【作者】 陈捷；

【导师】 刘春晓；

【作者基本信息】 南方医科大学 ， 泌尿外科学， 2010， 硕士

【摘要】 研究背景肾脏是人体重要的生命器官,肾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。血液透析和肾脏移植是目前临床上常用的治疗终晚期肾病与急性肾衰的方法,透析只能替代肾脏的部分功能,肾脏移植则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,针对终晚期肾病和肾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,运用组织工程技术研制可供移植的肾脏组织为肾病的治疗带来了新的希望。组织工程学是一门近年来新兴的综合性学科,是一门运用细胞、工程材料学方法和创造适当生化、理化因素,以改善或替代组织的生物学功能的学科,是人类治疗组织、器官的功能衰竭和缺失的研究方向。其基本原理和方法是令少量组织细胞通过体外培养扩增达到一定数量后,接种到具有一定空间结构的支架上,构成有生命活力的细胞支架复合物,并移植到体内,以达到修复或替代组织损伤的目的。近年来,组织工程学有了迅猛的发展,对多种组织均有相应的组织工程器官面世的报道,如骨、软骨、血管、膀胱等,尤其在泌尿系统组织工程学上,国外学者更已将组织工程膀胱投入临床试验,成为首个植入人体内的真正意义上的组织工程人造器官。它既为众多研究者们建立了典范,同时也开启了更为广阔的未知领域：是否所有的人体组织均能通过组织工程学的方法创造出人造器官?截至本研究完成前,国内外尚未有组织工程肾体外构建成功的案例,并且学者们的研究重点均在于如何体外培育肾细胞,而合适的细胞支架则一直鲜有提及。我科实验性应用灌注法制备大鼠全肾脱细胞基质支架,目前未见其它相关报道,制备的脱细胞基质是否具有良好的细胞相容性尚不确切。研究目的1.探讨灌注法制备大鼠的全肾脏脱细胞基质(ACM)支架的条件和方法。通过对多种细胞支架的比较发现,脱细胞基质(Acellular Matrix)是一种良好的细胞支架。它具有原组织的宏观及微观结构,并且富含胶原、弹力纤维,并含微纤维蛋白、纤维结合素、层粘连蛋白、蛋白多糖、透明质酸、硫酸软骨素等。经脱细胞后留下的细胞外基质成分具有良好的生物相容性,并且各种组成的成分分别携带或接收不同的信号,诱导不同的细胞,因而对于细胞类型中基因表达方式,细胞的粘附、移行、转移等都有极重要的影响。目前制备脱细胞基质的方法基本局限于以酶或去污剂浸泡洗脱,而对实质性脏器基本无法达到脱细胞的目的。本研究以去污剂为洗脱细胞的溶剂,引入灌注法,通过血管、肾小球、肾小管这一自然平台达到将肾脏内部细胞洗脱的目的,并在研究过程中探讨、建立一套系统的制备方法。2.观察并证明所制备的全肾脏脱细胞基质具有规则的三维空间结构,良好的孔隙率,且已将细胞完全洗脱。由于已将在移植中产生抗原性的最重要成分——细胞洗脱,余下的脱细胞基质不存在MHC抗原。后者所诱发的免疫反应为类Th-2反应,与异种移植诱发的典型Th-1反应不同。而这种反应对植入的脱细胞基质并无排斥作用,表明了脱细胞基质具有种属差异性小、生物相容性佳等优点。经本研究建立的方法所制备的脱细胞基质,在肉眼观察下呈均一白色半透明,我们通过扫描电镜观察检测,从而说明其具有良好的微观孔隙结构以利于细胞生长,且已将细胞完全洗脱。3.检测鼠肾脱细胞基质的细胞相容性；应用L929细胞检测鼠肾脱细胞基质的细胞相容性；探讨灌注法制备的肾脱细胞基质作为细胞支架,构建泌尿系实质性组织工程器官的可行性。研究方法1.大鼠全肾脏脱细胞基质的制备。取12周龄的Whistar大鼠共20只,10%水合氯醛腹腔注射麻醉下完整切除两侧肾脏,分别保留输尿管、肾静脉及肾动脉。每对肾脏随机取1只作对照组,另1只作实验组。所有肾脏均沿肾动脉插入留置针建立灌注通道。实验组以肝素化PBS溶液轻推测试通道密闭性完整后,依次灌注肝素化PBS溶液,1%十二烷基磺酸钠(SDS)溶液,去离子水,1％TritonX-100溶液以及含青链霉素的PBS溶液。调整灌注压强及灌注时间,经修正获得最佳方案。2.采用扫描电镜观察支架微观结构；经2.5%戊二醛灌注固定,梯度法丙酮脱水、乙酸异戊酯置换、临界点法干燥、真空喷镀后扫描电镜下观察标本。3.检测鼠肾脱细胞基质与L929细胞(鼠成纤维细胞)的细胞相容性；(1)培养L929细胞作为种子细胞,设立空白组(无细胞)、阴性对照组(培养基)和实验组(鼠肾脱细胞基质浸提液)及阳性对照组(含0.64%苯酚溶液的培养基)。取对数生长期的L929细胞,4×103/孔接种于96孔板中,每组各5孔,于接种24,72,120 h,通过MTT法显色,于酶标仪490nm波长下检测吸光度值,计算相对增殖率。统计方法：实验结果以平均值士标准差表示((?)±s),应用SPSS13.0统计软件,对第24、72和120小时三个时间各组实验结果进行重复测量的方差分析(a=0.05)。(2)设立对照组(培养基)、实验组(鼠肾脱细胞基质浸提液)及阳性对照组(含0.64%苯酚溶液的培养基),培养48 h后应用流式细胞技术检测细胞凋亡情况。统计方法：实验结果以平均值士标准差表示(i±s),采用SPSS13.0统计软件包分析数据,统计学分析采用单因素方差分析(a=0.05)研究结果1.大鼠全肾脏脱细胞基质的制备及灌注过程。共对20只大鼠完成实验。开始灌注液流速慢,约10~40滴/min,随后逐渐加快。更换1%SDS后肾颜色从外至内分段逐渐变白色半透明,120~150min颜色变化逐渐明显,基本可在肉眼下看清肾内分支状管脉结构,直至约180~240min肾完全呈均一白色半透明状,灌注液滴速保持在100~120滴/min,此时测量灌注液总使用量约400~600ml。2.大鼠全肾脏脱细胞基质的微观检测结果。扫描电镜观察结果：对照组可见肾小球及近曲小管等结构完整；实验组可见肾小体、肾小管周围的基膜及胶原蛋白等细胞外基质形成网状结构,其余空间均为形状规则的孔隙,内无细胞。3.大鼠全肾脏脱细胞支架细胞相容性实验结果。(1)第24、72和120小时实验组吸光度值分别为0.620±0.032、0.957±0.007和1.336±0.011,阴性对照吸光度值分别为0.602±0.017、0.965±0.005和1.347±0.006,与阴性对照组比较无统计学差异(P=0.902>0.05)。L929细胞在实验组第24,72,120小时相对增殖率分别为102.99%,99.17%,99.18%,脱细胞基质支架在各个时间段(24,72,120小时)的细胞毒性评分为(0—1级),符合生物材料的医用标准,说明灌注法制备的大鼠肾脱细胞基质无明显细胞毒性,对细胞增殖无影响。而阳性对照组的细胞毒性评分为3级或4级。根据该实验数据绘制细胞生长曲线可看出实验组和对照组两条生长曲线几乎平行,说明实验组和阴性对照组对细胞生长的影响无显著差别。(2)应用L929细胞与大鼠肾脱细胞基质浸提液共培养48 h,进行流式细胞仪检测,与阴性对照组比较,正常细胞的比例差异无显著性意义,说明灌注法制备的大鼠肾脱细胞基质支架对细胞的凋亡无显著影响。研究结论灌注法用于制备全肾脏脱细胞基质支架,简便可靠,该支架无细胞成分残留,它具有完整的物理结构,提供良好的三维环境以利于细胞生长,种属差异性小、不易引起排斥反应、生物相容性好,是一种理想的细胞支架。更多还原

