纳米银涂层的胆道塑料支架研制及其抗菌效果和延长支架通畅期的实验研究

发布时间：2018-06-26 03:51

本文选题：纳米银 + 胆道支架　；参考：《浙江大学》2016年博士论文

【摘要】：一、研究背景自1980年Sohendra N首次报道成功应用胆道塑料支架进行胆道梗阻引流以来,胆道支架已经被广泛应用在各种病因引起的胆道梗阻。胆道支架能安全有效地进行胆道减压引流,姑息性的胆道支架植入现已经取代外科姑息胆肠内引流或者放置T管外引流手术。然而,支架梗阻,随之而发生的黄疸再发,并且常常伴有急性胆管炎是胆道支架术后的主要并发症,发生率为10%～30%,经常是发生急性梗阻性化脓性胆管炎,败血症而危及生命。胆道支架梗阻的原因包括支架的设计、支架材料的性质、支架表面的不平整形成微生物被膜和胆泥淤积。而微生物聚集形成微生物膜后导致的胆泥淤积、胆道感染是最常见原因和后果。至今为止,临床尚缺乏有效抗菌胆道塑料支架能够减少支架植入后的胆道感染、延长支架通畅期提高生存率。目前,纳米材料已成为材料科学领域中的热点,纳米银(Nano Silver)就是将粒径做到纳米级的金属银单质。纳米银粒径大多在25纳米左右,对大肠杆菌、淋球菌、沙眼衣原体等数十种致病微生物都有强烈的抑制和杀灭作用,而且不会产生耐药性。动物试验表明,这种纳米银抗菌微粉即使用量达到标准剂量的几千倍,受试动物也无中毒表现。同时,它对受损上皮细胞还具有促进修复作用。值得一提的是,该产品遇水抗菌效果愈发增强,更利于疾病的治疗。纳米银杀菌具有以下特点：1.广谱抗菌。纳米银颗粒直接进入菌体与氧代谢酶(-SH)结合,使菌体窒息而死的独特作用机制,可杀死与其接触的大多数细菌、真菌、霉菌、孢子等微生物。2.强效杀菌。据研究发现,Ag可在数分钟内杀死650多种细菌。纳米银颗粒与病原菌的细胞壁/膜结合后,能直接进入菌体、迅速与氧代谢酶的巯基(-SH)结合,使酶失活,阻断呼吸代谢使其窒息而死。独特的杀菌机理,使得纳米银颗粒在低浓度就可迅速杀死致病菌。3.渗透性强。纳米银颗粒具有超强的渗透性,可迅速渗入皮下2mm杀菌,对普通细菌、顽固细菌、耐药细菌以及真菌引起的较深处的组织感染均有良好的杀菌作用。4.修复再生。纳米银可促进伤口愈合,促进受损细胞的修复与再生,去腐生肌,抗菌消炎改善创伤周围组织的微循环,有效地激活并促进组织细胞的生长,加速伤口的愈合,减少疤痕的生成。5.安全无毒。早在《本草纲目》中记载：生银,无毒；美国公共卫生局1990年《关于银毒性的调查报告》中说明：银对人体无明显毒副作用；纳米银是局部用药,银含量少,是最安全的用药方式。6.无耐药性。纳米银属于非抗菌素杀菌剂：纳米银能杀灭各种致病微生物,比抗菌素更强,10nm大小的纳米银颗粒独特抗菌机理可迅速直接杀死细菌,使其丧失繁殖能力,因此,无法生产耐药性的下一代,能有效避免因耐药性而导致反复发作久治不愈。二、目的研发新型的纳米银涂层胆道塑料支架并研究此新型支架在体内、外抗菌效果和延长支架通畅期情况。三、方法本研究以抗凝血性肝素作为聚阴离子,以具有抗菌作用的壳聚糖作为聚阳离子复合具有长效广谱抗菌性能的纳米银,采用旋涂层层组装方法构建成纳米银涂层的胆道塑料支架。通过多层膜的表面形貌用AFM和SEM观察,材料表面分析及紫外分光光度计表征。体外纳米银释放检测和抗菌实验。体内实验：64只中国巴拿实验猪,雌雄不限,体重为30kg-40kg,随机分为A组(对照组,control) 32只：植入临床用的COOK公司特氟龙胆道塑料支架(临床常用)；B组(实验组,AgNP)32只：植入纳米银涂层的胆道塑料支架。每组各8只用作观察支架通畅期和猪生存期。每组另24只按照在术后第4、12、24、48周经猪耳缘静脉抽血作血常规、生化、细胞因子检测分析猪的生存状态。上述每一节点6只猪再手术抽门静脉血、胆总管抽胆汁作细菌培养,取出胆道支架,切取胆管吻合口组织作检测。猪均单笼饲养在浙江大学实验动物中心,专人饲养,观察猪一般情况,如果猪出现胃纳差,发热,皮肤黄染等症状,立即终止实验观察,再手术取标本检测。再手术从原切口进腹,观察腹腔粘连情况及胆管吻合口情况。剪开吻合口观察大体标本后,取胆管吻台口及上下1cm左右胆管组织分为4份,一份浸泡于10%福尔马林中固定,进行HE染色、Masson染色；一份用2.5%戊二醛固定行电镜检查；其余二份保存在一80"C冰箱内备用。取肝脏组织一块行HE染色.实验猪采用肌松剂法处死。整个动物实验严格遵守浙江大学实验动物中心伦理要求。四、统计学方法统计学分析采用SPSS 19.0 for Windows软件进行。数据以均数±标准差表示。组间比较采用Students' T-Test检验,独立样本采用Mann. Whiimey U检验,配对样本采用Wilcoxon符号秩检验。P值小于0.05为两组间有显著性差异,P值小于0.01时两组间有非常显著性差异。猪生存率采用Kaplan-Meier生存曲线分析。五、结果紫外检测用来表征纳米银的合成结果,肝素和壳聚糖呈近乎严格层层交替排列,这可能是由于基材表面最外层能被完全涂覆,而仅受到次外层的少量穿插。其中奇数层的接触角比偶数层的接触角要小,由于肝素的亲水性要比壳聚糖的亲水性要好,说明奇数层肝素含量相对较多,偶数层壳聚糖含量相对较多。紫外分光光度仪检测用来跟踪载纳米银的壳聚糖与肝素组装过程。由于纳米银在433nm处的紫外吸收,多层膜在该处有最大吸收。看到,最大吸收值均随组装双层膜数增加而增加并呈线性变化,说明壳聚糖纳米银是逐层组装的。随着组装双层膜数的增加,多层膜的载银量相应增加,因此纳米银的吸收值增加。用AFM的敲打模式观察了分别采用旋涂法和浸涂法组装的多层膜表面形貌,结果显示组装方法对表面粗糙度有很大的影响,采用浸涂法组装时的表面较粗糙(RMS=22.37nm),而通过旋涂法组装的多层膜则表面较平坦(RMS=14.73nm).同时旋涂使组装时间缩短了近5个小时,这对于工艺化的实现大大节约了时间,提高了效率。体外抗菌测试：壳聚糖纳米银／肝素多层膜(PET-1)则体现出了很强的抗菌性能,相同组成的多层膜在PBS缓冲溶液中放置1个月后(PET-2)的抗菌结果显示了多层膜的长效抗菌性。多层膜在PBS中放置了近一个月,仍体现出较好的抗菌性能。手术情况：所有手术均顺利完成,术中麻醉平稳,无术中死亡发生,术后恢复顺利,无围手术期死亡。无术后肠梗阻及明显胆瘘发生。术后第4周,对照组和实验组猪生活状况良好,胃纳可,无发热和黄染。超声扫描肝内外胆管无扩张,支架在胆道内,位置良好。对比对照组(40.13±3.30 weeks),实验组(72.37±4.23 weeks)支架通畅期明显延长(p0.0001)。实验组和对照组生存率比较结果：在用于观察生存率的各8只猪中,实验组8只猪平均生存时间是73.88±4.06周,而对照组8只猪平均生存时间是41.38±3.24周。Kaplan-Meier生存曲线显示实验组生存率明显延长。实验组和对照组胆道细菌感染情况：在每一节点4、12、48周,每批6只猪抽的胆汁细菌培养结果显示,对照组细菌培养阳性率逐步升高,分别为0%,33.33%,50%和100%。而实验组从4周至48周,胆汁细菌培养均为阴性。在抽到的胆汁培养阳性细菌中,主要是大肠埃希氏菌,金黄色葡萄球菌,鹌鹑鸡肠球菌D,阴沟肠杆菌,肺炎克雷白氏杆菌,粪肠球菌。其中,又以大肠埃希氏菌最多见,占6/11(54.55%)。证实了纳米银涂层的胆道塑料支架良好的抗菌性。胆管吻合口组织HE染色、Masson's trichrome染色在术后48周胆管吻合口组织切片结果显示,对照组吻合口疤痕增生明显,胶原纤维组织增多,肌纤维组织减少,炎症细胞浸润明显。术后肝功能变化：因为胆道支架梗阻会导致血液总胆红素升高,以直接胆红素为主。同时胆道梗阻,伴随着Y-GT升高。对照组和实验组猪每4、12、24、48周均经耳缘静脉抽血生化检查,结果显示：对照组血液Y-GT从4-48周持续升高；总胆红素、直接胆红素从12-48周进行性升高；而实验组对比对照组数值明显减少(P0.01)。六、结论我们研发出的纳米银涂层的胆道塑料支架,在体外、体内实验均证实对肠道常见的多种细菌有强的抗菌性,对比临床常用的塑料支架有更长的通畅期,能够延长实验动物的生存率。
