【作者】 袁铭；

【导师】 李汉忠；

【作者基本信息】 中国协和医科大学 ， 泌尿外科， 2007， 博士

【摘要】 第一部分：猪膀胱无细胞基质生物支架材料的制备及其相关生物学特性研究【目的】通过检测猪膀胱无细胞基质(PBAM)的物理和生物学特性及其有关组成成分的含量，探讨PBAM作为膀胱替代生物支架材料或组织工程用支架材料的可行性。【方法】采用低渗-去污剂洗涤-核酸酶消化脱细胞方法去除猪膀胱中的细胞成分，制备成膀胱无细胞基质，通过H-E染色及Massons染色光镜检查观察去细胞效果；分子生物学方法测定PBAM中DNA含量；采用ELISA方法测定PBAM中转化生长因子β1(TGF-β1)的含量；应用万能电子拉伸机进行PBAM生物力学测定。【结果】经脱细胞处理后，光镜观察无细胞残留，基质三维网状结构完整，但PBAM中DNA的含量仅减少至2％～5％，后经进一步重复双核酸酶消化及加强换液次数后，DNA的含量均低于1％。PBAM中仍含有一定量的生长因子TGF-β1；生物力学性能基本稳定，仍有一定的弹性和韧度。【结论】1、我们所制备的PBAM物理性状及生物力学性能稳定。2、国内外首次采用ELISA方法检测到PBAM中仍含有一定量的生长因子TGF-β1，表明其在组织工程膀胱的构建中具有潜在的积极作用。3、我们所制备的PBAM具有无菌、无毒和良好的组织相容性，能长期储存，可以作为“现成品”用于组织工程、器官替代及修补用生物支架材料的应用研究。第二部分：猪膀胱无细胞基质在异种移植中的免疫原性及生物安全性研究【目的】进一步对我们所制备的猪膀胱无细胞基质移植物进行免疫原性及生物安全性检测。【方法】通过采用免疫组化方法检测此去细胞过程对猪膀胱中结构细胞和内皮细胞表达的主要组织相容性复合体-Ⅰ(Major histocompatibility complex，MHC-Ⅰ)抗原和α-半乳糖基抗原(α-galactosyl residues，α-Gal)表达情况的影响；通过分子生物学方法观察此去细胞过程对猪膀胱中的PERV的影响，以及把PBAM作为支架材料进行犬膀胱替代修复实验，观察异种移植前后PERV的表达情况。【结果】新鲜猪膀胱有猪α-Gal和MHC-I抗原的高表达，经脱细胞处理后，PBAM中α-Gal和MHC-I抗原的活性表达均明显降低或基本无表达；新鲜猪膀胱基因组内存在PERV基因序列，经去细胞处理所制备的PBAM未发现有PERV基因序列。应用PBAM进行异种移植或者说作为支架材料进行异种膀胱替代没有发现活性PERV传染的证据。【结论】1、新鲜猪膀胱存在有与异种移植免疫排斥反应直接有关的主要抗原α-Gal和MHC-I，PBAM中α-Gal和MHC-I抗原的活性表达均明显降低或基本无表达。2、新鲜猪膀胱基因组内存在PERV基因序列，PBAM中无PERV基因序列。3、应用PBAM进行异种移植或作为支架材料制备组织工程膀胱进行异种膀胱替代可能具有良好的免疫耐受性及可靠的生物安全性。第三部分：猪膀胱无细胞基质在异种动物犬体内进行膀胱修补替代性实验研究【目的】观察PBAM在异种动物体内的生物相容性、生物降解情况及生物安全性问题，同时观察单纯应用PBAM进行膀胱替代修补实验是否可行，修补面积大小对膀胱再生是否有影响，为下一步构建组织工程膀胱及尿路组织缺损的修复和替代提供实验依据。【方法】杂种犬分四组，A组为对照组，切除50％膀胱后原位直接缝合，B组、C组和D组为实验组，分别切除50％膀胱后用用PBAM替代修补(修补面积约为原膀胱的30％、40％、50％，PBAM大小分别约为16cm~2、30cm~2、64cm~2)。术前及术后查血常规、电解质及血肌酐情况。术前及PBAM移植后1个月检测PERV。术前及术后12周行膀胱测压及膀胱顺应性的检查，并行腹部平片及膀胱造影检查。术后12周及24周每组分别处死一条犬进行大体标本及病理学检查。【结果】有8条犬安全度过围手术期，术后血常规及肾功能检查对照组与实验组无差别；其中D组两条犬分别在术后28天和34天时死亡，尸体解剖死亡原因均为修补中心处膀胱穿孔、漏尿、腹腔内感染所致。术后12周时，A组、B组和C组膀胱容量分别恢复到术前63％，85％，89％，膀胱顺应性分别恢复到原来的67％，88％，87％。腹平片均未见有明显膀胱结石形成；12周时的膀胱标本很难找到支架与膀胱吻合处，支架明显萎缩，面积减小至75-85％，已经被新生的膀胱组织取代，但壁较薄，已修复如正常的膀胱外观。组织学检查显示基质支架替代部分膀胱区域基本形成多层移行上皮细胞，与正常的泌尿上皮细胞差异不大，有少量白细胞和淋巴细胞浸润，可见散在平滑肌肌束。24周的标本，已经找不到支架，支架已经被完全吸收，支架内表面光滑，有肉眼可见的血管网，外表面触之较粗糙。组织学检查显示膀胱各层组织基本完全再生，基本完成膀胱组织替代重建。对照组的标本观察符合切口愈合过程。在12周时分别处死的B组和C组1条犬的膀胱标本修补处膀胱发现有泥沙样颗粒附着，24周时所有膀胱标本均未见有结石形成。【结论】1、PBAM作为生物支架材料生物相容性良好，支架材料可完全吸收，并可基本完成膀胱组织替代重建。2、PBAM异种移植生物安全性良好。3、初步大动物实验证实所制备PBAM可用于膀胱的修补替代治疗，为进一步实验及临床应用奠定了基础。4、较大面积的PBAM用于膀胱替代治疗可能会出现膀胱穿孔、支架材料萎缩及微结石形成等并发症，期待构建组织工程膀胱用于实验及临床应用研究。更多还原

【Abstract】 PartⅠ: Preparation of porcine bladder acellular matrix and research on its relative biocharacteristics【Objective】To investigate the feasibility of the porcine bladder acellular matrix as biomaterial scaffold for bladder substitution or for tissue engineering application by detecting the physics and biology characteristics and other components.