膀胱移行上皮细胞种植改性脱细胞小肠粘膜下基质在家兔尿道重建中的应用

发布时间：2018-04-28 23:12

本文选题：过氧乙酸 + 小肠粘膜下基质　；参考：《华中科技大学》2016年博士论文

【摘要】：尿道缺损是泌尿外科的最常见的疾病之一,可发生于先天性疾病如尿道下裂、尿道上裂等疾病,也可以发生于尿道损伤、感染,常伴有损伤后的尿道纤维化瘢痕狭窄。短段的尿道狭窄也可以通过尿道扩张、尿道狭窄切开及尿道狭窄段切除加端端吻合术予以治疗。然而,长段的尿道狭窄或者尿道缺损,需要进行移植物进行修补。目前,临床上常用的移植物包括舌粘膜、颊粘膜及阴茎皮肤。然而,口腔粘膜的取材会带来一定的并发症,如张口困难、感染、溃疡、麻木、疼痛等。另外,长段的尿道狭窄术后易复发,对于该类患者,往往面临取材来源不够。因此,需要寻求更广泛来源的尿道替代移植物。组织工程技术的迅速发展,为尿道重建提供了新的治疗途径。然而,Chapple等最近认为在组织工程技术大规模的应用于临床之前应该继续研究以寻找出更加合适的组织工程材料。目前,报道较多组织工程材料的主要包括化学合成材料、天然生物基质类材料、天然生物材料的提取物,以及不同类材料的聚合物。其中,小肠粘膜下基质(SIS)属天然生物基质类材料,具有天然的细胞与组织兼容性,且含括胶原、纤维连接蛋白、GAG及生长因子和信号蛋白等,能促进细胞生长与迁移。但其结构致密,不利于营养液的渗透与代谢物质的交换。另外,该类基质常规脱细胞后仍具有少量的异体细胞核成分的残余,会引起宿主的慢性炎症反应。理想的组织工程支架材料应该具有三维(3D)多孔结构及最少的异体细胞核成份的残余。本实验通过过氧乙酸氧化SIS制作出具有3D多孔结构的改性SIS,同时也最大限度地去除了异体的细胞核成分残余。我们进一步评价膀胱移行细胞种植这种经过氧乙酸(Peracetic acid, PAA)改性的小肠粘膜下基质(SIS)在家兔尿道重建中的应用效果。本研究共分以下三部分：第一部分：PAA改性脱细胞SIS基质的制备目的：制作出3D多孔的SIS支架材料,并评估其表征、力学性能方法：取新鲜的猪小肠组织,机械方法游离出粘膜下层及浆肌层,去除上皮层,分离出小肠粘膜下基质,置入蒸馏水预脱细胞处理,PAA改性SIS组加5%过氧乙酸(PAA),而无PAA改性组加PBS液,然后两组均置入1%Triton X-100。最后75%乙醇消毒,无菌纯净水保存备用。取材行HE、MASSON染色、扫描电镜观察。应用密度瓶法测定两组基质材料的空隙率。取材大小5mmx60mm,使用万能试验机测定其力学性能,包括最大拉长、最大强度及杨模量大小。统计分析两组SIS材料在形态学和力学性能间的差异。结果：未经PAA处理的SIS结构致密,且有少量细胞成分残余,而经PAA处理的SIS呈3D多孔状,几乎没有细胞核成分残余。SIS的上皮面电镜结果显示经5%PAA处理SIS表面孔径明显增加,经过测量分析后,脱细胞新鲜SIS的孔径值为1.75±0.32μm,而经过PAA氧化处理后的SIS孔径值为5.16±1.831μm,无PAA处理的SIS空隙率为18.5±2.6%,而经过PAA处理后的SIS空隙率66.8±3.9%,两者差异均具有显著统计学意义(P0.01)。经万能拉伸仪测定,脱细胞无PAA处理SIS及经PAA处理的SIS的最大拉伸率分别为16.3±1.6%和15.0±1.2%,最大拉伸强度分别为35.1±7.2 Mpa和24.3±5.6 Mpa,杨氏模量分别为150.4±39.2Mp和118.5±28.4Mpa.结论：经PAA改性的后的SIS具有3D多孔状结构,几乎无异体细胞核成分残余,且仍能维持一定的机械性能,可以作为理想的组织工程材料。第二部分：组织工程膀胱移行上皮细胞-SIS复合物的体外构建目的：体外构建膀胱移行上皮细胞-改性SIS复合物,观察其形态、评估改性SIS的细胞兼容性。方法：取成年新西兰大白兔膀胱壁0.6x0.6 cm,置入PBS液漂洗。将膀胱组织块置入含有DMEM培养基的pronase E蛋白酶中,消化过夜,小心刮取膀胱粘膜层移行上皮细胞。加含有5%胎牛血清、1%青霉素-链霉素的ECM培养基培养,0.05%胰酶传代。制作细胞爬片,应用抗AE1/AE3抗体进行细胞免疫组织化学鉴定。制作PAA改性SIS支架材料和非PAA改性SIS支架材料浸提液,行MTT实验记录两组细胞生长的OD值,了解两组支架材料的细胞兼容性。收集细胞并种植于预先制好的PAA改性SIS支架材料和非PAA改性的SIS支架材料上,继续培养至2周,取材行HE染色、电镜观察。利用RT-PCR技术和Western-blot技术检测Uroplakin基因在UC-经PAA改性SIS复合物的表达变化。结果：膀胱移行上皮细胞在无PAA处理SIS及5%PAA处理后SIS浸提液中生长状态良好,且均优于单纯ECM培养基的趋势,但是差异均无统计学意义(p=0.35)。无PAA处理SIS组细胞生长以单层结构为常见,而在5%PAA处理后SIS组中,细胞生长连接成片,形成2-3层的复层结构。电镜下,无PAA处理SIS上细胞呈多角形,扁平状,伸出伪足,紧密贴附于支架材料上。而在5%PAA处理SIS支架上,细胞分布更加紧密,相互融合呈多层面三维立体结构,表层细胞多呈圆形、椭圆形及立方形。经PAA改性SIS-UC复合物较无PAA改性-UC复合物高表达Uroplakin基因。结论：采用酶消化法联合机械的刮擦法能分离培养具有活性的膀胱移行上皮细胞,阳性表达AE1/AE3。未经PAA处理的脱细胞SIS与经5%PAA改性处理SIS支架材料与膀胱移行上皮细胞均具有良好的细胞兼容性。经5%PAA改性处理SIS能更好的促进细胞在支架材料上生长并形成复层结构,且能促进膀胱移行上皮向终末分化。第三部分：组织工程膀胱移行上皮-SIS复合物修补家兔尿道缺损模型的建立与重建效果观察目的：观察组织工程膀胱移行上皮-SIS复合物修补家兔尿道缺损的效果方法：健康成年新西兰家兔18只,分成三组,膀胱移行上皮细胞种植PAA改性的SIS支架组、膀胱移行细胞种植无PAA改性的SIS材料组,无膀胱上皮细胞种植的经PAA改性SIS组,每组各6只。按照第二部分的方法,构建组织工程膀胱上皮细胞-SIS复合物。再次麻醉家兔,游离腹侧尿道海绵体至粘膜层,建立家兔尿道粘膜缺损模型1.5×0.8cm2,修剪支架材料大小为1.7×1cm2,以补片的方式进行修补。术后抗感染、尿管冲洗,手术后半年行尿道造影检查,大体病理观察及HE、MASSON、免疫组化检测。结果：上皮细胞-5% PAA改性SIS复合物组均存活,尿道通畅,再生尿道粘膜完全再生。病理见复层移行上皮、上皮下平滑肌增生,血管增生。而上皮细胞-无PAA改性SIS复合物组,有尿瘘发生,再生尿道粘膜不完整。病理见上皮不规则,及慢性炎症细胞浸润。单纯PAA改性SIS组有尿道狭窄,尿道粘膜苍白、僵硬、挛缩,病理提示大量纤维结缔组织增生,平滑肌和血管少见。上皮细胞-PAA改性SIS组的上皮及平滑肌表达定量显著优于上皮细胞-无PAA改性组(P0.05)。平滑肌及血管均优于单纯PAA改性SIS组(P0.05)结论：膀胱移行上皮细胞种植PAA改性SIS是进行长段尿道缺损修补的理想替代物。
