【作者】 梁岳龙；

【导师】 蔡秀军；

【作者基本信息】 浙江大学 ， 外科学， 2012， 博士

【摘要】 第一部分聚癸二酸丙二醇聚酯的制备及吻合支架的制造研究目的制备一种新型生物可降解高分子材料,并以此制造一系列空腔脏器吻合支架。研究方法将癸二酸、1,3-丙二醇、1,2-丙二醇以2：3.75：1.25的摩尔比通过熔融缩聚二步法完成材料制备,经无水甲醇提纯。制备的材料采用凝胶渗透色谱(GPC)法测定其分子量,采用红外图谱表征合成产物。根据吻合支架设计模型铸造相应模具,然后通过注塑的方法制备相应的支架。结果通过熔融缩聚的方法成功制备了目标产物聚癸二酸丙二醇聚酯(PSPP),重均分子量为67000左右,数均分子量为30000左右,分布宽度为2.2。红外谱图中分别在1161cm-1、1728cm-1、2927cm-1、2851cm-1出现吸收峰。以制备的PSPP材料通过注塑的方法制得各种空腔脏器吻合支架。结论以癸二酸、1,3-丙二醇、1,2-丙二醇为单体,采用熔融缩聚法成功制备了PSPP。通过注塑法制造了一系列空腔脏器吻合支架。第二部分聚癸二酸丙二醇酯的生物相容性实验研究研究目的通过体外和体内生物相容性实验,系统评价新型生物可降解聚酯材料PSPP的生物相容性,为进一步动物实验和临床研究提供实验依据。研究方法依据我国颁布的医疗器械生物学评价指南(GB/T16886.2008),选择细胞毒性试验、Ames试验作为体外试验项目,全身毒性试验、皮内反应、迟发型超敏反应、植入试验和直肠黏膜刺激试验作为体内试验项目来对PSPP的生物相容性进行评估。结果(1)体外试验：细胞毒性试验据24h、48h和72h观察,PSPP浸提液组的细胞相对增殖率RGR值均在75%以上,毒性判定均为1级；Ames试验在活化或非活化代谢条件下,PSPP组TA97、TA98、TA100和TA102的菌株回变菌落数均与阴性对照组相接近,无统计学差异(P>0.05)且PSPP组回复突变菌落数均未超过阴性对照组2倍,判断为阴性。(2)体内试验：皮内注射24h、48h、72h后观察每只动物在每一规定的观察时间内的原发性刺激记分均为0；全身毒性反应试验分别自尾静脉注射供试液和空白对照液4h、24h、48h和72h后,两组动物未出现死亡,进食正常,无呕吐、呆滞、运动机能减退、呼吸困难等症状出现；迟发型超敏反应试验PSPP加和不加弗氏完全佐剂情况下,在致敏后第3天和第7天及激发后24h和48h,皮肤过敏反应评分均为0分；直肠黏膜刺激试验连续直肠灌注1周后通过肉眼观察,结合组织病理学检查,未见直肠给药部位出现充血、红肿及坏死现象；植入试验大体标本观察,实验组和阴性对照组2w时可见周围组织轻度水肿,4w、12w均可见材料被一薄层囊壁包绕,与周围组织无明显粘连,未发现周围组织有充血、水肿、坏死等异常情况,组织学观察2w时两组炎症细胞反应均为IV级,囊腔反应均为IV级,4w时炎症细胞反应均为III级,囊腔反应均为II级,12w时炎症细胞反应均为I级,囊腔反应均为I级。结论PSPP无论在体外和体内均表现出良好的生物相容性。第三部分聚癸二酸丙二醇酯的生物降解性能研究研究目的研究聚癸二酸丙二醇酯(PSPP)在体外和动物体内不同介质中的降解规律,明确影响PSPP降解速度的主要因素,了解PSPP在消化道不同部位的降解时间。研究方法体外降解选择人新鲜胆汁、模拟胃液和模拟肠液为降解介质,PSPP支架为样本,每种介质各设立48个样本。每个支架独立浸泡于装有10ml降解介质的平底试管内,37℃,100转／分钟持续振荡,每日更换浸提介质。每周每种介质分别取出3个样本用来观察外观和完整性,检测重量和重均分子量,扫描电镜下观察表面形态改变,观察期为4个月或至支架崩解。体内降解以巴马小型猪为实验对象,选择小肠、胃、结肠三个部位进行试验,样品5个为1串,分别悬挂在相应的消化道内。小肠每4天处死一头动物,共4头,胃和结肠每周处死1头,各6头。取出样本后分别观察形态变化,分子量变化,扫描电镜观察表面形态改变。结果在体外人新鲜胆汁中,PSPP支架的降解周期约为3.5个月,观察期内总失重将近60%,重均分子量从原来的67000左右降低到31000左右,扫描电镜发现支架表面逐渐呈鱼鳞样改变伴脱落；在模拟胃液中,4个月内支架外形保持完整,支架的重量,重均分子量及支架表面形态改变均不明显；在模拟肠液中降解周期约为2.5个月,失重46%左右,重均分子量降至30000左右,扫描电镜下,材料表面呈鱼鳞样改变。体内降解中,空肠内的降解周期约为2周,重均分子量变化不明显,扫描电镜下材料表面明显被腐蚀；胃内降解6周支架厚度变化不明显,重均分子量稍有下降,扫描电镜下材料表面变得粗糙伴少量空洞；结肠内5周左右降解,重均分子量呈逐渐下降趋势,扫描电镜下材料表面被腐蚀。结论在体外人新鲜胆汁中,PSPP支架的降解周期约为3.5个月,在模拟肠液中降解周期约为2.5个月,在模拟胃液中降解周期在4个月以上,碱性性环境有助于PSPP降解,酸性环境中,PSPP表现为相对生物惰性,胆汁的乳化功能和胰酶均对PSPP降解起到明显促进作用。在体内,PSPP支架在空肠内降解速度快,时间约为2周,和小肠吻合口愈合时间相匹配；结肠内约为5周左右；胃内降解速度慢,表现相对惰性。第四部分内支架联合大网膜修复胆道热损伤的动物实验研究研究目的通过动物实验验证内支架联合大网膜修复胆道热灼伤伴穿孔的可行性和安全性。研究方法以巴马小型猪为实验对象,随机分为两组,每组16头。手术构建胆总管侧壁热灼伤伴穿孔的模型,实验组采用内支架联合大网膜修补胆管：修剪灼伤胆管后将PSPP支架与胆管两端荷包捆绑连接,缺损部分由大网膜覆盖；对照组采用胆管间断缝合。术后观察实验动物黄疸和胆漏情况；各组分别于术后2周、1月、3月和6月处死4头动物并取材,观察两组的吻合口愈合情况,比较大体观、胆道造影情况及组织病理形态学改变,包括HE染色和Masson染色,采用免疫组化和定量PCR法测定吻合口a-SMA.TGF-β1和b-FGF的相对含量。结果两实验组均顺利完成手术,术后无黄疸及胆漏发生。将两组术后肝功能和胆红素的变化值进行比较分析,发现对照组术后2周和1月时ALP变化明显大于实验组,术后3月AST升高明显高于实验组,术后6月DBiL变化明显高于实验组。胆道造影显示实验组胆管无明显狭窄,对照组术后3月有一头实验动物吻合口线样狭窄伴胆总管扩张,对照组术后6月有2头动物见近端胆管和肝内胆管扩张,吻合口可触及疤痕狭窄。HE染色和Masson染色均显示术后各时段实验组的吻合口炎症和纤维增生情况较对照组轻。免疫组化显示术后各时间段实验组的a-SMA和TGF-β1评分低于对照组,b-FGF评分高于对照组。荧光定量PCR结果显示术后2周时实验组较对照组b-FGF、TGF-β1mRNA表达略增加,α-SMAmRNA表达明显减少；术后1月时实验组较对照组b-FGF mRNA表达增加,TGF-β1、α-SMAmRNA表达明显减少；术后3月时实验组较对照组b-FGF、α-SMAmRNA表达略减少,TGF-β1mRNA表达明显减少；术后6月时实验组较对照组b-FGF和a-SMAmRNA表达相似,TGF-β1mRNA减少。结论内支架联合大网膜修复胆道热灼伤是一种安全可行的胆道修复方式,可有效降低术后胆道狭窄。更多还原