【Methods】The method of hypotension and detergent and nucleic acid enzymatic digestion extraction process has been developed to remove all the cellular components from the porcine bladder to prepare the bladder acelluar matrix. The PBAM was then examined by H-E and Massons staining to confirm no cell elements remained. The methods of the molecular biology and enzyme linked immunosorbent assay (ELISA) were used to determine the contents of the DNA and Transforming Growth Factor-β1 (TGF-β1) respectively in PBAM. The biomechanics of the PBAM were testing by omnipotent electronic material testing machine.【Results】There were no cell elements remained for the PBAM under the examination of optical microscope. The matrix of this scaffold material still have complete three diamensions network structure. The contents of the DNA in PBAM only reduced from 2% to 5% after the initial decellular method. But through the repetition of the double nucleic acid enzymatic digestion and strengthen for the water exchange, the DNA contents were all less than 1%. There were still some growth factor such asTGF-β1 in PBAM. It had stable biomechanics characteristic and had good elasticity and toughness.【Conclusion】The PBAM had good physical rationality and stable biomechanics characteristic. The growth factor for the TGF-β1 in PBAM was first discovered by the method of the ELISA at home and abroad. The discovery of the TGF-β1 in PBAM indicate that the TGF-β1 may have a latent active effects for the construction of the tissue engineering bladder. The PBAM which we made has the character of sterility and asepsis and good biocompatibility and can be stored for long time, so it can be used as ready-made product bioscaffold materials for the tissue engineering and organ substitute or mending.PartⅡ: The immunogenicity and biologic safety in xenotransplantation with porcine bladder aceHular matrix【Objective】To observe the immunogenicity and biologic safety of the bladder acellular matrix graft as biomaterial scaffold for tissue engineering application in exnotransplantation.【Methods】The antigen of Major histocompatibility complex andα-galactosyl residues in the porcine structure cell and endothelial cell were detected by using the method of the immunohistochemistry to observe the effect on this decellularization method. The molecular biology method were used to observe the influence for the PERV in porcine bladder with this decellularization method. The expression of the PERV in canine with bladder substitution for PBAM were still detected.【Results】There are high expressions for the antigens of theα-Gal and MHC-Ⅰin fresh porcine bladder. But the expression of theα-Gal and MHC-Ⅰcut down significantly or nothing after the decellularization course. The gene order of the PERV exist in the fresh porcine bladder genome, but could not found in the PBAM. There were no proof for the alive PERV in exnotransplantation with PBAM.