组织工程医用生物补片用于阴道重建的动物实验研究

目的 观察应用组织工程医用生物补片(一种脱细胞真皮基质材料)在实验动物体内进行阴道重建的效果.方法 对12只中国小型实验猪行阴道全切除术,应用组织工程医用生物补片行阴道重建术,分别于术后1、2、4、6、8、12周每次选择2只实验猪切除全层阴道,并以其中2只实验猪阴道切除术中切除的正常阴道组织为对照.对阴道重建术后不同时间切除的阴道组织行HE染色和Van Gieson(VG)染色,以观察和评价阴道各层组织的生长状态;采用上皮广谱角蛋白单克隆抗体AE1/AE3、平滑肌α肌动蛋白(α-actin)单克隆抗体进行免疫组化染色,以证实重建阴道是否存在上皮组织和平滑肌组织;于重建术后1、12周行透射电镜检查,观察阴道组织超微结构的变化;于重建术后12周应用组织浴槽对离体阴道组织进行药物和电刺激,以评价阴道平滑肌的收缩功能.结果 (1)阴道重建术后1周,可见阴道黏膜覆盖近2/3,光镜下观察,阴道上皮层数少,仅1～2层;电镜下观察,上皮细胞排列疏松,不规则.阴道重建术后4～6周,可见黏膜上皮层数增多,达4～5层.重建术后12周,大体上很难与正常阴道区分,上皮广谱角蛋白单克隆抗体AE1/AE3免疫组化染色结果与HE染色结果一致;电镜下观察,阴道上皮细胞分层分化良好,排列有序,细胞的相邻面有桥粒连接.(2)VG染色、免疫组化染色显示,阴道重建术后4周,开始出现散在的平滑肌细胞,此后逐渐形成排列均匀的肌束.(3)阴道重建术后12周,重建的阴道与正常阴道组织对KCl刺激均能产生收缩波,收缩反应强度变化相似,分别为(2.96±0.29)和(3.14±0.30)g;对于不同电压、不同频率的电刺激,当电压固定、频率逐渐升高时,两者收缩反应强度变化分别为(3.43±0.34)和(4.65±0.73)g;当频率固定、电压逐渐升高时,两者收缩反应强度变化则分别为(4.92±0.38)和(4.89±0.44)g.结论 组织工程医用生物补片是在实验动物体内进行阴道重建较理想的材料,重建术后3个月基本可以完成组织重建.

Experimental study on vagina reconstruction with tissue-engineering biological material

Objective To investigate the effect of vagina reconstruction using tissue-engineering biological material (acellular dermal matrix) in an animal model.Methods Vagina excision and vagina reconstruction with tissue-engineering biological material were performed in 12 Chinese experimental miniature pigs.The control group was matched with two of normal vagina specimens resected.At week 1,2,4,6,8,12 after surgery,the animals were sacrificed,respectively,and the neovaginas were prepared for immunohistochemical and Van Gieson (VG) staining to evaluate the status of various layer growth of vagina.Epithelial broad spectrum of monoclonal antibodies of AE1/AE3 and α-actin were used to test the existence of epithelial and smooth muscle tissue by immunohistochemical staining.The ultrastructure of neovagina was studied by transmission electron microscope at week 1 and 12 after surgery.Contractile function of isolated smooth muscle of neovagina was evaluated by chemical and electronic stimulation after 12 weeks' reconstruction.Results (1) Epithelization of 2/3 neovaginal mucosa was observed within 1 week.Only 1-2 layer epitheliums were observed under the light microscopy and epithelial cells with characteristics of loose and disarrangement were shown with the electron microscopy.Within 4-6 weeks,epithelization in mucosa of neovaginal canal was intensified to 4-5 layers.After 12 weeks,the differences between the neovagina and the native vagina were harldy noted either in the gross or microscopically.(2) After 4 weeks,a few smooth muscle cells were observed with VG and immunohistochemical staining,and homogeneous muscle bundle was formed.(3) After 12 weeks,similar contractile responses between neovagina and native vagina were observed when KCI and electrical stimulation with different frequency and voltage were given (2.96±0.29)g vs.(3.14±0.30)g,(3.43±0.34)g vs.(4.65±0.73)g,(4.92±0.38)g vs.(4.89±0.44)g].Conclusion The tissue-engineering biological material might be an ideal graft used in the reconstruction of vagina.