【作者】 程远；

【导师】 潘明新； 汪艳；

【作者基本信息】 南方医科大学 ， 普通外科学， 2011， 硕士

【摘要】 研究背景：我国是肝病大国,对于终末期肝衰竭患者十分常见。然而,到目前为止,针对终末期肝病的根治性方法只有肝脏移植。然而,受到器官短缺及长期服用抗排斥药物等不利因素,这种治疗方法远远不能满足临床上的需求,许多需要肝脏移植的病人在等待肝源的过程中死去。生物人工肝系统作为一种有效手段已经用于急性肝衰竭(ALF)治疗,或作为等待肝源及肝移植后恢复阶段的肝脏替代治疗。但是受到技术限制、生物安全性及成本较大等因素的影响,现有的人工肝系统还有待进一步的改进。总之,现有的肝脏替代治疗手段都未能完全满足临床的需要,寻求一种安全、经济、有效的肝脏功能替代疗法已经十分迫切,肝脏组织工程是构建可移植肝脏的一条创新型途径,其核心内容是利用生物学和工程学的原理,通过将大量肝细胞(或肝样细胞)、内皮细胞等细胞材料与生物支架材料相复合,构建出一个可供移植的组织工程肝脏,用以对病损肝脏进行形态、结构及功能上的永久性替代。因此,组织工程肝脏的研究对缓解现有肝脏治疗措施的不足具有重大的研究意义。由于肝脏的组织结构及生理功能复杂,组织工程化肝脏对于种子细胞和支架材料的要求就更为苛刻。良好的支架材料应有足够的表面积用于细胞粘附,也应有足够的空间使细胞集落扩展,增殖,足够的孔隙以利于物质与气体的交换,具有可降解性,良好的生物相容性。然而,相对于复杂的肝脏结构,以往所使用的人工合成支架材料都显得过于简单,无法满足肝细胞高密度生长所需营养供应的要求,更无法模拟肝脏组织复杂的细胞生长环境。因此,寻找最大限度模拟体内肝细胞生长微环境的三维支架材料用于肝细胞培养,保证细胞的数量、形态及功能的维持,是解决组织工程化肝脏构建的首要问题。去细胞化支架的出现,从根本上打破了传统支架材料结构简单的局限。去细胞化支架是指通过化学或物理的方法将细胞破坏并从组织中完全移除,完好地保留了细胞外基质(ECM)及组织器官的三维支架结构,早已成为组织工程研究中的热点。去细胞化肝脏生物支架与以往的支架材料相比有着明显的优势。首先,它保留的三维网状结构为肝细胞生长提供了所需的微环境。去细胞化肝脏支架保留了肝脏的大部分细胞外基质包括胶原,弹性纤维,蛋白聚糖等组分。这些成分不仅对于肝细胞的粘附、生长等生物学行为过程中起到重要作用,而且能够与周围的细胞发生交互作用,为细胞的分化、迁移及细胞之间相互作用提供信号。第二,支架中保留的血管网络解决了肝细胞生长所必需的营养输入与废物排出的难题,从而使肝细胞的组织化培养成为可能。另外,由于细胞外基质成分在不同的物种之间具有高度保守性,所以去细胞化技术得到的细胞外基质支架具有良好的生物相容性及低免疫原性。最后,细胞外基质中可能遗留的生物活性成分具有促进细胞生长、增殖及分化的生物特性。至今,去细胞化支架已成功地应用于心血管、输尿管、膀胱等组织器官的研究中。由于去细胞化技术在肝脏组织工程中的应用起步较晚,再加上肝脏结构极其复杂,利用去细胞化肝脏支架进行可移植组织工程化肝脏的重建还很长时间的探索。另一方面,去细胞化肝脏支架所构建的组织工程化肝脏必须通过异体移植的形式发挥作用。去细胞化肝脏支架移植到受体体内,组织相容性是考察支架能够移植的检测标准之一。如细胞成分去除不彻底则可引发严重的免疫排斥反应,炎症反应不仅可以导致组织细胞降解,同时也是组织钙化的又一个重要原因。然而,既使去除了组织细胞表面的大部分高免疫源性物质如组织相容性(MHC)Ⅰ和Ⅱ类蛋白,由于在去细胞化过程中可能残留了少量的细胞碎片和脂质成分,以及去细胞过程中引发的支架结构的改变,因此仍然存在引发免疫排斥反应或导致支架结构钙化的可能。至今,还未见关于去细胞化肝脏支架异体组织相容性的相关报道。因此,积极探索利用去细胞肝脏支架构建具有肝脏功能的组织工程化肝脏,同时进行去细胞化肝脏支架组织相容性研究都具有重要的意义。本课题组在既往工作中已成功使用化学去垢剂方法制备去细胞化全肝脏支架。在此基础上,本实验进一步优化去细胞化大鼠肝脏的制作流程,使去细胞化效果更加稳定。紧接着,我们利用去细胞化肝脏支架保留的管道结构组合到体外的循环装置上,并与肝脏肿瘤细胞系C3A细胞进行体外循环共培养。实验证实细胞在流动的状态下与支架结构紧密地粘附,并能够在较长时间里保持一定的功能与活性。实验说明：去细胞化肝脏支架保留的肝内管道结构为在其上进行干细胞的高密度培养创造了条件,在去细胞化肝脏支架上种植肝细胞并进行细胞循环灌注培养甚至肝脏再生是现实可行的。另外,通过去细胞化肝脏支架组织异体皮下植入实验我们也可以看出：利用Trixton-100及SDS化学去垢剂制备的去细胞化肝脏支架脱细胞化效果完全,去细胞化后的肝脏支架结构具有良好的组织相容性,这说明使用去细胞化肝脏支架作为支架材料适合异体移植,为进一步构建可移植的组织工程化肝脏的研究提供了理论基础。第一章去细胞化肝脏支架体外循环灌注培养实验目的：利用去细胞全肝脏生物支架进行体外肝细胞循环灌注培养,并对所得到生物支架进行功能测定及组织学观察,为进一步的体外肝脏组织重建奠定实验基础。方法：以SD大鼠作为研究对象,利用化学去垢剂方法制备全肝脏生物支架。首先取SD大鼠肝脏,将所得肝脏经门静脉插管后依次灌注曲拉通X100(TritonX100),十二烷基磺酸钠(SDS),用时约8小时,并用O.01mM的磷酸盐缓冲液(PBS)灌洗支架上残留SDS 210h,将支架结构中的肝静脉、胆管及肝动脉以1#丝线结扎。去细胞化肝脏支架经过氧乙酸(0.1%)消毒后使用PBS溶液反复冲洗干净,使用1ml注射器将5mlLO2细胞(约为2×107个/ml)经去细胞化肝脏支架门静脉注入支架中。再利用去细胞化肝脏支架保留的门静脉系统连接到体外自制的循环培养装置(附带恒温箱、氧合器、蠕动泵、混合气体罐及培养基等)进行体外循环培养10天,每2天换一次培养液,培养期间定期检查细胞的功能状态及循环系统的污染状况。最后取出标本经多聚甲醛脱水固定,石蜡包埋后切片行HE染色观察组织细胞的生长及分布状况。结果：利用去细胞化肝脏支架构建的体外循环培养系统共进行了11次体外循环培养。其中共有2次培养过程中放生污染,主要原因可能为在构建体外循环装置和培养换液过程中的操作不当导致。其余9次培养过程中未出现污染,其中最初5次培养过程中发现培养期间细胞生长状况较差,培养3天可见培养液中大量细胞碎片,生化功能检测显示肝细胞生长状态差,经病理切片检测可见大部分细胞脱落,支架上残留大量细胞残片及少量肝细胞。分析原因可能为氧气供给不合理,循环灌注速度过快导致细胞受剪切力影响受损等多种原因。后来通过逐渐地摸索和改进循环培养方法,培养的结果有了很大的改善。结论：利用化学去垢剂法制备去细胞化全肝脏支架重复性好。体外循环灌注培养是利用去细胞化肝脏支架构建组织工程化肝脏的可行性方法,但还需要进一步的试验摸探索。第二章去细胞化支架异体植入局部反应试验目的：评价化学去垢剂法制备去细胞化肝脏支架生物相容性,探究去细胞化肝脏支架植入异体组织后的局部反应状况,为进一步利用去细胞化肝脏支架重建组织工程化肝脏并实现异体内移植奠定实验基础。方法：以SD大鼠去细胞化肝脏支架作为研究对象,首先利用化学去垢剂法制备去细胞化肝脏支架并分组：实验组参照实验一中的去细胞化方法制备去细胞化肝脏支架,阳性对照组在制备过程中省略SDS灌洗过程。制成后使用使用过氧乙酸(0.1%)消毒(同方法一),彻底消毒后再使用PBS液反复冲洗10便,最后将支架浸泡在PBS溶液中并放置-20℃冰箱保存。实验前取出冰冻组织并将其分别制成直径1cm、厚1mm左右的薄片。选取5、14、21、28、35共5个植入期,每一植入期用3只巴比西小白鼠(雌雄不限),阳性对照组使用3只小白鼠。所有动物采用乙醚麻醉,将支架埋置于小鼠背部皮下与肌肉之间。术后通过大体观察、组织学观察评价支架组织炎症情况,有无血管形成,有无纤维囊等。根据炎性反应程度和包膜形成评级。结果：实验组去细胞化肝脏支架脱细胞完全,呈白色半透明状,显微镜下未见明显细胞成分残余；阳性对照组支架组织呈土黄色,镜下可见细胞碎片残余。皮下埋植实验生物学反应评价结果：可见阳性对照组术后5天可见红肿、化脓性表现。实验组动物实验期间无一例死亡,切口愈合良好,无红肿、溃烂等感染症状。组织学观察可见阳性对照组术后5天可见大量中性粒细胞浸润。而各个植入期实验组炎症反应轻微,无钙化、硬结形成,组织反应结果复合组织相容性的评定标准(合格)结论：化学去垢剂可以有效地移除肝脏内的细胞成分而保留低免疫源性的细胞外基质成分,具有良好的组织相容性好,可以作为肝脏组织工程的备选支架材料。更多还原

