【作者】 李文春；

【导师】 宋健；

【作者基本信息】 武汉大学 ， 人体解剖与组织胚胎学， 2013， 博士

【摘要】 研究背景：胆道手术是外科手术的难点,术中损伤胆管的修复、修复后发生再狭窄以及胆道恶性狭窄的治疗一直是腹部外科的难题。人们一直在试图设计一种可以代替胆管结构和功能的人工胆管,来解决胆管修复与重建的难题。同种异体移植已经在临床工作中广泛开展,但术后对于排斥反应的预防和治疗仍有很多问题需要解决,而且对于胆管的移植仍然无法完全解决术后狭窄梗阻和Oddi括约肌功能恢复的问题。近年来,由于人类器官短缺,异种移植已经成为当今器官移植学的新领域。为有效解决器官短缺问题,这就有必要探讨新的供体材料。猪是目前被认为最有可能进行人类异种器官移植的动物。国内外学者虽已将猪到人异种移植进行了实验研究和临床应用,但对异种移植所涉及的猪胆管的力学特性的比较研究国内外报道很少。转基因动物作为器官移植的供体受到重视,培育表达人类补体调节蛋白的转基因猪已告成功,超急性排斥反应已被克服,从而使猪到人异种器官移植极具吸引力和挑战性。异种移植供源,除了满足生理功能和克服免疫原性的障碍外,其解剖学和生物力学的匹配性也是移植成功与否的重要因素,这就有必要从生物力学角度探讨猪胆总管作为异种移植器官和作为组织工程胆管材料可行性。第一部分猪胆管作为人胆管移植替代材料的生物力学评价目的：通过对猪与人胆总管生物力学的比较研究,探讨其生物力学特性之间的关系,从生物力学方面评价猪胆总管作为人胆管移植替代材料的可行性。方法：健康2月龄湖北白猪50头(雄性30头,雌性20头),随机分为10组。普食饲养,分别于3、4、5、6、7、8、9、10、11、12月龄时用氯胺酮肌肉注射(5mg/kg)麻醉宰杀5头(雄性3头,雌性2头),测量胆总管在体长度后取出。人胆总管取自无肝胆疾病、年龄在20-40岁意外死亡的5例成年尸体。每个标本做5μm冰冻横断面切片,H-E法染色。用计算机图像分析仪测量胆总管的壁厚和直径等几何形态学指标。用e-ruler计算机软件测量胆总管的张开角。用生物力学试验机对胆总管进行压力—直径关系试验,计算出增量弹性模量(identical incremental elastic modulus, Einc),压力-应变弹性模量(pressure-strain elastic modulus, Ep),容积弹性模量(volume elastic modulus, Ev)和顺应性(compliance, C)等力学特性参数。免疫组织化学的方法染色,用激光共聚焦显微镜检测胆总管壁胶原纤维、平滑肌和弹性纤维的荧光定量表达,计算显微结构成份的含量。结果：1.猪的体重、肝的重量和体积随月龄的变化：猪的体重、肝的重量和体积随着月龄的增加而增大,6-7月龄猪的体重、肝的重量和体积与成人的相近(F=109.15,P=0.08)。2.猪与人胆总管几何形态的比较：人与各月龄组猪胆总管的壁厚(F=6.46,P=0.00)、直径(F=9.56,P=0.00)和长度(F=13.29,P=0.00)在总体上差异有统计学意义；3-6月龄猪胆总管的壁厚和直径比成人的小(P<0.01),7-10月龄猪胆总管的壁厚和直径与成人的相近(P>0.05)；各月龄组猪胆总管的长度均比成人的短(P<0.01)。3.猪与人胆总管张开角的比较：3-12月龄猪胆总管的张开角随月龄增加而逐渐增大(P<0.05),7月龄时趋于稳定。7-12月龄猪胆总管的张开角变化不明显,且与成人的相近(P>0.05)。4.猪和人胆总管的弹性模量与压力的关系：猪与人胆总管的Einc、Ep和Ev均随压力的上升而增大,当压力达4kPa时,胆总管的直径不再变化,弹性模量也随之不变或变化很小,曲线趋于平缓。5.人与猪胆总管弹性模量的比较：单因素方差分析显示,人与各月龄组猪胆总管的Einc(F=502.08,P=0.00)、Ep(F=137.42,P=0.00)和Ev(F=134.59,P=0.00)在总体上差异有统计学意义；3-6月龄及11、12月龄猪的弹性模量比成人的大(P<0.01),7-10月龄猪胆总管的弹性模量与成人的相近(P>0.05)。6.各月龄猪胆总管顺应性的变化：猪胆总管的顺应性先随月龄增加而增大,10月龄后又减小。猪胆总管顺应性随月龄的变化与弹性模量相反。7.人与猪胆总管顺应性的比较：单因素方差分析显示,人与各月龄组猪胆总管的顺应性在总体上差异有统计学意义(F=62.93,P=0.00)；7-10月龄猪胆总管的顺应性与成人的相近(P>0.05),而3-6月龄及11、12月龄猪胆总管的顺应性明显小于成人(P<0.01)。8.人与猪胆总管的显微结构成分的含量：猪与人胆总管组织结构相似。各月龄组猪胆总管的平滑肌(F=89.85,P=0.001)、胶原纤维(F=273.69,P=0.003)和弹性纤维(F=182.60,P=0.006)的含量随月龄的而变化。其中平滑肌和弹性纤维的含量随月龄的增加而增多,胶原纤维的含量和胶原纤维/弹性纤维比值(Collagen/Elastin, C/E值)随月龄的增加而减少。7-10月龄猪胆总管的平滑肌、胶原纤维和弹性纤维的含量与成人的差异不明显(P>0.05)。结论：猪胆总管的生物力学特性随年龄的变化而变化。7-10月龄猪胆总管的几何形态、张开角、弹性模量、顺应性和显微结构成分等生物力学特性与成人的比较匹配。从猪胆总管的年龄和生物力学特性的相关性提示,7-10月龄猪的胆总管作为人异种移植胆管供源是可能的。第二部分猪胆管脱细胞支架的制备及组织学和生物力学评价目的：用不同的脱细胞方法对猪胆总管进行处理,从猪胆总管脱细胞前后的组织学和生物力学特性方面进行评价,探索一种合适的脱细胞方法,为组织工程胆管支架材料的应用提供理论和实验依据。方法：1.分组：30段猪胆总管,沿纵轴制作成30mm×12mm的样本,随机分为5组(每组6例)：新鲜对照A组及脱细胞B组、脱细胞C组、脱细胞D组和脱细胞E组。2.脱细胞处理：分别用四种方法脱细胞(B组：0.05%胰蛋白酶+核酸酶,C组：0.1%SDS+核酸酶,D组：1.0%Triton X-100+核酸酶,E组：1.0%Triton X-100+0.1%SDS+核酸酶)。处理过程为37℃恒温下持续振荡24h.PBS液反复漂洗。3.脱细胞效果观察：HE染色,光镜观察脱细胞基质的组织结构和细胞残留情况。4.脱细胞基质的核酸(DNA)含量测定：用紫外分光光度法检测脱细胞基质的DNA含量,计算脱细胞率5.生物力学试验：将标本均沿纵轴裁成20mm×10mm的样本条,在TestResources生物力学试验机上进行加载一卸载试验和极限抗张强度试验。计算出生物力学材料常数(α1、β1、α2、β2)、弹性模量、极限抗张强度和断裂伸长率等指标。结果：1.光镜观察证实,四种方法脱细胞后,猪胆总管壁的细胞不同程度被去除。脱细胞B组有少量细胞残留,纤维有损伤；脱细胞C组、D组、E组的细胞均被去除,纤维无明显损伤。2.A组的DNA含量为71.24±2.56μg/100mg。B、C、D、E4个脱细胞组的DNA含量与A组的差异有统计学意义(F=15.29,P=0.00),4个组均有明显的脱细胞效果(P<0.01),B组的脱细胞率为77.03%,比C组、D组、E组的脱细胞效果稍差(P<0.05),E组的脱细胞率高达99.03%。3.D、E2个脱细胞组胆总管的生物力学材料常数(α1、β1、α2、β2)与A组的差异无统计学意义(F=12.21,P=0.06),B组、D组比A组、C组和E组的小(P<0.01),D组和E组2个脱细胞组之间差异不明显(P>0.05),E组的更接近A组。4.D组、E组2个脱细胞组胆总管的弹性模量比A组的稍增大,但差异不明显(P>0.05),B组、C组比A组的小(P<0.05),D组和E组2个脱细胞组之间差异不明显(P>0.05)。5.脱细胞D组、E组的UTS值和SOF值与A组差异不明显(P>0.05)；脱细胞B组、C组的UTS值明显小于A组(P<0.05),SOF值明显大于A组(P<0.05)；脱细胞D组、E组的UTS值和SOF值之间无明显差异(P>0.05)。结论：1.0.05%胰蛋白酶+核酸酶组的脱细胞效果欠佳,DNA剩余量较多,胆总管ECM结构完整性受到了破坏,生物力学特性变化表现为材料常数变小,刚度变小、强度明显降低和伸展性增加。2.0.1%SDS+核酸酶组的脱细胞效果虽然好,但生物力学特性发生了与B组相似的改变。3.1.0%Triton X-100+核酸酶和1.0%Triton X-100+0.1%SDS+核酸酶2个组的脱细胞效果好,且不会影响猪胆总管的生物力学特性,前者的脱细胞率为95.42%,而后者的脱细胞率高达99.03%,能更好地降低胆总管的免疫原性,是一种比较理想的猪胆总管脱细胞方法。4.从免疫原性和生物力学角度考虑,1.0%Triton X-100+0.1%SDS+核酸酶的脱细胞方法制成的脱细胞猪胆总管,是构建组织工程胆管的良好支架材料。更多还原

