【作者】 胡泉；

【导师】 王常勇；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2009， 硕士

【摘要】 肝脏是人体重要的生命器官,肝脏疾病严重影响着患者的健康和生活质量,针对这种疾病的治疗一直受到国内外研究人员的广泛关注。肝脏移植是目前临床上治疗肝功能衰竭、终末期肝病和代谢性肝病的有效方法。但由于肝脏移植受到移植器官来源有限及移植后引发免疫排斥反应等因素的限制。因此,针对终晚期肝病和肝衰的治疗迫切需要寻找新的技术和手段。随着组织工程研究的不断深入,运用组织工程技术构建可供移植的肝脏组织,并降低其免疫原性已经成为现代移植外科领域研究的热点,为肝病的治疗带来了新的希望。鉴于肝脏组织结构和功能的复杂性,如何在体外成功地再造组织工程化肝脏是组织工程研究面临的重要挑战之一。近年来,肝脏组织工程的相关研究取得了较大进展,如:使用三维细胞装配技术构建具有活性的组织工程化肝脏,使得构建的肝组织在明胶/壳聚糖三维结构中培养6天具有部分肝脏生理功能。另有学者设计了一种均一连续的肝脏组织工程片层,培养于温度敏感材料表面,移植皮下200天后仍然具有部分生理功能,这些研究结果显示了肝脏细胞具有在体外培养形成功能性组织的能力。但对于移植过程中的引发的免疫排斥问题相关研究工作并未作更深的探讨。目前器官移植术后免疫排斥反应机制已经较明确,主要为T细胞及抗体介导的排斥反应,并以前者为主。杀伤性T细胞主要通过通过两种机制来攻击靶细胞,第一种是通过释放Fas-L诱导表面有Fas受体的靶细胞进入凋亡,第二种是依赖于穿孔素传递杀伤因子。临床研究发现在急性排斥、慢性排斥、不排斥病例中,两种机制发挥作用不同,第二种机制主要是在急性排斥病例发现,第一种机制中Fas/Fas-L途径在急、慢性病例中都有发挥主要作用。现阶段临床上,术后病人使用全身免疫抑制剂来控制免疫排斥反应,但该疗法存在以下缺点:1、全身毒副作用大;2、剂量难以掌握;3费用昂贵等。针对以上问题,国内外大量学者寻求一种新的方法来降低器官移植术的免疫排斥反应,从而减少或短期内不使用全身免疫抑制剂。众所周知,睾丸组织是人和动物体内的天然的免疫豁免区。睾丸异体移植成活率较高。有学者发现将大鼠的睾丸组织植于普通部位(皮下)可产生免疫耐受。以往的实验局限于对睾丸组织整体的研究。近年来,对睾丸细胞进行分离研究,发现睾丸支持细胞(Sertoli Cell,SC)天然稳定高表达Fas-L ,是产生免疫耐受的功能细胞。睾丸支持细胞是一种很好的种子细胞,它不仅可以通过1、细胞间接触经Fas/Fas-L途径杀伤淋巴细胞;2、通过释放Fas-L杀伤淋巴细胞及中性细胞;3、吸引炎性细胞胞浸润,可使联合移植物免受攻击。还能够分泌多种生长因子具有营养支持的作用。此外,睾丸支持细胞贴壁生长,有利于胶原片层的收缩塑性。因此,关于它的研究近年来一直受到众多研究人员的关注:2003年,英国科学家Rahman等通过体外实验结果证实睾丸支持细胞与HepG2微囊包被共移植可以提高HepG2细胞活性,降低免疫排斥反应,移植后对急性大鼠肝衰竭具有良好的改善作用。另有研究发现将SC移植到成年大鼠脑内能够显著使已病变的的神经元细胞恢复功能。因此,睾丸支持细胞是对共培养细胞具有营养作用以及移植后降低免疫排斥反应作用。使用睾丸支持细胞复合肝细胞在细胞外基质凝胶中三维共培养构建组织工程化肝脏片层并移植的研究国内外至今未见报道。本研究中,我们在体外分离、培养肝细胞和睾丸支持细胞,并对肝细胞和睾丸支持细胞的形态和功能进行相关检测;大鼠肝细胞和SC在细胞外基质凝胶中复合三维共培养构建组织工程化肝脏片层,对其形态和功能上进行相关检测。在此基础上,组织工程化肝脏片层进行SD大鼠皮下移植,取材后将移植片层进行组织学观察和免疫组织化学检测。通过上述研究,我们探讨了睾丸支持细胞在细胞外基质凝胶环境对肝细胞的活性与生理功能的促进作用;肝细胞/SC复合片层在移植的过程中SC细胞发挥的营养支持作用和免疫抑制作用。本论文的主要研究内容包括以下四个部分:第一部分、大鼠肝细胞的分离、培养与鉴定本实验采用门静脉灌注的方法,经过两次灌注后消化分离获得成年SD大鼠肝细胞,经过免疫组化检测。结果表明,分离的细胞具有良好的活力,细胞呈ALB、CK18免疫组化染色阳性。为了探讨大鼠肝细胞的糖原合成能力和白蛋白分泌能力。我们采用PAS糖原染色法,表明分离的肝细胞具有糖原合成能力;在培养液上清的肝细胞功能检测实验中,我们也证明,体外培养的肝细胞具有分泌ALB的生理功能。第二部分、睾丸支持细胞的分离、培养与鉴定本研究通过V型胶原酶单次消化法分离获得SC。通过台盼蓝活性检测和免疫组化检测。结果表明,原代分离的细胞具有良好的活力,细胞呈Fas-L免疫组化染色阳性。为探讨SC的功能,我们制备了SC的条件培养基,并用其培养淋巴细胞。通过流式细胞仪检测淋巴细胞的凋亡情况。结果表明,SC在体外能够诱导淋巴细胞的凋亡。对SC条件培养基培养的肝细胞进行培养液上清功能检测实验,检测结果表明,SC条件培养基培养的肝细胞相对与没有使用SC条件培养基培养的肝细胞对照组来说,ALB分泌明显提高。第三部分、基于肝细胞/SC的组织工程化肝脏体外再造的实验研究本研究以原代大鼠肝细胞和乳鼠睾丸支持细胞为种子细胞,以添加10%Matrigel的Ⅰ型液态胶原为支架材料,体外构建组织工程化肝细胞/SC复合片层,并在培养过程中施加静态拉伸力作用。观察片层细胞生长的情况,并在培养一定时间内取材,进行组织学检测、免疫组织化学染色以及片层的功能检测。结果表明,组织工程化肝脏复合片层中肝细胞具有良好的细胞活性,细胞之间可以相互接触、粘附、聚集并形成球状体结构而且具有分泌ALB能力。第四部分、组织工程化肝脏体内移植的实验研究本研究以第三部分体外构建的组织工程化肝脏片层,培养2天后片层收缩具有一定韧性后将其移植入成年SD大鼠腹部皮下。这部分实验研究根据移植物、移植动物的不同分为4组,G1组:肝细胞/SC复合片层移植入注射免疫抑制剂的SD大鼠体内;G2组:肝细胞/SC复合片层移植入未注射免疫抑制剂的SD大鼠体;G3组:单纯肝细胞片层移植入注射免疫抑制剂的SD大鼠体内;G4组:单纯肝细胞片层移植入未注射免疫抑制剂的SD大鼠体内。以上4组SD大鼠移植3周后取材,将片层进行组织学检测和免疫组织化学鉴定。结果表明,G1、G2组移植片层组织学检测形态良好,肝细胞层于肌细胞层界限明显,肝细胞紧密接触生长。G3组移植片层内肝细胞数量不多,活力良好。G4组移植片层内肝细胞存活数量不多。综上所述,本研究以原代大鼠肝细胞、SC细胞为种子细胞,以添加Matrigel的Ⅰ型胶原为支架材料,体外构建了组织工程化肝脏片层,并在培养过程中施加静态拉伸力作用。研究结果表明了睾丸支持细胞在细胞外基质凝胶环境对肝细胞的活性与生理功能的促进作用。片层内细胞活性良好,细胞之间可以相互接触、粘附、聚集,形成了球状体结构。将片层体外移植过程中,SC细胞发挥了营养支持作用和免疫抑制作用,为下一步组织工程化肝脏体内原位移植提供了一种新的参考方法。体外构建的组织工程化肝细胞片层可以作为肝脏发育研究以及肝脏疾病治疗药物筛选的模型,深入研究也将具有潜在的临床应用前景。本研究所采用的工程化组织构建方法也为其它复杂生命器官的体外再造提供了思路。更多还原

【Abstract】 Since liver is one of the vital organs of human body, liver diseases have severe effect on patients’health and quality of life. Therefore treatment of liver diseases has drawn the attention of researchers both domestic and abroad. Currently, bioartificial liver and liver transplantation is the most effective approach in the treatment of liver failure, end-stage liver disease and metabolic liver diseases. Bioartificial liver could not perform an overall liver function .Constrained by the shortage of organ supply and the immunological rejection after transplantation, liver