【Abstract】 Since kidney is one of the vital organs of human body,renal diseases have severe effect on patients’health and quality of life.Researchers explored many methods for the treatment of renal diseases.In them,dialysis and renal allotransplantation are most effective to replace kidney function of end-stage renal disease as well as the acute renal failure.However,dialysis replaces only a small fraction of normal kidney function. Whereas,renal allotransplantation is restrained by donor shortage,allograft failure,and long-term immunosuppression.Therefore,it is urgent to explore new methods to give a supplement to current methods.The emergence of tissue engineering brings new hopes for the functional and biological replacement of end-stage renal disease as well as acute renal failure.There are many tissues having been engineered until now.Tissue engineering is a rising subject in recent years, which applies cells, engineering material methods, and creates suitable biochemical and physio-chemical factors to improve or replace biological functions of tissues. It is the study direction of treating function failure and decline of human tissues and organs. The basic theory and method is culturing cells to a certain amount in vitro, after which the cells are inoculated to a scaffold with certain space structure, to construct a cell-scaffold complex that is later transplanted in vivo to repair or replace injured tissue. In recent years, rapid development can be seen in tissue engineering, especially in the field of the urologic system. There are reports of the emergence of tissue engineered organ corresponding to various tissues, such as bones, cartilages, blood vessels and bladders. Oversea scholars even have put tissue engineered bladder into clinical experiments already, which become the first artificial tissue engineered organ implanted in human body. This not only established an example for numerous researchers, but also unsealed a wider unknown domain:Can all human tissues have artificially-created organs through approaches of tissue engineering? Up to the end of this research, no success in construction of tissue engineered kidney in vitro is found domestically or overseas. Emphasis of researches has been put on how to grow renal cells in vitro. There is little mention of suitable cellular scaffold. Our department produced a whole-kidney acellular matrix (ACM) scaffolds in rats by perfusion.At present,there is little related report about it. The cellular biocompatibility of the ACM is poorly understood.Objective1. To explore and discuss the conditions and approaches of preparation of whole-kidney acellular matrix (ACM) scaffold in rats by perfusion.After comparison of various cellular scaffolds, ACM is found a favorable supporting scaffold. It possesses the macro and micro structure of original tissues, and it is rich in collagen, elastic fibers. Moreover, it contains fibrillin, fibronectin, laminin, protein polysaccharides, hyaluronic acid