聚乳酸支架皮下活化用于组织工程输尿管构建研究

发布时间：2018-07-21 12:15

【摘要】：目的：探讨螺旋形聚乳酸支架皮下诱导结缔组织薄膜形成的规律及包埋时间对尿路上皮细胞增殖的影响，最终体外构建出具有一定生物活性的组织工程输尿管。方法： Wistar大鼠膀胱活检获得泌尿道粘膜标本，酶消化法原代分离尿路上皮细胞。用分子量为1.5×10~5Da的聚乳酸制备螺旋形输尿管内支架和片状薄膜，前者用于探讨皮下诱导结缔组织薄膜形成的规律，后者用于探讨包埋时间对尿路上皮细胞增殖的影响。将螺旋形聚乳酸输尿管内支架和片状聚乳酸薄膜包埋到Wistar大鼠皮下，在不同的时间点（1、2、3周）将螺旋形聚乳酸输尿管内支架和片状聚乳酸薄膜连同表面结缔组织一起取回，经脱细胞处理清除结缔组织层中的细胞成分。将体外扩增的尿路上皮细胞按4×10~4cells/cm~2的密度接种到螺旋形聚乳酸输尿管内支架和片状聚乳酸薄膜表面的脱细胞结缔组织层，检测尿路上皮细胞在结缔组织层的附着、增殖情况。结果：成功原代分离膀胱尿路上皮细胞，经角朊上皮无血清培养基（DSFM）培养后免疫组织化学染色证实为纯化的尿路上皮细胞；细胞排列为典型的铺路石样外观，4代以前的细胞状态良好，可作为组织工程输尿管构建的理想种子细胞；包埋1周，在螺旋形聚乳酸输尿管内支架表面形成了一层菲薄的组织薄膜，炎症反应轻微，可见少量多型核白细胞存在，无新生血管存在；包埋2周，在螺旋形聚乳酸输尿管内支架表面可见薄层结缔组织形成，在结缔组织周边可见少量毛细血管存在；包埋3周，螺旋形聚乳酸输尿管内支架表面结缔组织中有纤维增生表现，可见大量毛细血管存在，炎症反应完全消失。各包埋时间组，，结缔组织经脱细胞处理后可见大量胶原组织残留，经免疫组织化学鉴定其内含有丰富的Ⅰ型胶原蛋白、Ⅳ型胶原蛋白、层粘连蛋白、纤维连接蛋白，这些蛋白组成与尿路上皮基底层主要成分相近。在各包埋时间组，扫描电子显微镜观察发现脱细胞结缔组织表面均呈三维立体结构；与包埋2、3周组相比，包埋1周组的脱细胞结缔组织较薄，部分区域暴露出内层的聚乳酸支架部分；脱细胞结缔组织种植尿路上皮细胞3天后，尿路上皮细胞附着良好，细胞周边有伪足样结构伸出，并沿结缔组织表面延伸。MTT结果显示，在不同包埋时间组，尿路上皮细胞在脱细胞结缔组织层均能连续增殖。体外孵育5、7天，与包埋1周组相比，尿路上皮细胞在包埋2、3周的脱细胞结缔组织层中具有更高的增殖活性（p0.05）。结论：体外扩增的尿路上皮细胞可种植于体内诱导形成的脱细胞结缔组织上构建出具有一定生物活性的组织工程输尿管。综合结缔组织薄膜脱细胞前后组织病理学检测结果及尿路上皮细胞在脱细胞结缔组织层中的增殖情况，我们认为包埋2、3周的脱细胞结缔组织是体外构建组织工程输尿管的良好介质；该研究结果可为皮下诱导形成结缔组织薄膜、种植自身尿路上皮细胞后构建组织工程输尿管奠定实验基础。尽管如此，该组织工程输尿管用于输尿管损伤修复、重建的有效性和安全性，仍待进一步动物实验研究证实。
[Abstract]:Objective: To investigate the regularity of the formation of connective tissue film under the subcutaneous induction of spiral polylactic acid stent and the effect of embedding time on the proliferation of urinary tract epithelial cells. Finally, a tissue engineering ureter with certain biological activity was constructed in vitro. Methods: urinary tract mucosa specimens were obtained by bladder biopsy in Wistar rats, and the original urinary tract was separated by enzyme digestion. Epithelial cells. A spiral ureteral stent and flake thin film were prepared with a molecular weight of 1.5 * 10~5Da polylactic acid. The former was used to investigate the formation of subcutaneous induced connective tissue film. The latter was used to explore the effect of embedding time on the proliferation of urinary tract epithelial cells. The spiral polylactic ureteral stent and flaky polylactic acid film were retrieved together with the surface connective tissue at different time points (1,2,3 weeks) to remove the cell components in the connective tissue layer at different time points (Wistar weeks). The cells expanded in vitro were inoculated into the spiral shape at 4 x 10~4cells/cm~2 density. Polylactic ureteral stent and the decellularized connective tissue layer on the surface of flaky polylactic acid film to detect the attachment and proliferation of urinary tract epithelial cells in connective tissue layer. Results: successful primary separation of bladder urothelial cells, and immuno histochemical staining of the serum-free culture medium of keratinocyte (DSFM) proved to be the purified urinary tract. Epithelial cells; cells arranged as a typical paving stone appearance. The 4 generation of cells were in good condition and could be used as ideal seed cells for the construction of the tissue engineered ureter. A thin layer of thin tissue film was formed on the surface of the spiral polylactic ureteral stent for 1 weeks, and a slight inflammatory reaction was found, and a small amount of polymorphonuclear leukocytes were found. No neovascularization existed; 2 weeks of embedding, a thin connective tissue formed on the surface of the spiral polylactic ureteral stent, and a small amount of capillaries were found around the connective tissue; for 3 weeks, fibrous proliferation in the surface connective tissue of the spiral polylactic acid ureteral stent showed a large number of capillary and inflammatory reactions. Complete disappearance. A large amount of collagen tissue remained in the connective tissue after acellular treatment. It was identified by immuno histochemistry as a rich type of collagen type I, type IV collagen, laminin, fibronectin, and these proteins were similar to the main components in the bottom of the urinary tract. The scanning electron microscope showed that the surface of the acellular connective tissue was three-dimensional structure. Compared with the embedded 2,3 week group, the decellated connective tissue was thinner in the 1 week group, and the inner layer of the polylactic acid scaffold was exposed in some areas. After 3 days after the acellular connective tissue planted the urinary tract cells, the urinary tract epithelial cells attached well and fine. The perimeter of the cell was protruded and extended along the connective tissue surface.MTT results showed that the urinary tract epithelial cells were able to proliferate continuously in the acellular connective tissue layer at different embedding time groups. In vitro incubation for 5,7 days, the urinary tract epithelial cells had a higher proliferation activity in the 1 week embedded connective tissue layer embedded in the embedded 2,3 week group. P0.05. Conclusion: in vitro expanded urinary tract epithelial cells can be planted in the acellular connective tissue induced by the body to construct a tissue engineered ureter with certain biological activity. The histological examination of the tissue film before and after the dehydration of the connective tissue film and the proliferation of the urinary tract epithelial cells in the decellular connective tissue layer In addition, we believe that the acellular connective tissue embedded in 2,3 weeks is a good medium for constructing the tissue engineered ureter in vitro. The results can provide an experimental basis for subcutaneous induction of connective tissue film and the construction of tissue engineered ureter after the cultivation of its own urinary tract epithelial cells. The effectiveness and safety of wound repair and reconstruction remain to be confirmed by animal experiments.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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