[Abstract]:First of all, the biliary stents have been widely used in various causes of biliary obstruction since the first report of Sohendra N in 1980. The biliary stents have been widely used for biliary obstruction caused by various causes. Biliary stents are safe and effective for biliary tract decompression and drainage. Palliative biliary stent implantation has now replaced surgical palliative choledochus. Internal drainage or external drainage of T tube. However, stent obstruction, followed by jaundice recurrence, and often accompanied by acute cholangitis is a major complication after biliary stenting, the incidence of 10% to 30%, often acute obstructive suppurative cholangitis, septicemia and life-threatening. The cause of biliary stents obstruction includes a branch. The design of the frame, the nature of the scaffold material, the unevenness of the scaffold surface forms the siltation of the microorganism membrane and the bile mud, and the cholestasis of the bile duct caused by the microbial aggregation and the formation of the microorganism membrane is the most common cause and consequence. So far, the clinical lack of effective antibacterial biliary ductile scaffold can reduce the biliary tract infection after the stent implantation. At present, nanomaterials have become a hot spot in the field of material science. Nano silver (Nano Silver) is a metal silver monomer with nanometer size. The size of nano silver particles is mostly around 25 nanometers, and many kinds of pathogenic microorganism such as Escherichia coli, gonococcus and trachomatis trachomatis are strongly suppressed. In animal experiments, animal tests showed that the nano silver antibacterial micropowder had no poisoning performance even if the dosage reached the standard dose. It also promoted the repair of damaged epithelial cells. Nano silver sterilization has the following characteristics: 1. broad-spectrum antibacterial. Nano silver particles directly enter the bacteria and oxygen metabolism enzyme (-SH) combined, the bacteria asphyxiate the unique mechanism of action, can kill most bacteria, fungi, mold, spores and other microorganisms.2. strong bactericidal action. According to the research, the research found that Ag can kill more than 650 within a few minutes. The nano silver particles are combined with the cell wall / membrane of the pathogenic bacteria and can enter the bacteria directly and quickly combine with the sulfhydryl group (-SH) of the oxygen metabolizing enzyme to inactivate the enzyme and block the respiratory metabolism to suffocate. The unique bactericidal mechanism makes the nano silver particles quickly kill the pathogenic bacteria,.3., with strong permeability. Strong osmosis can quickly infiltrate into the subcutaneous 2mm to sterilize, and have good bactericidal effect on.4. repair and regeneration for