【Abstract】 Background:Biliary tract surgery is one of the difficult points in surgical operation, and the intraoperative repair of bile duct, post-repair restricture and treatment of malignant stricture are always the major concerns in abdominal surgery. Efforts have been made to design a type of artificial bile duct to replace the diseased bile duct as a solution to bile duct repair and reconstruction. Allograft transplantation has been widely used in clinical practice. However, there are still many problems in the prevention and treatment of rejection after operation. The postoperative stricture and obstruction together with functional restoration of Oddi sphincter could not be completely addressed by bile duct transplantation. Xenotransplantation has become a new field of organ transplantation due to the shortage of human organs in recent years. It is necessary to discover new donor materials to effectively overcome the shortage of organs. Pig is considered as the animal most suitable for human xenotransplantation. Pig has been used in experimental research and clinical practice of xenotransplantation by domestic and overseas scholars. However, there have been few reports on comparative research of the mechanical characteristics of porcine bile duct in xenotransplantation. Genetically modified animals are the candidate donors of human organs. The genetically modified pig which can express human complement regulatory protein has been cultured successfully, and the problem of hyperacute rejection has been solved. This makes pig very attractive and challenging to serve as donors for human xenotransplantation. It is necessary for xenotransplantation donor to possess the physiological functions and overcome the immunogenicity, in addition to the anatomical and biomechanical compatibility. Therefore, the study on the feasibility of porcine common bile duct to be used as human xenotransplantation donor or in tissue engineering should be carried out from the biomechanical perspective.Part1Biomechanical evaluation of porcine bile duct as human bile duct graft substitute materialsObjective:Through the comparative study of biomechanics of common bile duct in pig and human, to explore the relationship of biomechanics of the common bile duct between the human and pig, and evaluation from the aspects of biomechanics porcine bile duct as human bile duct transplant feasibility of alternative materials.Methods:Totally50healthy Chinese Hubei white pigs aged2months (30males and20females) were randomly divided into10groups.50pigs were housed under standard husbandry conditions,5pigs (3males,2females) were killed via an intramuscular injection of ketamine (5mg/kg) at3,4,5,6,7,8,9,10,11and12months respectively. The common bile duct was cut after anatomical isolation and in situ measurement of respective in vivo length. The bile ducts of humans were obtained from5deceased donors (3human men,2human women) without hepatic diseases, who were between20and40years old. Each sample sliced into5μm frozen sections was stained with