【Conclusion】The major antigen of theα-Gal and MHC-Ⅰdirectly related to the immunological rejection for the xenotransplantation exist in the fresh porcine bladder, but their expression activity cut down significantly or nothing after the decellularization course. There were no expression of the PERV in PBAM. The PBAM have good immunotolerance and reliable biologic safety in xenotransplantation.PartⅢ: Bladder repair and replacement experiment with porcine bladder acellular matrix in vivo for xenogeneic animal canine【Objective】To observe the biocompatibility and biodegradation and biologic safety of the PBAM in different species animal body. We still want to observe the results of the PBAM as the bladder replacement experiment and the influences for bladder regenerate with mending areas. This results can be provided to the next construction the tissue engineering bladder and repair or replacement for the urinary defects.【Methods】hybrid canine were divided into 4 groups and marked with A, B, C and D. Group A with 50% bladder incised and sutured in site directly was control group. As experiment groups, group B, C and D were all incised 50% bladder and replaced using the PBAM respectively (their area was about 30%, 40%, 50% of the overall canine bladder). The blood routine, the electrolytes, the serum creatinine, PERV, cystometry and compliance, plain abdominal radiograph and cystography were detected before and after the operations. One canine in each group will be killed to observe the replacement specimen at 12 weeks and 24 weeks aider the operations.【Results】There were 8 canines living surround the operation stage. The Blood routine and the renal function before and after operations had no differences between the control group and the experimental group. 2 canies in group D died at 28 days and 34 days after the operation. The causes of the death were all induced by vesical perforation, leakage of urine and intra-abdominal infection. After 12 weeks, the vesical capacity regained to 63%, 85%, 89% of their preoperative capacity respectively, and the bladder compliance recovered to 67%, 88% and 87% from their native bladder respectively. Plain abdominal radiograph indicated no bladder stone formation. The bladder specimens couldn’t almost find the anastomosis of the scaffold and the native bladder. The scaffold shrinked significantly to 75% to 85%, but new generated bladder tissue with thin wall still could be found. Histology revealed multiplayer urothelial cells and scattered smooth muscle bundle on the replacement bladder. After 24 weeks, the replacement bladder with PBAM regenerated the bladder multiplayer tissue on the whole. After 12 weeks, one canine killed in group B and C could be found the sediment grains cohereing to the replaced bladder with PBAM, but no stone formation in bladder specimens after 24 weeks【Conclusion】As a biomaterial scaffold, PBAM has good biocompatibility and good biodegradable and good biologic safety in xenotransplantation. The initial big animal experiment proved that the PBAM can be used to mend and replace that bladder. But the larger area PBAM still can be induced complications with vesical perforation and scaffold shrink and micro stone formation.更多还原