transplantation could not be applied widely. Therefore, it is urgent to find new techniques and tools for the treatment of end-stage liver disease .The development of liver tissue engineering brought new hope for the treatment of liver diseases.In view of the complexity of the liver structure and function ,it is a great challenge to construct engineered liver invitroly . In recent years, great progresses have been achieved in liver tissue engineering research, such as: the application of three-dimentional cell assembly technology to construct engineered liver tissue, which present physiological activity in the gelatin /chitosan three-dimensional structure after cultivation for 6 days. Another scholar has designed a uniform layer of engineered liver patch, cultivated on temperature-sensitive surface. which can survive for 200 days after subcutaneous transplantation. However,the problem of immunal rejection aroused during transplantation still remained for further investigation.The mechanism for immunal rejection after organ transplantation is identified,which is the rejection of mediated by T cell and antibody.The T cell attacked the target cell mainly through two mechanisms:1.release of Fas to induce apoptosis of target cell expressing Fas-Lon celluar membrane;2.to transmit killing factors through perforin.Clinically, we applied eneral immune depressant to control the immunal rejection.However,there are disadvantegous :1.great sys-toxicity;2.dosage control;3.great expense.People are exploring new approaches to reduce the immunal rejection after organ transplantation in order to avoid the application of sys-immunal depressant.It is known to all that the didymus tissue was the native immune-privileged site . Survival rate of didymus allograft was high.Studies showed that when transplanted subcutaneously,the didymus could generate immune tolerance.Former studies focused on the didymus as whole.Recently,the didymus cells were isolated for study.The SC expressed Fas-L stably ,resulting in immune tolerance.SC could kill lymphocyte through cell to cell contaction by Fas/Fas-L; 2.kill lymphocytes and neutrophilic cell by releasing Fas-L;3.attracting inflammatory cell infiltrate and help the graft to avoid attacking.Besides,it could also secret various of growth factors providing nutritions.Furthermore,the SC adhered to grow,which make it easy for collagen contraction.Therefore,the study of SC is becoming more and more popular.2003,Rahman from England demonstrated that co-transplantation of SC and HepG2 microcapsule could increase HepG2 activity,reduce immunal rejection,and showed an improvement of liver failure.The SC was also proved to have an effect on the affected neurone.Therefore,the SC could provide nutrition to cocultured cells as well as reduce immunal rejection.There are’t any report about the construction of engineered liver tissue using SC and hepatocytes as seeding cells.In this study, we isolated and cultured hepatocytes and Sertoli cells (Sertoli Cell, SC) invitroly. The morphology and function of hepatocytes and SC were detected .We also constructed engineered liver tissue with rat hepatocytes and SC in three-dimensional hydrogel. On this basis, the tissue-engineered hepatocytes /SC patch was transplanted subcutaneously in liver failure rat model. The mortality of transplanted and untransplanted ones were determined .After a certain period of time, rats were sacrificed to harvest the implanted materials for histological and immunohistochemistry assessments.Through the researches above, we successfully constructed engineered liver patch which survive well after subcutaneous