hematoxylin-eosin (H&E). Geometric morphological indices (wall thickness and diameter) of common bile duct were measured with a computer image analysis system. The opening angles of common bile duct were measured using computer software of e-ruler. The pressure-diameter experiment of common bile duct was tested with the biomechanical test equipment. The corresponding parameters were calculated, including incremental elastic modulus (Einc), pressure-strain elastic modulus (Ep), volume elastic modulus (Ev), and compliance. The common bile duct was stained with immunohistochemical methods, the content of microstructural components (collagen, smooth muscle and elastin) of common bile duct were detected and calculated with laser confocal microscope.Results:1. Changes in the body weight of pig, weight and volume of porcine livers at different month ages. The body weight of pig, weight and volume of porcine livers gradually increase with the increased age. There was no significant difference in body weight of pig, weight and volume of porcine livers between pigs aged6-7months and adult human (F=109.15, P=0.08).2. Comparison of geometric morphological indices of common bile duct between pigs and humans. There were significant differences in wall thickness(F=6.46, P=0.00), diameters (F=9.56, P=0.00), and length (F=13.29, P=0.00) of the common bile ducts between humans and pigs in different ages. The wall thickness and diameters of common bile duct of pigs aged3-6were smaller than that of adult human (P<0.01). There was no significant difference in the wall thickness and diameters of common bile duct between pigs aged7-10months and adult humans (P>0.05). The length of the porcine common bile ducts in different ages was shorter than that of adult human (P<0.01).3. Comparison of opening angles of common bile duct between pigs and humans. The opening angles of porcine common bile duct increased gradually from3to12months (P<0.05), and tended to be stabilize when the pigs aged7months. There was no significant difference in the opening angles of common bile duct between pigs aged7-10months and adult humans (P>0.05). 4. Relation between elastic modulus of the common bile duct of humans and pigs and pressure. The Einc, Ep and Ev of common bile duct of pigs and humans increased with increasing pressure. When the pressure reached4kPa and higher, the diameters of common bile duct did not change any more, and the elastic modulus did not change or slightly changed, and the slopes of the curves become gentle.5. Comparison of elastic modulus of the common bile duct between humans and pigs. The one-factor analysis of variance showed that there were significant differences in Einc (F=502.08, P=0.00),Ep (F=137.42,P=0.00),andEv(F=134.59, P=0.00) of the common bile ducts between humans and pigs in different ages. The Einc, Ep, and Ev of common bile duct of pigs aged3-6,11-12months were higher than that of adult humans (P<0.01). There was no significant difference in the elastic modulus of the common bile duct between pigs aged7-10months and adult humans (P>0.05).6. Changes in compliance of porcine common bile duct at different ages. The compliance of porcine common bile duct gradually increase with increased age, however, after the pigs aged10months, the compliance gradually decrease with increased age. The changes in compliance of porcine common bile duct with age were opposed to the