common bacteria, stubborn bacteria, drug-resistant bacteria and fungi. Nano silver can promote wound healing, promote the repair and regeneration of damaged cells, go to saprophytic muscle, antiseptic anti-inflammatory and improve the microstructure around trauma. Circulation, effectively activating and promoting the growth of tissue cells, accelerating the healing of wounds, reducing the formation of.5. scar formation, is safe and non-toxic. Early in < Compendium of Materia Medica >, it was recorded: silver, non-toxic; the US Public Health Bureau, 1990 < survey of silver toxicity > shows that silver has no toxic side effects on human body; nano silver is a local drug, silver containing Nano silver is a non antibiotic agent: nano silver is a non bactericidal agent: nano silver can kill various pathogenic microorganisms, stronger than antibiotics. The unique antibacterial mechanism of nano silver particles size of 10nm can quickly kill bacteria directly and make it lose reproduction ability. Therefore, it can not produce the next generation of drug resistance and can be effective. It can be effective and can be effective. Two, aim to develop a new type of nano silver coated biliary plastic scaffold and study the new scaffold in the body, the external antibacterial effect and the prolongation of the stent patency. Three. Method this study used anticoagulant heparin as polyanion and Antibacterial Chitosan as polycation. Nano silver with long-acting and broad-spectrum antibacterial properties was composed of silver coated with silver coating by spin coating layer assembly. The surface morphology of multilayer films was observed by AFM and SEM, material surface analysis and UV spectrophotometer. In vitro silver release test and antibacterial experiment in vitro. In vivo experiment: 64 Chinese Pakistan The experimental pigs, male and female, 30kg-40kg, were randomly divided into group A (control group, control) 32: implanted clinical COOK company Teflon biliary duct plastic stent (clinical common use); B group (experimental group, AgNP) 32: implantation of nano silver coated biliary plastic stent. Each group of 8 only used to observe stent patency period and pig survival period. Another 24 in each group. The survival status of pigs was analyzed by blood routine, biochemical and cytokine detection by blood routine, biochemical and cytokine detection in the porcine auricular vein at week 4,12,24,48 after the operation. 6 pigs of each node were reoperated for the portal vein blood, the bile duct bile was used as the bacterial culture, the biliary stent was removed, and the bile duct anastomotic tissue was taken for examination. The pigs were kept in the Zhejiang University in a single cage. Animal Center, special people feed, observe the general situation of pigs, if the pig appears stomach deficiency, fever, skin yellow dye and other symptoms, immediately terminate the experimental observation, reoperation for specimen detection. Reoperation from the original incision into the abdomen, observe the abdominal adhesion and bile duct anastomosis. After cutting the anastomotic mouth to observe the general specimen, take the