transplantation, with cell aggregate into a spherical structure. The results showed that tissue-engineered liver patch is of great significance in liver transplantation.In this paper, the main research contents include the following four parts:Part I:the isolation ,cultivation and identification of rat liver cellsThis experiment used the method of portal vein perfusion, after perfusion for two times ,the hepatocytes of SD rat was derived. Immunohistochemical results demonstrated a good vitality of separated cells, cells were ALB、CK18 positive. In order to assesst the ability of glycogen synthesis and albumin secretion of the isolated hepatocytes,We used PAS staining, which result showed that the isolated hepatocytes has a capacity of glycogen synthase; in the assessment of culture medium supernatant, we also demonstrated that the liver cells have the capability of secreting ALB.PartⅡ:isolation, cultivation and identification of Sertoli cellsIn this study, we derived SC by V-collagenase digestion. Immunohistochemical results showed a good vitality of isolated cells, cells were Fas-L positive. To study the function of SC, we prepared the SC conditioned medium which was also applied in the cultivation of lymphocytes and hepatocyte.We detected the cell apoptosis of lymphocytes through flow cytometry. The results showed that, SC could induce the apoptosis of lymphocytes. The assessment of supernatant of hepatocyte cultivated in conditioned medium showed that secretion of ALB in coculture system was increased significantly compared with hepatocytes cultured without SC conditioned media.PartⅢ, construction of engineered liver based on hepatocytes/SCIn this study, primarily isolated rat hepatocytes and Sertoli cells were mixed with 10% Matrigel and liquid typeⅠcollagen as scaffold material to construct engineered liver patch and exert statistic stretching force while in cultivation. We observed cell growth in the patch, and after a certain period of time, the patch was subjected to histological, immunofluorescent staining, as well as the function assessments. The results showed that the primarily isolated rat hepatocytes present a good vitality in three-dimensional gel matrix environment, secreted ALB, and formed a spherical structure through mutual contacts ,adhesion and aggregation.Part IV study of tissue-engineered liver transplantation in vitroIn this study, the patch constructed above was grafted into the adult hepatic failure rat abdominal subcutaneous. There were 4 groups, G1:hepatocyte/SC patch transplanted to immune depressed SD rats; G2: hepatocyte/SC patch transplanted to non-immune depressed SD rats;G3:hepatocytes only patch transplanted to immune depressed SD rats;G4:hepatocytes only patch transplanted to non-immune depressed SD rats. These four groups above were harvested 3 weeks later and subjected to histology and immunohistochemical study. The results showed that, cells in G1、G2groups survive well and attached to each other. Cells in G3 grow well with normal activity .Almost no cells survived in G4 .In conclusion, primary rat hepatocytes, SC cells were mixed with the typeⅠcollagen and Matrigel to construct engineered hepatic tissue which was exerted a static stretching force during cultivation. The results showed that in the three-dimensional extracellular matrix gel, hepatocytes present good vitality, the cells from a spherical structure through mutual contacts adhesion, aggregation. The engineered liver tissue can be used as model for the study of liver development as well as drug screening. More important, this study provided a strategy for the reconstruction of other vital organs.更多还原