【Abstract】 PART I Synthesis of Poly [sebacic acid-co-(1,3-propanediol)-co-1,2-propanediol)] elastomers and manufacture of anastomosis stentsObjective To synthesize a new type of bio-degradable high molecular polymer, to develop and manufacture a series of anastomosis stents for hollow viscus.Method It was synthesized using1,3-propanediol,1,2-propanediol and sebacic acid with the molar ratio of2:3.75:1.25by Two Step Melt Polycondensation Synthetic Method, then purified by anhydrous methanol. The molecular weight of the material synthesized was determined by gel permeation chromatography(GPC). And the material was identified according to the Infrared Spectroscopy (IR) spectra. The mould was modified on the basis of original design model, and the stents were manufactured by injection molding with the novel material.Result We successfully synthesized the target product Poly [sebacic acid-co-(1,3-propanediol)-co-1,2-propanediol)](PSPP) by Melt Polycondensation Synthetic Method, with Weight-average Molecular Weight of about67000, Number-average Molecular Weight of about30000and Distribution Width of2.2. There were four absorption peaks including1161cm-1,1728cm-1,2927cm-1and2851cm-1in the Infrared Spectroscopy spectra. Then we successfully manufactured a series of anastomosis stents with PSPP by injection molding for hollow viscus.Conclusion With sebacic acid,1,3-propanediol and1,2-propylene glycol as monomers, we successfully synthesized PSPP by Melt Polycondensation Synthetic Method, and manufactured a series of anastomosis stents by injection molding for hollow viscus. PART Ⅱ Experimental study of the biocompatibility of PSPPObjective To systematically assess the biocompatibility of the novel bio-degradable polyester PSPP in vitro and in vivo, providing experimental base for further animal experiments and clinical researches.Method Based on the medical equipment biology assessment guide promulgated in china (GB/T16886.2008), we chose cytotoxicity test, Ames test as items of in vitro experiments and systemic toxicity tests, intradermoreaction, delayed type hypersensitivity, implant test as well as rectal mucous membrane stimulation test as items of in vivo experiments.Result (1) In vitro experiments:Cytotoxicity test demonstrated that RGR results in24h,48h and72h are all above75%, the scale was1. Ames test demonstrated that the colonies numbers in bacterial strains TA97, TA98, TA100and TA102in PSPP group were similar to those of the control group with no statistical difference (P>0.05), and no more than2times that of the control group, the result was negative.(2) In vitro experiments:After intradermal injection of PSPP leach liquor, all experimental animals had a primary stimulate scoring of0in24h,48h and72h. Systemic toxicity tests:After tail vein injection of PSPP leach liquor in experimental group and saline in control group, observations in4h,24h,48h and72h all showed no death in both groups, and animals in both group ate normally, without symptoms such as vomiting, dull-looking, hypocinesis or dyspnea. Animals gained weight in both groups, and there was no significant difference. Delayed type hypersensitivity:After injected by PSPP leach liquor and PSPP leach liquor combining with Freund’s complete adjuvant, assessing skin allergy score on the third day and seventh day after allergic sensitization,24h and48h after stimulation showed0,0,0,0respectively. Rectal mucous membrane stimulation test:After one week continuous rectal infusion of PSPP leach liquor, both macroscopic observation and histopathological examination showed no manifestation of congestion, swelling or necrosis on the rectum mucosa. Implant test:Two weeks after implantation, gross specimen adjacent to the stent revealed mild surrounding tissue edema in both experimental group and control group. Results in four weeks and twelve weeks were similar in both groups, with stent surrounded by a thin layer of bursa wall, no obvious