bile duct kissing mouth mouth and up and down 1cm The left and right bile duct tissues were divided into 4 parts, one was soaked in 10% formalin, was stained with HE and Masson, and one was fixed with 2.5% glutaraldehyde for electron microscopy. The rest two were stored in the 80 "C refrigerator, and the liver tissue was stained with HE. The experimental pigs were killed by muscle relaxant. The whole animal experiment strictly observed the Zhejiang University. The ethical requirements of experimental animal center. Four, statistical methods and statistics analysis were carried out using SPSS 19 for Windows software. The data were represented by mean number + standard deviation. The group was compared with Students'T-Test test, independent samples were tested by Mann. Whiimey U test, and the paired sample used Wilcoxon symbol rank test for.P value less than 0.05 as two groups. The difference, there is a very significant difference between the two groups when the P value is less than 0.01. The survival rate of the pig is analyzed by the Kaplan-Meier survival curve. Five, the UV detection is used to characterize the synthesis of nano silver, and the heparin and chitosan are near the strict layers of alternately arranged, which may be due to the full coating of the outer layer of the substrate surface, but only the outer layer. The contact angle of the odd number layer is smaller than the contact angle of the even number layer, because the hydrophilicity of the heparin is better than the hydrophilic property of the chitosan. It shows that the odd number of heparin content is relatively more and the even number of chitosan content is relatively more. The UV spectrophotometer is used to track the assembly process of chitosan and heparin carrying nano silver. The maximum absorption of the multilayer film is increased with the increase of the number of the double layer membrane, which indicates that the chitosan nano silver is assembled layer by layer. With the increase of the number of the double layer films, the silver content of the multilayer film increases correspondingly, so the absorption value of the nano silver is increased. The surface morphology of the multilayer film assembled by spin coating method and dip coating method was observed by the knocking mode of AFM. The results showed that the assembly method had a great influence on the surface roughness. The surface of the assembly was relatively rough (RMS=22.37nm), while the surface of the multilayer film assembled by the spin coating was flat (RMS=14.73nm). The loading time is shortened by nearly 5 hours, which greatly saves time and improves the efficiency. In vitro antibacterial test: chitosan nano silver / heparin multilayer film (PET-1) shows strong antibacterial properties. The antibacterial results of the same composite multilayer film in the PBS buffer solution (PET-2) show the multilayer film. The multi-layer membrane was placed in PBS for nearly a month and still showed good antibacterial performance. Operation condition: all operations were successfully completed, anesthesia was smooth, no intraoperative death occurred, postoperative recovery