本文選題：納米銀 + 膽道支架　； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：一、研究背景自1980年Sohendra N首次報道成功應(yīng)用膽道塑料支架進(jìn)行膽道梗阻引流以來,膽道支架已經(jīng)被廣泛應(yīng)用在各種病因引起的膽道梗阻。膽道支架能安全有效地進(jìn)行膽道減壓引流,姑息性的膽道支架植入現(xiàn)已經(jīng)取代外科姑息膽腸內(nèi)引流或者放置T管外引流手術(shù)。然而,支架梗阻,隨之而發(fā)生的黃疸再發(fā),并且常常伴有急性膽管炎是膽道支架術(shù)后的主要并發(fā)癥,發(fā)生率為10%～30%,經(jīng)常是發(fā)生急性梗阻性化膿性膽管炎,敗血癥而危及生命。膽道支架梗阻的原因包括支架的設(shè)計、支架材料的性質(zhì)、支架表面的不平整形成微生物被膜和膽泥淤積。而微生物聚集形成微生物膜后導(dǎo)致的膽泥淤積、膽道感染是最常見原因和后果。至今為止,臨床尚缺乏有效抗菌膽道塑料支架能夠減少支架植入后的膽道感染、延長支架通暢期提高生存率。目前,納米材料已成為材料科學(xué)領(lǐng)域中的熱點,納米銀(Nano Silver)就是將粒徑做到納米級的金屬銀單質(zhì)。納米銀粒徑大多在25納米左右,對大腸桿菌、淋球菌、沙眼衣原體等數(shù)十種致病微生物都有強烈的抑制和殺滅作用,而且不會產(chǎn)生耐藥性。動物試驗表明,這種納米銀抗菌微粉即使用量達(dá)到標(biāo)準(zhǔn)劑量的幾千倍,受試動物也無中毒表現(xiàn)。同時,它對受損上皮細(xì)胞還具有促進(jìn)修復(fù)作用。值得一提的是,該產(chǎn)品遇水抗菌效果愈發(fā)增強,更利于疾病的治療。納米銀殺菌具有以下特點：1.廣譜抗菌。納米銀顆粒直接進(jìn)入菌體與氧代謝酶(-SH)結(jié)合,使菌體窒息而死的獨特作用機(jī)制,可殺死與其接觸的大多數(shù)細(xì)菌、真菌、霉菌、孢子等微生物。2.強效殺菌。據(jù)研究發(fā)現(xiàn),Ag可在數(shù)分鐘內(nèi)殺死650多種細(xì)菌。納米銀顆粒與病原菌的細(xì)胞壁/膜結(jié)合后,能直接進(jìn)入菌體、迅速與氧代謝酶的巰基(-SH)結(jié)合,使酶失活,阻斷呼吸代謝使其窒息而死。獨特的殺菌機(jī)理,使得納米銀顆粒在低濃度就可迅速殺死致病菌。3.滲透性強。納米銀顆粒具有超強的滲透性,可迅速滲入皮下2mm殺菌,對普通細(xì)菌、頑固細(xì)菌、耐藥細(xì)菌以及真菌引起的較深處的組織感染均有良好的殺菌作用。4.修復(fù)再生。納米銀可促進(jìn)傷口愈合,促進(jìn)受損細(xì)胞的修復(fù)與再生,去腐生肌,抗菌消炎改善創(chuàng)傷周圍組織的微循環(huán),有效地激活并促進(jìn)組織細(xì)胞的生長,加速傷口的愈合,減少疤痕的生成。5.安全無毒。早在《本草綱目》中記載：生銀,無毒；美國公共衛(wèi)生局1990年《關(guān)于銀毒性的調(diào)查報告》中說明：銀對人體無明顯毒副作用；納米銀是局部用藥,銀含量少,是最安全的用藥方式。6.無耐藥性。納米銀屬于非抗菌素殺菌劑：納米銀能殺滅各種致病微生物,比抗菌素更強,10nm大小的納米銀顆粒獨特抗菌機(jī)理可迅速直接殺死細(xì)菌,使其喪失繁殖能力,因此,無法生產(chǎn)耐藥性的下一代,能有效避免因耐藥性而導(dǎo)致反復(fù)發(fā)作久治不愈。