【Abstract】 China is a big country of liver diseases,so the patients with end-stage liver failure are very common.However, liver transplantation is still the only radical trement for end-stage liver diseases.Nevertheless because of the shortage of organ source and the long-term use of anti-rejection drugs after surgery,liver transplantation can not meet the clinical need, many patients who need liver transplantation were dying in the process of waiting for the liver donor.Bioartificial liver system has been used as an effective means for acute liver failure (ALF) treatment, also working as a temporary liver substitution while waiting for the liver or in the recovery phase of the liver after liver replacement therapy.However,being limited by the negative factors of technology, bio-safety and so on,the existing artificial liver system remained to be further improved. In short, the existing liver replacement therapy have failed to meet the clinical needs,and finding a safe,economical and effective alternative method is very urgent.Tissue engineering is an innovative way to build a transplanted liver tissue.The major task is to build a artificial liver by compounding of a large number of liver cells (or liver-like cells), endothelial cells and other cell material with the scaffold.The aim is to construct a tissue engineering liver for the patients whose liver has lost its morphology, structure and function,to abide by the use of biological and engineering principles.Therefore,it is of a great significance for liver tissue engineering research to alleviate the existing problem of lacking suitble treatment for liver failure.Because of the complex function and physiological structure of liver.Liver tissue engineering has more slashing demanding on seeding cells and the scaffold material.The perfect scaffold material should have enough surface area and space for cell mass to attach and proliferation,having the capability for shaping specific structures.In addition, the scaffold must be bio-compatible,degradable and most importantly having the function of exchange for Nutrition and oxygen.However, comparing with the complicated liver structure,the previously used synthetic scaffolds are all obviously too simple to meet the nutritional requirements for high-density growth of liver cells,let alone the imitation of the comlex structure and the micro-environment of liver.Therefore,it is urgent to find a kind of scaffold that can provide a three-dimensional micro-environment,which utmostly imitating the vivo environment liver cells lives. Only in this way can we ensure the number and function of liver cells.The emergence of decellularized scaffold breaks through the limitation of