changes in elastic modulus.7. Comparison of compliance of common bile duct between humans and pigs. The one-factor analysis of variance showed that there was significant differences in compliance (F=62.93, P=0.00) of the bile ducts between humans and pigs in different ages. There was no significant difference in compliance of the common bile duct between pigs aged7-10months and adult humans (P>0.05), but the compliance of common bile duct of pigs aged3-6,11and12months was lower than that of adult humans (P<0.01).8. Content of microstructural components of common bile duct of humans and pigs. The organization structure of porcine common bile duct was similar to that of human. Changes in content of smooth muscle (F=89.85, P=0.001), collagen (F=273.69, P=0.003) and elastin (F=182.60, P=0.006) of porcine common bile duct with ages. The content of smooth muscles and elastins increased with the increased age, whereas the content of collagens and collagen/elastin (C/E) ratio decreased gradually with the increased age. There were no significant differences in the content of smooth muscles, collagens and elastins of the common bile duct between pigs aged7-10months and adult humans (P>0.05).Conclusions:Changes in biomechanical properties of porcine common bile duct with age. The biomechanical properties of geometric morphological indices, opening angles, elastic modulus, compliance, and microstructural components of the common bile duct of pigs aged7-10months match that of adult human. The correlation between age and biomechanical properties of common bile duct in pigs could imply that it was possible for the common bile duct of pigs aged7-10months to be used as the donor for bile duct xenotransplantation.Part2Preparation and histological and biomechanical evaluation of decellularized porcine bile ductObjective:The common bile ducts were treated with different acellular matrix. The histological and biomechanical characteristics of porcine common bile ducts before and after acellular matrix treatment were evaluated, and an appropriate acellular matrix was explored to provide theoretical and experimental basis for the application of bile duct scaffold materials for tissue engineering.Methods:1. Grouping:Thirty porcine common bile ducts were made into samples of30mm x12mm along the vertical axis and were divided into5groups (6in each) randomly:fresh control group A, acellular matrix group B, acellular matrix group C, acellular matrix group D and acellular matrix group E.2. Acellular matrix treatment:Four different acellular matrix treatments were used (group B:0.05%trypsin+nuclease, group C:0.1%SDS+nuclease, group D:1.0%triton X-100+nuclease, group E:1.0%triton X-100+0.1%SDS+nuclease). All the steps were performed under constant temperature of37℃with continuous agitation, and the samples were washed repeatedly using PBS solution.3. Observation of acellular matrix treatment effect:After HE dyeing, the tissue structure of acellular matrix and the residual cells were observed under light microscope.4. Determination of nuclear acid (DNA) content of acellular matrix:The DNA content of acellular matrix was detected using UV spectrophotometry, and the acellular rate was calculated.5. Biomechanical experiment:The samples were cut into stripes of20mm×10mm along the vertical axis. The loading-unloading experiment and ultimate tensile strength experiment were performed using