二、目的研發(fā)新型的納米銀涂層膽道塑料支架并研究此新型支架在體內(nèi)、外抗菌效果和延長支架通暢期情況。三、方法本研究以抗凝血性肝素作為聚陰離子,以具有抗菌作用的殼聚糖作為聚陽離子復(fù)合具有長效廣譜抗菌性能的納米銀,采用旋涂層層組裝方法構(gòu)建成納米銀涂層的膽道塑料支架。通過多層膜的表面形貌用AFM和SEM觀察,材料表面分析及紫外分光光度計表征。體外納米銀釋放檢測和抗菌實驗。體內(nèi)實驗：64只中國巴拿實驗豬,雌雄不限,體重為30kg-40kg,隨機(jī)分為A組(對照組,control) 32只：植入臨床用的COOK公司特氟龍膽道塑料支架(臨床常用)；B組(實驗組,AgNP)32只：植入納米銀涂層的膽道塑料支架。每組各8只用作觀察支架通暢期和豬生存期。每組另24只按照在術(shù)后第4、12、24、48周經(jīng)豬耳緣靜脈抽血作血常規(guī)、生化、細(xì)胞因子檢測分析豬的生存狀態(tài)。上述每一節(jié)點6只豬再手術(shù)抽門靜脈血、膽總管抽膽汁作細(xì)菌培養(yǎng),取出膽道支架,切取膽管吻合口組織作檢測。豬均單籠飼養(yǎng)在浙江大學(xué)實驗動物中心,專人飼養(yǎng),觀察豬一般情況,如果豬出現(xiàn)胃納差,發(fā)熱,皮膚黃染等癥狀,立即終止實驗觀察,再手術(shù)取標(biāo)本檢測。再手術(shù)從原切口進(jìn)腹,觀察腹腔粘連情況及膽管吻合口情況。剪開吻合口觀察大體標(biāo)本后,取膽管吻臺口及上下1cm左右膽管組織分為4份,一份浸泡于10%福爾馬林中固定,進(jìn)行HE染色、Masson染色；一份用2.5%戊二醛固定行電鏡檢查；其余二份保存在一80"C冰箱內(nèi)備用。取肝臟組織一塊行HE染色.實驗豬采用肌松劑法處死。整個動物實驗嚴(yán)格遵守浙江大學(xué)實驗動物中心倫理要求。四、統(tǒng)計學(xué)方法統(tǒng)計學(xué)分析采用SPSS 19.0 for Windows軟件進(jìn)行。數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差表示。組間比較采用Students' T-Test檢驗,獨立樣本采用Mann. Whiimey U檢驗,配對樣本采用Wilcoxon符號秩檢驗。P值小于0.05為兩組間有顯著性差異,P值小于0.01時兩組間有非常顯著性差異。豬生存率采用Kaplan-Meier生存曲線分析。五、結(jié)果紫外檢測用來表征納米銀的合成結(jié)果,肝素和殼聚糖呈近乎嚴(yán)格層層交替排列,這可能是由于基材表面最外層能被完全涂覆,而僅受到次外層的少量穿插。其中奇數(shù)層的接觸角比偶數(shù)層的接觸角要小,由于肝素的親水性要比殼聚糖的親水性要好,說明奇數(shù)層肝素含量相對較多,偶數(shù)層殼聚糖含量相對較多。紫外分光光度儀檢測用來跟蹤載納米銀的殼聚糖與肝素組裝過程。由于納米銀在433nm處的紫外吸收,多層膜在該處有最大吸收。看到,最大吸收值均隨組裝雙層膜數(shù)增加而增加并呈線性變化,說明殼聚糖納米銀是逐層組裝的。隨著組裝雙層膜數(shù)的增加,多層膜的載銀量相應(yīng)增加,因此納米銀的吸收值增加。