再造组织工程尿路移行上皮组织的实验研究

RECONSTRUCTION OF UROTHELIUM TISSUE USING ISSUE-ENGINEERING TECHNIQUE

目的采用组织工程技术,将培养扩增的膀胱移行上皮细胞与可降解的聚乳酸/聚羟基乙酸共聚物(polylactical/glycolicacidcopolymer,PLGA)支架材料复合并植入裸鼠体内进行培育,探讨构建尿路移行上皮组织的可行性。方法取幼兔膀胱,机械分离与酶消化法获取膀胱移行上皮细胞,并在体外行原代培养与传代扩增,将第4～5代扩增的上皮细胞接种至8个PLGA聚合材料的表面作为实验组,4个单纯支架材料作为对照组。实验组:细胞材料复合体培育4周和8周各4个,对照组:单纯支架培育4周和8周各2个,分别植入各组裸鼠体内进行培育,按各时间点取出标本后进行大体观察、组织学及免疫组织化学检测。结果实验组标本经HE和马森染色,4周显示在材料表面形成数层上皮细胞;8周移行上皮细胞进一步增殖形成上皮组织,抗角蛋白免疫组织化学染色,胞浆呈棕黄色阳性。对照组4周和8周经HE染色材料表面见少量成纤维细胞沉积,马森染色呈蓝色,经抗角蛋白免疫组织化学染色为阴性。结论采用组织工程技术可再造人尿路移行上皮组织,为尿路组织工程进一步实验研究奠定了基础。

Objective To evaluate the feasibility of reconstruction of urothelium tissue in vivo using tissueengineering technique. Methods The urothelium cells were obtained from young rabbit, bladder by mechanical and enzyme digested methods. After expanded in vitro, the 4th to 5th generation urothelium cells were seeded onto the surface of 8 Polylatical/glycolic acid copolymer polymer,the polymer matrix without seeding cells served as control group. A total of 8 cell-polymer scaffolds and 4 simply scaffolds were separately implanted into subcutaneous pockets of athymic mice. The experiment groups included cell-polymer scaffolds 4 weeks and cell-polymer scaffolds 8 weeks. The control group included simply scaffold 4 weeks and simply scaffold 8 weeks. After 4 and 8 weeks, the specimens were obtained and examined by gross inspection, histologically and immunohistochemically. Results The results of HE and Masson staining showed that the polymer were covered by urothelium cells layers and cells layers increased markly in experimental group. Immuocytochemical studies revealed that the cells were stained positively for anti-cytokeratins （AE1/AE3） in experimental group. Fiber tissue deposition were found on the surface of polymers in control group by HE and Masson staining. Immunocytochemical staining of implants showed the negative result for cytokeratins in control group. Conclusion It is feasibility that reconstruction of urothelium tissue using tissue-engineering technique,which provides basic understandings for further development of the bladder and ureteral tissue engineered research.