and chondroitin sulfate, etc. After decellularization, the remaining extracellular matrix components have good bio-compatibility. Respectively, every component carries or receives different signals and attracts different cells, and therefore has significant influences on the adherence, migration and transfer of cells. At present, the method of preparing ACM is basically limited to soaking and eluting cells with enzyme and eradicator, which normally can not achieve decellularization for parenchymal organs. Using eradicator as the dissolvent for elution, the research introduces perfusion as the method to elute the internal renal cells via natural platforms including blood vessels, renal glomerulus and renal tubules. This study also involves exploring and discussing the approaches of preparation, and finally the establishment of a systematic method.2. To observe and prove the prepared ACM has a regular three-dimensional space structure and good porosity, and cells are completely eluted.As cell, the most important component to engender antigenicity during transplants, is eluted, no MHC antigen exists in the remaining ACM. The immunoreaction ACM triggers is Th-2 reaction. Different from the typical Th-1 reaction triggered by heterotransplantation, this reaction has no rejection to the implanted ACM, which shows that ACM contains merits including little otherness between species and excellent bio-compatibility. The ACM prepared with the methods established by the research is evenly white and semitransparent under macroscopic observation. It is examed by scanning electron microscope,,to prove ACM has favorable micro hole structure that facilitates cell growth and complete decellularization.3. Evaluate the cytocompatibility of ACMEvaluate the cytocompatibility of ACM with the L929 cells invitro to assess the possibility of ACM as the cytoskeleton and tissue-engineered urinary organ construction.Methods1. Preparation of whole-kidney ACM in ratsBoth kidneys from twenty 12-week old whistar rats were removed under anesthesia at abdomens with 10% chloral hydrate, and the ureters, venae renales and renal arteries were kept. For every couple of kidneys, one is randomly put in the control group while the other in the experimental group. Intravenous catheters were inserted through renal arteries for all kidneys to construct channels for perfusion. After a slight push of heparinized PBS solution to test the obturation of the channels, the experimental group was perfused in order with heparinized PBS solution,1% sodium dodecylsulphate (SDS) solution, deionized water,1% TritonX-100 solution and PBS solution that contains mycillin. The best approach was achieved after adjustments of the pressure and time of perfusion.2. The micro observation of whold-kidney ACM in ratsThe whold-kidney ACM was observed under scanning electron microscope after being fixed with 2.5% glutaraldehyde, dehydrated with acetone using the gradient method, replaced by isoamyl acetate, dried with critical point method, and finally sprayed in vacuum.3. Cytocompatibility