用AFM的敲打模式觀察了分別采用旋涂法和浸涂法組裝的多層膜表面形貌,結(jié)果顯示組裝方法對表面粗糙度有很大的影響,采用浸涂法組裝時的表面較粗糙(RMS=22.37nm),而通過旋涂法組裝的多層膜則表面較平坦(RMS=14.73nm).同時旋涂使組裝時間縮短了近5個小時,這對于工藝化的實現(xiàn)大大節(jié)約了時間,提高了效率。體外抗菌測試：殼聚糖納米銀／肝素多層膜(PET-1)則體現(xiàn)出了很強的抗菌性能,相同組成的多層膜在PBS緩沖溶液中放置1個月后(PET-2)的抗菌結(jié)果顯示了多層膜的長效抗菌性。多層膜在PBS中放置了近一個月,仍體現(xiàn)出較好的抗菌性能。手術(shù)情況：所有手術(shù)均順利完成,術(shù)中麻醉平穩(wěn),無術(shù)中死亡發(fā)生,術(shù)后恢復(fù)順利,無圍手術(shù)期死亡。無術(shù)后腸梗阻及明顯膽瘺發(fā)生。術(shù)后第4周,對照組和實驗組豬生活狀況良好,胃納可,無發(fā)熱和黃染。超聲掃描肝內(nèi)外膽管無擴(kuò)張,支架在膽道內(nèi),位置良好。對比對照組(40.13±3.30 weeks),實驗組(72.37±4.23 weeks)支架通暢期明顯延長(p0.0001)。實驗組和對照組生存率比較結(jié)果：在用于觀察生存率的各8只豬中,實驗組8只豬平均生存時間是73.88±4.06周,而對照組8只豬平均生存時間是41.38±3.24周。Kaplan-Meier生存曲線顯示實驗組生存率明顯延長。實驗組和對照組膽道細(xì)菌感染情況：在每一節(jié)點4、12、48周,每批6只豬抽的膽汁細(xì)菌培養(yǎng)結(jié)果顯示,對照組細(xì)菌培養(yǎng)陽性率逐步升高,分別為0%,33.33%,50%和100%。而實驗組從4周至48周,膽汁細(xì)菌培養(yǎng)均為陰性。在抽到的膽汁培養(yǎng)陽性細(xì)菌中,主要是大腸埃希氏菌,金黃色葡萄球菌,鵪鶉雞腸球菌D,陰溝腸桿菌,肺炎克雷白氏桿菌,糞腸球菌。其中,又以大腸埃希氏菌最多見,占6/11(54.55%)。證實了納米銀涂層的膽道塑料支架良好的抗菌性。膽管吻合口組織HE染色、Masson's trichrome染色在術(shù)后48周膽管吻合口組織切片結(jié)果顯示,對照組吻合口疤痕增生明顯,膠原纖維組織增多,肌纖維組織減少,炎癥細(xì)胞浸潤明顯。術(shù)后肝功能變化：因為膽道支架梗阻會導(dǎo)致血液總膽紅素升高,以直接膽紅素為主。同時膽道梗阻,伴隨著Y-GT升高。對照組和實驗組豬每4、12、24、48周均經(jīng)耳緣靜脈抽血生化檢查,結(jié)果顯示：對照組血液Y-GT從4-48周持續(xù)升高；總膽紅素、直接膽紅素從12-48周進(jìn)行性升高；而實驗組對比對照組數(shù)值明顯減少(P0.01)。六、結(jié)論我們研發(fā)出的納米銀涂層的膽道塑料支架,在體外、體內(nèi)實驗均證實對腸道常見的多種細(xì)菌有強的抗菌性,對比臨床常用的塑料支架有更長的通暢期,能夠延長實驗動物的生存率。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R657.4;R318.08

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