自体尿源性干细胞结合3D多孔小肠粘膜下基质用于兔尿道重建的实验研究
发布时间：2018-12-18 00:41
【摘要】：目的:探索应用自体兔尿源干细胞细胞(rabbit urine-derived stem cells,rUSCs)复合小肠黏膜下基质(Small intestinal submucosa,SIS)构建兔组织工程尿道的可行性,为临床构建组织工程尿道,用于尿道下裂残疾、长段尿道缺损等复杂、疑难情况尿道重建奠定基础。方法:收集24只雄性新西兰大白兔尿液及膀胱冲洗液,分离、培养得到rUSC,检测其自我更新及体外多向分化(平滑肌和上皮方向)能力;用5%过氧乙酸(peracetic acid,PAA)处理SIS材料,对其微结构、免疫原性、生物相容性进行研究,并将PKH67标记的rUSCs与SIS材料复合;随机将24只雄性新西兰大白兔分为两组,并建立尿道缺损模型,缺损长度约为2.0cm,对照组应用单纯5%PAA处理后的SIS材料修复(12只),实验组应用5%PAA处理后并复合rUSCs的SIS材料修复(12只)。均于术后2周、3周、4周、12周行组织学和Western Blot检测,12周行尿道造影。结果:原代培养第4天即可在镜下观察到rUSC单克隆细胞,在第6天可形成克隆团,在第11天出现细胞融合,形态成“米粒”状,大小均一、排列紧凑;rUSCs经体外诱导分化后,能高效的分化为平滑肌细胞和上皮细胞。经5%PAA处理后,SIS材料表面变得疏松多孔,且DNA含量进一步降低(P0.05);组织学提示实验组平滑肌束再生在各个时间点均优于对照组(P0.05),且更有序;上皮层数和新生血管数量在早期(2、3、4周)好于对照组(P0.05),在晚期(12周)差异无统计学意义(P0.05);对照组基质出现炎性细胞浸润和纤维化;尿道造影显示实验组仅一只出现轻微尿道狭窄,而对照组三只均出现轻微尿道狭窄。免疫荧光显示rUSCs在体内可转化为平滑肌和上皮细胞。结论:1)rUSCs能够通过简单无创的方法获取,具有良好的自我更新能力,体外易于培养和扩增,并且具有分化成平滑肌细胞和上皮细胞的能力,是一种理想的组织工程种子细胞来源;2)SIS材料经5%PAA处理后,SIS支架材料孔径增大,孔隙率增高,且细胞成分进一步减少,可作为组织工程支架材料来源;3)复合rUSCs的3D多孔SIS材料能促进尿道组织再生,减少炎症反应、尿道狭窄等并发症。
[Abstract]:Objective: to explore the feasibility of using autologous rabbit urethral stem cells (rabbit urine-derived stem cells,rUSCs) combined with small intestinal submucosal matrix (Small intestinal submucosa,SIS) to construct tissue engineered urethra in rabbits. For hypospadias, long urethral defects, complex, difficult cases of urethral reconstruction laid the foundation. Methods: urine and bladder flushing fluid were collected from 24 male New Zealand white rabbits. RUSC, was obtained to detect the ability of self-renewal and differentiation (smooth muscle and epithelial direction) in vitro. SIS material was treated with 5% peracetic acid (peracetic acid,PAA), and its microstructure, immunogenicity and biocompatibility were studied. PKH67 labeled rUSCs was combined with SIS material. Twenty-four male New Zealand white rabbits were randomly divided into two groups, and the urethral defect model was established, the length of the defect was about 2.0 cm. The control group was repaired with SIS material treated with 5%PAA alone (12 rabbits). The experimental group was repaired with SIS material (12 rats) treated with 5%PAA and combined with rUSCs. Histology and Western Blot were detected at 2 weeks, 3 weeks, 4 weeks and 12 weeks after operation, and urethrography was performed at 12 weeks. Results: the rUSC monoclonal cells could be observed under microscope on the 4th day of primary culture, and the clones could be formed on the 6th day. On the 11th day, the cells were fused in the shape of "rice grain", uniform in size and compact in arrangement. RUSCs can effectively differentiate into smooth muscle cells and epithelial cells after differentiation in vitro. After treated with 5%PAA, the surface of SIS materials became loose and porous, and the content of DNA decreased further (P0.05). Histology showed that the regeneration of smooth muscle tract in the experimental group was better than that in the control group at all time points (P0.05), and the content of DNA in the experimental group was more orderly than that in the control group. The number of epithelial layer and the number of neovascularization in the early stage (2 ~ 3 weeks) was better than that in the control group (P0.05), but there was no significant difference in late (12 weeks) (P0.05), in the control group, inflammatory cell infiltration and fibrosis appeared in the matrix. Urethrography showed only one slight urethral stricture in the experimental group and slight urethral stricture in the control group. Immunofluorescence showed that rUSCs could be transformed into smooth muscle and epithelial cells in vivo. Conclusion: 1) rUSCs can be obtained by simple and non-invasive method. It has good self-renewal ability, easy to be cultured and expanded in vitro, and can differentiate into smooth muscle cells and epithelial cells. It is an ideal source of seed cells for tissue engineering. 2) after SIS was treated with 5%PAA, the pore size and porosity of SIS scaffold increased, and the cell composition decreased further, which could be used as the source of scaffold material for tissue engineering. 3) 3D porous SIS combined with rUSCs can promote urethral regeneration and reduce complications such as inflammation and urethral stricture.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R699