traditional scaffolds which have simple structure.Decellularization was a process of removing all the cells from organs and leaving ECM and three-dimensional structure without being damaged.It is usually accopmlished by means of chemical or physical methods.Decellularized scaffold have been become a hot research in tissue engineering in recent years.Decellularized liver scaffolds have a great deal of advantages compared with previous liver scaffolds.First,it retained the three-dimensional network structure of liver which provides the necessary micro-environment for cell growth and prolification.In addition it also retained the most part of the liver extracellular matrix, including collagen,elastic fibers, proteoglycans and other components,which play an important role not only in the liver cell adhesion,growth and other biological behaviors,but also in the process of interacting with the surrounding cells for cell differentiation,migration and interaction by providing signals between cells.Second, the retained blood vessels network of the scaffold resolve the problem of nutrition input and waste discharges,that is very necessary to hepatocyte growth and make the organization culture of liver cells as possible.Third,because extracellular matrix components are all highly conserved between different species,extracellular matrix scaffold has good bio-compatibility and low immunogenicity.Finally,there may be some active components left in the extracellular matrix which will be benefit for the cell growth, proliferation and differentiation. So far,decellularized scaffolds have been successfully used in the study in cardiovascular, ureter, bladder, liver tissues and other organs.On the other hand, tissue engineering liver constructed by decellularized scaffold must be transplanted into allogeneic body to play a role in the final form.The compatibility test is one of the importment criteriaits Whether or not decellularized liver scaffold can be tansplanted into the allogeneic recipient.It will cause severe immune response and serious inflammation if the cellular components can not be completely removed from the tissue,and result in the degradation of tissue and calcification.However, even though the most highly immunogenic substances, such as histocompatibility (MHC) I and II-like protein had been removed from the scaffold,a small amount of cellular debris and lipid composition left in the scaffold still can cause immune rejection and lead to calcification.So far,there are few relevant reports concerning the allogeneic histocompatibility of decellularized liver scaffold.Therefore,it is of important significance to carry out the research of making tissue engineering liver with decellularized scaffold and simultaneously testing the tissue compatibility of scaffold. In previous work, our group has successfully made decellularized scaffold by chemical methods.On the basis, we have combined the scaffold that preserved the pipeline structure after the decellularization into the Circulation devices constructed by ourself and co-culturing with the liver tumor cell-lines C3 A in vitro.Experiments result confirmed that the cells could