of whole-kidney ACM in rats with L929 cells in vitor(1)Samples were randomly divided into blank group(without any cells),negative control group(culture media), experimental group(rat kidney ACM leaching liquor),and positive control group(culture media containing 0.64%phenol).L929 cells in the logarithmic phase were seeded in 96-well plates at the density of 4×103/well,with 5 wells in each group.At 24,72,and120 hours after incubation,cells were stained with MTT method to detect absorbance at 490 nm and calculate relative growth rate.(2)Control group(culture medium),experimental group(rat kidney ACM leaching liquor),and positive control group(culture media containing 0.64% phenol)were set up to detect cell apoptosis at 48 hours after culture using flow cytometry.Results1. The process of perfusion for preparing whole-kidney ACM in rats Twenty rats were used in the experiment. During the process, the speed of perfusion was slow at the beginning, about 10 to 40 drops per minute, and it increased gradually afterwards. After the solution was changed to 1% SDS, the color of kidneys gradually changed to white and semitransparent from external to internal, and it was segmented and lobate. After 120 to 150 minutes, the change of color was obvious, and the branching and canal structure was seen clearly with unaided eyes. When it reached 180 to 240 minutes, the kidney was evenly white and semitransparent. The speed of perfusion was kept at 100 to 120 drops per minute. The use of solution was measured to be 400 to 600 ml at this time.2. Results of micro observation of whole-kidney ACM in ratsUnder the scanning electron microscope, renal glomerulus and proximal convoluted tubule was found to have complete structure in the control group. As for the experimental group, a reticulate structure was found that was formed by basilar membranes around renal glomerulus and tubules and extra cellular matrix including collagen protein. All other spaces were regular-shaped, cell-free holes.3. Results of the cellular biocompatibility of whole-kidney ACM in rats(1)The actual absorption coefficient of experimental groups was0.620± 0.032、0.957±0.007 and 1.336±0.011,after24,72 and 120 hours.The actual absorption coefficient of negative control group was 0.602±0.017、0.965±0.005 and 1.347±0.006.There was no significant difference between the experimental group and negative control((P=0.902>0.05)). The grade of the cytotoxicity of the whole-kidney ACM in rats was 0-1 at every time intervals(24,72,and 120 hours).(2) At 48 hours after incubation.There was no significant difference in cell apoptotic rate between experimental group and negative control group(P>0.05).ConclusionsPerfusion is a simple and reliable approach for preparation of whole-kidney ACM scaffold. This scaffold is ideal as it has no cell residue, possesses complete physical structure, provides nice three-dimensional environment in favor of the growing of cells. Moreover, it is bio-compatible, not easy to induce rejection, and it has little otherness between species.更多还原