[Abstract]:Urethral defect is one of the most common diseases in the Department of urology. It can occur in congenital diseases such as hypospadias, hypospadias and other diseases. It can also occur in urethral injury, infection, and urethral fibrosis scar stricture after injury. Short segment urethral stricture can also be treated with urethral dilatation, urethral stricture incision and urethral stricture excision. However, long segment urethral strictures or urethral defects need to be repaired by graft. Currently, the commonly used graft includes the mucosa of the tongue, the buccal mucosa, and the penis skin. However, the selection of the oral mucosa will bring some complications, such as oral difficulty, infection, ulcers, numbness, pain and so on. Long segment urethral stricture is easy to relapse. For this type of patients, it is often faced with insufficient sources of material. Therefore, a more extensive source of urethral replacement graft is needed. The rapid development of tissue engineering technology provides a new way of treatment for urethral reconstruction. However, Chapple and so on are most recently considered to be applied in a large scale in tissue engineering. The bed should continue to be studied to find more appropriate tissue engineering materials. At present, most of the reports of tissue engineering materials include chemical synthetic materials, natural biomaterials, extracts of natural biomaterials, and polymers of different materials. Among them, the submucosal matrix of the small intestine (SIS) is a natural biomaterial. It has natural cell and tissue compatibility, including collagen, fibronectin, GAG and growth factors and signal proteins, which can promote cell growth and migration. But the structure is dense and is not conducive to the infiltration of nutrient solution and the exchange of metabolic substances. In addition, this type of matrix often has a small number of allogenic cell nuclear components. The residual, cause the chronic inflammatory response of the host. The ideal tissue engineering scaffold material should have a three-dimensional (3D) porous structure and the least residual cell nuclear components. In this experiment, the modified SIS with 3D porous structure was produced by oxidation of peroxy acetic acid, and the residual cell nuclear components were removed to the maximum extent. We further evaluate the effect of Peracetic acid (PAA) modified small intestinal submucosal matrix (SIS) in the reconstruction of urethra in rabbits. This study is divided into three parts: Part I: preparation of PAA modified decellular SIS matrix: the preparation of 3D porous SIS scaffold materials, and evaluation of SIS Its characterization and mechanical properties: taking fresh pig small intestine tissue, removing the submucosa and muscularis layer mechanically, removing the epithelial layer, separating the submucous matrix of the small intestine, placing the distilled water preconditioning, the PAA modified SIS group and 5% peracetic acid (PAA), without the PAA modified group and PBS solution, and then placing the two groups in the final 75% of the X-100. 