adhere to the scaffolds closely and be able to keep certain hepatecytes activity and function for a long time even in the circulating state.It is proved once again that the acellular scaffold has good biocompatibility and reserve the pipeline stucture that stimulated the Intrahepatic structure of the liver at its Maximum.In addition, the Allograft implantation test result showed that the acellular scaffold has been comletely decellularized and resulted to minor minimal inflammation and good histocompatibility.The test made a basis for the further use of acellular liver scaffolds to construct a tissue engineering liver that can be transplanted.ChapterⅠ:Circulating co-culturing test by use of decellularized scaffold in vitro.Objective:In order to further construct the experimental basis for further liver tissue reconstruction,we performed the circulating perfusion co-culturing test by use of decellularized whole liver scaffold with C3A cells in vitro,and doing the function assay and histological examination on time.Methods:SD rats as the subjects,making whole liver decellularized scaffold by use of chemical detergents.Cannulated the portal vein and perfused sequentially withl%TritonX100 and 1%SDS for about 4h,and then perfused with phosphate buffered saline to dilute SDS residue.The pipe structure of the hepatic vein, bile duct and hepatic artery were ligated with silk thread l.The acellular scaffold were sterilized through oxygen acid (0.1%) and rinsed repeatedly with PBS solution.Injected about 5mlL C3A cells (about 106/ml) to the scaffold through the portal vein by using of Penicillium needle with a volume of 1ml.Then,the scaffold was connected to the self-construcred device (with incubators, oxygenator, peristaltic pumps, gas cans and mixed media, etc.),with the inlet connected to the preserved portal vein structure in vitro.The culturing period lasted for 10 days.The culture medium was changed for a half every 2 days.We insisted on regularly check of the functional status and whether there was pollution in the circulatory system.Finally, the specimens was dehydrated by paraformaldehyde and embedded in paraffin,HE staining and the biopsy tissue were made to show the cell growth and distribution. RESULTS:We constructed the circulation culture system with scaffolds successfully and made 11 times of culturing tests.Among them,culturing pollution were found in two times in the training process.The main reasons might be for the improper operation of fluid changing in the building process and training process and so on.Pollution does not appeared in other tests,among which the result of 5 times were not very well.A large number of cell debris were observed in the medium cultured in the 3th day,and the biochemical liver function tests showed poor cell growth and function state,the pathological examination showed Most cells fall from the scaffold and a large number of cell debris with a small amount of residual liver cells existed.The reasons leading to the cell damage maybe the unreasonable oxygen supply,too fast perfusion speed causing the shearing forces and other reasons.Afterwards,through further exploration and