人舌黏膜上皮细胞体外构建组织工程尿道的初步研究

目的 探索人舌黏膜上皮细胞和脱细胞支架体外复合构建组织工程尿道的可行性.方法 前尿道狭窄患者10例,手术切取0.5 cm×0.8 cm大小舌黏膜组织,分离获得舌黏膜上皮细胞,AE1/AE3抗体行细胞免疫荧光鉴定.收集第3代上皮细胞,按1×107/ml密度分别接种于脱水BAMG支架、液体保存BAMG支架以及4层脱细胞基质(SIS)支架表面,体外培养7d后行HE及扫描电镜检测.结果 10例患者术后1个月未出现舌部相关并发症.14 d后原代舌黏膜上皮细胞融合达90％～95％并呈典型的“鹅卵石”状生长.第3代时增殖速度达顶峰,平均7d达到90％～95％融合,第4代开始细胞逐渐老化.HE及扫描电镜检测液体保存BAMG支架表面仅复合极少量细胞,脱水BAMG以及SIS支架表面可见明显多层上皮细胞覆盖,其中4层SIS支架内部可见局部细胞浸润性生长迹象 结论 人舌黏膜上皮细胞可以作为组织工程尿道上皮种子细胞来源之一,与脱水处理后的SIS和BAMG有很好的组织相容性和黏附能力,二者的有效复合可以构建适合尿道修复重建需要的组织工程替代材料.

A preliminary investigation of constructing tissue engineering urethral graft using human lingual keratinocyes and natural derived scaffold

Objective To investigate the feasibility of constructing tissue engineering urethral substituted graft using human lingual keratinocytes and natural derived scaffold.Methods From Oct.2009to Jan.2010,ten patients with anterior urethral stricture were enrolled in this study.A 0.5 × 0.8 cm lingual mucosa was harvested during the operation.Lingual keratinocytes were then isolated and cultured from the mucosa.AE1/AE3 antibody was used to identify the lingual keratinocytes.Keratinocytes were collected and seeded onto three types of scaffold including dehydrated BAMG,liquid stored BAMG and 4-layer SIS product,with the density of 1 × 107/ml at passage three.After being cultured for seven days in vitro,H&E staining and Electronic Scan Microscopy were used to evaluate the compound matrixes.Results No complication occurred in the patients after operation.The primary passage of confluence lingual keratinocytes appeared in a typical "cobblestone" shape after being cultured for 14 days in vitro.The proliferation rate of these cells increased rapidly during the three passages.However,it decreased significantly in the 4th passage.H&E and Electronic Scan Microscopy examinations showed that few cells grew on the surface of the liquid stored BAMG.Nevertheless,multiple keratinocytes layers could be seen in dehydrated BAMG and 4-layer SIS.Meanwhile,cellular infiltration could be observed in SIS sections.Conclusions Human lingual keratinocytes could be the alternative of seeding cells for constructing tissues for engineering the urethra.These cells exhibited good compatibility and are adhesive to the SIS or BAMG.The compound matrix,which used human lingual keratinocytes and natural derived scaffold,may meet the clinical need of urethral disease in the future.