was smooth, no perioperative death. No postoperative intestinal obstruction and obvious biliary fistula occurred. Fourth weeks after operation, control group and experimental group pig birth. There was no fever and yellow dye in the stomach. The internal and external bile duct of the liver were not dilated by ultrasonic scanning. The stent was in the bile duct, and the position was good. Compared with the control group (40.13 + 3.30 weeks), the stent patency period of the experimental group (72.37 + 4.23 weeks) was obviously prolonged (P0.0001). The survival rate of the experimental and control groups was compared: in 8 pigs used to observe the survival rate. The average survival time of 8 pigs in the experimental group was 73.88 + 4.06 weeks, while the average survival time of the 8 pigs in the control group was 41.38 + 3.24 weeks.Kaplan-Meier survival curve showed that the survival rate of the experimental group was obviously prolonged. The infection of biliary tract bacteria in the experimental group and the control group: the result of the bile bacteria culture of 6 pigs per batch at the 4,12,48 week of each node showed the control results. The positive rate of bacterial culture increased gradually, 0%, 33.33%, 50% and 100%., respectively, while the bile bacteria culture was negative in the experimental group from 4 to 48 weeks. Among the positive bacteria in the bile culture, the main bacteria were Escherichia coli, Staphylococcus aureus, quail Enterococcus D, Enterobacteriaceae, Klebsiella pneumoniae, and Enterococcus faecalis. The majority of Escherichia coli, accounting for 6/11 (54.55%), confirmed the good antibacterial properties of the biliary tract plastic scaffold with nano silver coating. The bile duct anastomotic tissue HE staining, Masson's trichrome staining in the bile duct anastomotic tissue section of the bile duct at 48 weeks after the operation showed that the scar scar proliferation was obvious in the control group, the collagen fibrous tissue increased and the muscle fiber tissue was increased. Decrease, infiltration of inflammatory cells. Changes in liver function after operation: the obstruction of biliary stents resulted in the increase of blood total bilirubin and direct bilirubin. Biliary obstruction, accompanied by an increase in Y-GT. The control group and the experimental group were examined every 4,12,24,48 week by the ear vein blood biochemical examination, the results showed that the blood Y-GT of the control group was 4-48 weeks from the control group. The total bilirubin and direct bilirubin increased from 12-48 weeks, while the experimental group compared with the control group significantly decreased (P0.01). Six, the conclusion we developed the nano silver coated biliary plastic scaffold, in vitro, in vitro experiments confirmed the strong antibacterial properties of the common bacteria in the intestines, compared with the commonly used plastics. The stent has a longer patency period, which can prolong the survival rate of the experimental animals.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R657.4;R318.08

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