75%. HE, MASSON dyeing and scanning electron microscopy were used to determine the void ratio of two groups of matrix materials by density bottle method. The mechanical properties of the material were measured by the universal test machine, including the maximum elongating, maximum strength and young modulus. The morphology and force of the two groups of SIS materials were statistically analyzed. The results showed that the structure of SIS without PAA treatment was compact, and there was a small amount of residual cell components, while SIS treated with PAA was 3D multi pore, and there was almost no residual.SIS in the upper surface of the cell nucleus with electron microscopy. The pore size of SIS was obviously increased by 5%PAA treatment, and the pore size of the fresh SIS was 1.7 after measurement and analysis. 5 + 0.32 mu m, and after PAA oxidation, the SIS aperture value is 5.16 + 1.831 mu m, the SIS void ratio without PAA treatment is 18.5 + 2.6%, and the SIS void ratio after PAA treatment is 66.8 + 3.9%, and the difference is statistically significant (P0.01). The maximum tensile strength is 16.3 + 1.6% and 15 + 1.2% respectively, the maximum tensile strength is 35.1 + 7.2 Mpa and 24.3 + 5.6 Mpa respectively. The young's modulus is 150.4 + 39.2Mp and 118.5 + 28.4Mpa., respectively. After PAA modification, the SIS has 3D porous structure, and there is almost no residual cell nuclear composition, and still can maintain a certain mechanical properties, which can be used as an ideal group. Fabric engineering material. Second part: Construction of tissue engineering bladder transitional epithelial cell -SIS complex in vitro purpose: construct bladder transitional epithelial cell - modified SIS complex in vitro, observe its morphology, evaluate the cell compatibility of modified SIS. Methods: take 0.6x0.6 cm of adult New Zealand white rabbit bladder wall, put into the PBS solution and rinse the bladder group. The fabric was inserted into the pronase E protease containing the DMEM medium, digested for the night and carefully scraped the bladder mucosa layer to move the epithelial cells. The ECM medium containing 5% fetal bovine serum, 1% penicillin - streptomycin, 0.05% trypsin passages, and the cell climbing tablets were made, and the anti AE1/AE3 antibody was applied to the cellular immuno histochemical identification. The PAA modified SIS branch was made. The scaffold material and the non PAA modified SIS scaffold were extracted, and the MTT experiment was used to record the OD values of two groups of cell growth. The cell compatibility of the two groups of scaffolds was understood. The cells were collected and cultivated on the pre made PAA modified SIS scaffold material and the non PAA modified SIS scaffold material, and continued to be cultured for 2 weeks. R and Western-blot techniques were used to detect the changes in the expression of Uroplakin gene in UC- via PAA modified SIS complex. Results: the growth state of the bladder transitional epithelial cells in the SIS extract without PAA treatment SIS and 5%PAA was better than that of the pure ECM medium, but the difference was not statistically significant (p=0.35). Cell growth is common in single layer structure, and in SIS group after 5%PAA treatment, cell growth is connected to pieces to form 2-3 layers of complex layer structure. Under electron microscope, the cells on SIS without PAA treatment are polygonal, flat, protruding and attached to the scaffold material. On the 5%PAA treatment SIS scaffold, the cells are more closely