the number of bacteria with higher TP ascites was less.
2, the bactericidal effect of ozone on ascites is better than that of plasma, but there are different bactericidal effects on different ascites. It is proved that the effective bactericidal effect of ozone in the biological safe dose range (not more than 80 g/ml) in the fluid like ascites, which is lower than plasma, can be effective (even with the TP content of 16.6g/L in the ascites with 80 u g/ml ozone action 20min Its bactericidal rate can reach more than 90%, and the sterilization efficiency of ozone is low when the TP content of ascites is low.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R187

【参考文献】