TestResources biomechanical tester. Indexes such as biomechanical material constant (α1,β1,α2,β2), elastic modulus, ultimate tensile strength (UTS) and breaking elongation rate were calculated.Results:1. The cells of porcine common bile ducts were removed to different degrees after the four treatments as indicated by light microscope observation. Few residual cells and fiber damage were observed in group B. All the cells were removed and no obvious damages were done in group C, group D and group E.2. The DNA content of group A was71.24±2.56μg/100mg. The DNA contents of four acellular matrix treatment groups B, C, D, E were statistically different from that of group A (F=15.29, P=0.00). There was obvious acellular matrix treatment effect in the four groups (P<0.01). The acellular matrix treatment effect of group B was slightly worse that of group C, group D, group E (p<0.05); the acellular rate of group E was up to99.03%.3. There were no statistical differences in the biomechanical material constants (α1, β1,α2, β2) of the common bile ducts between group A and two acellular matrix treatment groups D and E (F=12.21, P=0.06). The biomechanical material constants of group B and group D were lower than that of group A, group C and group E (P<0.01). There was no obvious difference between the two acellular matrix treatment groups D and E (P>0.05), with group E closer to the level of group A.4. The elastic module of common bile ducts of the two acellular matrix treatment groups D and E were a little larger than that of group A, but without significant difference (P>0.05). The levels of group B and group C were lower than that of group A (P<0.05), and the difference between group D and group E was not significant (P>0.05).5. There were no significant differences in UTS value and SOF value between the acellular matrix treatment groups D, E, and group A (P>0.05). The UTS value of acellular matrix treatment groups B and C was significantly lower than that of group A (P<0.05), and the SOF value was significantly higher than that of group A (P<0.05). There were no obvious differences in UTS value and SOF value between acellular matrix treatment groups D and E (P>0.05).Conclusions:1. The acellular matrix treatment effect of0.05%trypsin+nuclease was unsatisfactory with high residue amount of DNA; the EMC structural integrity of common bile duct was damaged, and the changes of biomechanical characteristics were manifested as reduced material constants, lower stiffness, obviously decreased strength and the increased extensibility.2. The acellular matrix treatment effect of0.1%SDS+nuclease was better, but a similar changing trend occurred to the biomechanical characteristics as in group B.3. The acellular matrix treatment effect of1.0%Triton X-100+nuclease and1.0%Triton X-100+0.1%SDS+nuclease was quite satisfactory, and the biomechanical characteristics of porcine common bile duct were not affected. The acellular rate of the former was up to95.42%, the acellular rate of the later was up to99.03%, and the immunogenicity of common bile duct can be better reduced. Therefore, it is an ideal acellular method for porcine common bile ducts.4. From the views of immunogenicity and biomechanics, the porcine common bile ducts prepared by the acellular method of1.0%Triton X-100+0.1%SDS+nuclease provides a good scaffold material for the construction of bile duct by tissue engineering.更多还原