distributed and multilayered with each other. The surface cells are mostly round, oval and cubic. The PAA modified SIS-UC complex is more expressed than the Uroplakin gene without the PAA modified -UC complex. Conclusion: the enzyme digestion method combined with mechanical scraping can separate and culture the active cell cell of the bladder transitional epithelium, and the positive expression of AE1/AE3. has not been refined by PAA treatment. Cell SIS and 5%PAA modified SIS scaffold materials have good cellular compatibility with bladder transitional epithelial cells. 5%PAA modified SIS can better promote cell growth on scaffold material and form complex layer structure, and can promote the differentiation of bladder transitional epithelium to terminal differentiation. Third parts: tissue engineered bladder transitional epithelium -SIS compound Objective: To observe the effect of reconstruction and reconstruction of rabbit urethral defect model for repairing the rabbit urethra defect: observe the effect of tissue engineering bladder transitional epithelium -SIS complex to repair rabbit urethral defect: 18 healthy adult New Zealand rabbits were divided into three groups, the bladder transitional epithelial cells were planted with PAA modified SIS stents, and the implantation of bladder transitional cells was not modified by PAA. In the sex SIS group, no bladder epithelial cells were implanted in the PAA modified SIS group, each group was 6. The -SIS complex of the tissue engineered bladder epithelial cells was constructed according to the second part method. The rabbits were again anesthetized, free ventral urethral cavernous body to mucous layer, and the rabbit urethral mucosa defect model was set up 1.5 x 0.8cm2, the size of the pruning scaffold material was 1.7 X. 1cm2, repair with patch. After operation, anti infection, urethral rinse, half a year after the operation, urethrography examination, gross pathological observation and HE, MASSON, immunohistochemical detection. Results: epithelial cells -5% PAA modified SIS complex group survived, urethral smooth, regenerated urethral mucous membrane completely regenerated. Pathological findings of the epithelium, epithelial smooth epithelium, smooth epithelial smooth Muscle hyperplasia, vascular hyperplasia, and epithelial cells - no PAA modified SIS complex group, the occurrence of urinary fistula, regenerative urethral mucosa incomplete. Pathological epithelial irregular, and chronic inflammatory cell infiltration. Simple PAA modified SIS group with urethral stricture, urethral mucosa pale, rigid, contracture, a large number of fibrous connective tissue hyperplasia, smooth muscle, and less blood vessels The expression of epithelial and smooth muscle in the -PAA modified SIS group was significantly better than that of the epithelial cell without the PAA modified group (P0.05). The smooth muscle and blood vessels were superior to those of the PAA modified SIS group (P0.05). The implantation of PAA modified SIS in the transitional cell of the bladder was an ideal substitute for the repair of long segment urethral defects.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R699.6

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