improvement of culturing methods,the results of circular culturing of C3A cells had developed greatly.Conclusion:Perfusion with chemical detergents is a repeatable method to make decellularized scaffold.The circulating perfusion culture is also a feasible method to construct the tissue engineering liver with acellular scaffold, but it needs further test to explore.Chapter II Local reaction experiment by allograft implantation of acellular liver scaffoldObjective:To evaluate the biocompatibility of acellular liver scaffold made by chemical detergent perfusion,to make experimental basis for the further use of scaffold to reconstruct the tissue engineering liver and the following transplantation in vivo. Methods:SD rats decellularized scaffold as the research object. Experimental group scaffold was prepared by the chemical detergents in the light of experiment 1, and the positive control group was prepared except for omitting the perfusion process of SDS.After the decellularization, the sterilizing procenture was acomplished by use of peracetic acid (0.1%) (as the same method I)and thoroughly washed by repeated perfusion with PBS solution for 10 times, At last the scaffolds were immersed in PBS solution for preservation in-20℃refrigerator. Before the experiment the frozen scaffold tissue were cut into slices with a diameter of about 1cm and 1mm thick. Select 5d,14d,21d,28d,35d as the five different implanting periods. There were three Ba Bi Xi mice for each implanted period in contrast, the positive control group with three mice. All animals were anesthetized by diethylether.The scaffold slices were embedded in the place between the skin and muscles of the mice back.Gross observation and histological evaluation such as tissue inflammation degree, with or without blood vessel formation,and the condition of fibrous capsule formation and so on.Rating According to the degree of inflammatory reaction and capsule formation Results:The scaffold in the experimental group were completely decellularized.The scafffold looked translucent white and no obvious residual cellular components by microscopy; Positive control group remained opaque and some remnants of cell debris could be seen by microscope. The experimental biology results of the positive control group showed visible local swelling, purulen in the 5th day after surgery. No deaths occured in the experimental group animals during the experiment and the surgery scar healed well, without swelling,ulceration and other symptoms of infection.The histological result of the positive control group shows a large number of neutrophil infiltration.But,no serious inflammatory reaction and no calcificationin were observed in the experimental group after surgery. Tissue reaction results of the assessment were with one accord with the histocompatibility criteria (qualified) Conclusion: Chemical decellularized method can effectively remove the cellular components of the liver and retain low immunogenicity of the extracellular matrix components. So, the acellular liver scaffolds has a satisfying tissue compatibility and can be seem as a candidate for liver tissue engineering scaffolds.更多还原