本文编号：2385025
[Abstract]:Objective: to explore the feasibility of using autologous rabbit urethral stem cells (rabbit urine-derived stem cells,rUSCs) combined with small intestinal submucosal matrix (Small intestinal submucosa,SIS) to construct tissue engineered urethra in rabbits. For hypospadias, long urethral defects, complex, difficult cases of urethral reconstruction laid the foundation. Methods: urine and bladder flushing fluid were collected from 24 male New Zealand white rabbits. RUSC, was obtained to detect the ability of self-renewal and differentiation (smooth muscle and epithelial direction) in vitro. SIS material was treated with 5% peracetic acid (peracetic acid,PAA), and its microstructure, immunogenicity and biocompatibility were studied. PKH67 labeled rUSCs was combined with SIS material. Twenty-four male New Zealand white rabbits were randomly divided into two groups, and the urethral defect model was established, the length of the defect was about 2.0 cm. The control group was repaired with SIS material treated with 5%PAA alone (12 rabbits). The experimental group was repaired with SIS material (12 rats) treated with 5%PAA and combined with rUSCs. Histology and Western Blot were detected at 2 weeks, 3 weeks, 4 weeks and 12 weeks after operation, and urethrography was performed at 12 weeks. Results: the rUSC monoclonal cells could be observed under microscope on the 4th day of primary culture, and the clones could be formed on the 6th day. On the 11th day, the cells were fused in the shape of "rice grain", uniform in size and compact in arrangement. RUSCs can effectively differentiate into smooth muscle cells and epithelial cells after differentiation in vitro. After treated with 5%PAA, the surface of SIS materials became loose and porous, and the content of DNA decreased further (P0.05). Histology showed that the regeneration of smooth muscle tract in the experimental group was better than that in the control group at all time points (P0.05), and the content of DNA in the experimental group was more orderly than that in the control group. The number of epithelial layer and the number of neovascularization in the early stage (2 ~ 3 weeks) was better than that in the control group (P0.05), but there was no significant difference in late (12 weeks) (P0.05), in the control group, inflammatory cell infiltration and fibrosis appeared in the matrix. Urethrography showed only one slight urethral stricture in the experimental group and slight urethral stricture in the control group. Immunofluorescence showed that rUSCs could be transformed into smooth muscle and epithelial cells in vivo. Conclusion: 1) rUSCs can be obtained by simple and non-invasive method. It has good self-renewal ability, easy to be cultured and expanded in vitro, and can differentiate into smooth muscle cells and epithelial cells. It is an ideal source of seed cells for tissue engineering. 2) after SIS was treated with 5%PAA, the pore size and porosity of SIS scaffold increased, and the cell composition decreased further, which could be used as the source of scaffold material for tissue engineering. 3) 3D porous SIS combined with rUSCs can promote urethral regeneration and reduce complications such as inflammation and urethral stricture.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R699
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相关期刊论文 前4条
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