adhesion to surrounding tissues, no manifestation of congestion, swelling or necrosis. In histological, the inflammatory cells reaction level was Ⅳ and capsule chamber reaction level was IV in both groups two weeks after implantation the inflammatory cells reaction level was Ⅲ and capsule chamber reaction level was Ⅱ in both groups four weeks after implantation; the inflammatory cells reaction level was Ⅰ and capsule chamber reaction level was Ⅰ in both groups twelve weeks weeks after implantation.Conclusion The above results demonstrate that PSPP has satisfactory biocompatibility both in vitro and in vivo. PART Ⅲ Biodegradability of PSPP in vitro and in vivoObjective This study is to investigate the biodegradation of PSPP in vitro and vivo, the main factors that contribute to the degradation rate of PSPP, the degradation time of PSPP in different part of gastrointestinal.Method In vitro degradation we chose human fresh bile, simulated gastric and intestinal liquid as media, the PSPP stent as sample. Each individual media has48samples. Each stent was soaked in flat-bottomed test tube containing10ml degradation media which was changed daily, with37℃,100rev/min sustained oscillation. We took three samples from each media every week to observe their appearance and integrity, to measure remaining weight and weight-average molecular weight, and to study the morphology of the surface by scanning electron microscopy. The observation period is four months or to the disintegration of sample. The in vivo degradation use Bama miniature pig as experimental subject, every five samples as a group are placed in the stomach, small intestine and colon. We killed pigs and removed the sample on a regular basis (group every four days in small intestine group and a week in other groups) to observe morphological changes, weight average molecular weight changes, and study the morphology of the surface by scanning electron microscopy.Result In vitro fresh bile, the degradation cycle was about3.5months, the total weight loss was nearly60%, weight-average molecular weight was reduced to about31,000from the original67000, scanning electron microscopy revealed the sample surface gradually showed a fish scale-like change with shedding. In simulated gastric liquid, the appearance of sample kept intact, weight, weight-average molecular weight and the morphology of the surface nearly unchanged. In simulated intestinal liquid, the degradation cycle was about2.5months, the total weight loss was nearly46%, weight-average molecular weight was reduced to about30,000, scanning electron microscopy revealed the sample surface gradually showed a fish scale-like change. In small intestine group, the degradation cycle was about2weeks, weight-average molecular weight unchanged, scanning electron microscopy revealed the sample surface was obviously corroded. In gastric group, the thickness of sample did not change significantly in6weeks, weight average molecular weight decreased slightly, scanning electron microscopy revealed the sample surface became rough with a small amount of hole. The samples in the colon degradated in about5weeks, Weight average molecular weight decreased gradually, scanning electron microscopy revealed the sample surface was corroded.Conclusion In vitro in human fresh bile, the degradation cycle was about3.5months, In simulated intestinal liquid, the degradation cycle was about2.5months, In simulated gastric liquid, the degradation cycle was over4months. The alkaline