聚羟基乙酸作为支架材料体外构建复层膀胱壁结构的探讨

目的：探讨聚羟基乙酸（polyglycolic acid，PGA）作为支架材料并复合成年猪膀胱平滑肌细胞和尿路上皮细胞，体外构建复层膀胱壁结构的可行性。方法：采用PGA作为支架材料。并以聚乳酸（polylact acid，PLA）塑形。以酶消化法分别获得猪膀胱尿路上皮细胞和平滑肌细胞，并体外扩增。以MTT法分别检测P3代膀胱平滑肌细胞（SMC）和尿路上皮细胞（UC）的体外增殖能力。将P3代膀胱平滑肌细胞先接种在支架上，再接种尿路上皮细胞。将细胞一材料复合物体外培养1～2周并行组织学鉴定。结果：酶消化法获得的膀胱平滑肌细胞和尿路上皮细胞经过体外培养仍具有较强的体外增殖能力。细胞材料复合物体外培养1周后形成分层排列结构，包括黏膜上皮层和膀胱平滑肌层。免疫组织化学显示平滑肌层α-Smooth Muscle Actin表达阳性，黏膜上皮层广谱角蛋白表达阳性。而细胞材料复合物继续体外培养至2周，细胞从支架上大量脱落。结论：PGA适合作为复层膀胱壁结构体外构建的支架材料。采用PGA作为支架材料并复合膀胱平滑肌细胞和尿路上皮细胞，可以体外构建出类似复层膀胱壁结构。细胞材料复合物体外培养1周左右较适合进行体内回植修复。

Using Polyglycolic Acid as Scaffold to Construct Multilayer Structure of Bladder Wall in Vitro

Objective：To discuss the feasibility of using polyglycolic acid （PGA） as scaffold, adult porcine smooth muscle cell （SMC） and urothelial cell （UC） as seeding cells, to form a sandwich structure of bladder wall in vitro. Methods：We use PGA as scaffold with poiylact acid remodeling. Primary porcine SMC and UC were obtained by enzymatic digestion and expanded in vitro. The proliferation ability of P3 SMC and UC in vitro were tested their proliferation ability by MTT. The PGA scaffold was seeded with smooth muscle cells at first, then with urothelial cells above. The construction were cultivated in vitro for 1-2 weeks and evaluated by H＆E and immunohistochemistry. Results：Both SMC and UC can quickly expand in vitro after enzymatic digestion and proliferation. After seeded on the scaffold and cultured in vitro for one week, SMC and UC have formed continual multilayer structure on the serosal and lumen surface respectively. The mucosa layer showed pan-cytokeratin expression, while the underlying muscle layer had positive stain of α-Smooth Muscle Actin. The construction continued to culture in vitro for another week until numerous seeding cells fell off from the scaffold. Conclusions： As a scaffold, PGA is suitable for constructing multilayer structure in vitro combined with bladder SMC and UC. We can form a sandwich structure in vitro, which mimic the native bladder structure including the separate mucosa and smooth muscle layers. One week construction culture is feasible for further bladder regeneration in vivo.