environment contributed to the degradation of PSPP, and in the acidic environment, PSPP performance was relatively biologically inert. The emulsifying function of bile and trypsin played an import role to improve the degradation of PSPP. In vivo, The degradation rate of PSPP was fast in the small intestine, the degradation time is about2weeks, which match the healing time of the anastomotic stoma in small intestine. In the colon, the degradation time is about5weeks; In the gastric, PSPP’s degradation was very slow, performed relatively inert.PART Ⅳ Animal study of intraluminal stent repair of bile duct injury with omentum Objective To assess the feasibility and safety of intraluminal stent repair with omentum for bile duct thermal burn together with perforation.Methods A total of32Bama minipigs were randomly chosen and divided into two groups,16in each. Experimental models with common bile duct (CBD) injury (thermal burn together with perforation) were built by animal surgery, the experimental group was repaired by a intraluminal stent with greater omentum, and the control group was repaired by suture. The incidence of jaundice and bile leakage was evaluated in both groups. Animals were sacrificed at2weeks,1,3and6months after operation. The healing of anastomosis was observed. The status of scar formation, cholangiography, histologic findings by HE, Masson staining and electron microscope were evaluated. The expression of a-SMA, TGF-β1and b-FGF were also evaluated and compared.Result All operation were accomplished successfully. No obvious leakage happened in either experimental or control group. The change of postoperative liver function bilirubin value were comparison analysised, it found that two weeks and1month after operation the ALP change in the control group was significantly greater than the experimental group,3months after operation, the AST change was significantly higher in the control group.6months postoperation, the DBiL change was significantly higher in the control group. Cholangiography indicates that there was no restenosis in experimental group, in control group, there was one animal with linear narrow in the anastomosis with choledochectasia3months after operation, and2animals with cholangiectasis in the proximal duct and intrahepatic bile duct with scar stricture in the anastomosis6months after operation. Both HE stain and Masson stain showed that inflammatory reaction and hyperplasia of the bile duct wall in the experimental group was much more moderate than that in control group. Immunohistochemistry stain showed that the experimental group had less expression of a-SMA and TGF-β1while more expression of b-FGF than that of control group. Fluorescence quantitative PCR results show that2weeks after operation, the experimental group had slightly increased expression of TGF-β1and b-FGF mRNA compared to the control group, and less express of a-SMA mRNA; After one month, b-FGF mRNA had increased expression while TGFβ-1and a-SMA mRNA had less expression compared to the control group;3months after operation, b-FGF, a-SMA and TGF-β1mRNA express in the experimental group had less expression, especially TGF-P1mRNA;6months after operation, the expression of b-FGF and a-SMA mRNA were similar in the two groups, while TGF-β1mRNA expression was less in the experimental group.Conclusion Intraluminal stent covered with greater omentum is a safe and feasible approach of repair of bile duct injury, it could reduce binary restenosis of bile duct.更多还原