口腔黏膜细胞与膀胱黏膜下脱细胞基质复合物构建组织工程化尿道的实验研究

目的 探讨以兔口腔黏膜细胞与同种异体膀胱黏膜下脱细胞基质(BAMG)复合物构建组织工程化尿道的可行性.方法 新西兰雄性兔24只,距尿道外口2.0 cm剥离尿道黏膜(2.0 cm×0.8 cm)后,随机分实验组和对照组,每组12只.切取实验组兔口腔黏膜组织分离细胞,在有灭活的3T3细胞培养皿上进行培养扩增,将培养获得的第2代口腔黏膜细胞种植于BAMG(2.2 cm×1.0 cm)上,植入实验组兔尿道缺损区域;对照组单纯采用无细胞植入的BAMG修复尿道.分别于术后1、2、6个月观察动物排尿情况,行尿道造影,8 F尿管插管确定有无狭窄;随后处死实验兔,取修复段尿道黏膜组织行组织学检查.结果 细胞培养获得的口腔黏膜细胞形态均一,生长良好;组织形态学、扫描电镜观察见口腔黏膜细胞与BAMG具有良好的相容性.实验组兔术后1、2、6个月伤口愈合良好、排尿通畅,无尿瘘发生,组织学和尿道造影检查显示带细胞修复的尿道形态完整、清晰宽敞,无狭窄发生;术后6个月植入的口腔黏膜细胞仍然存在,并明显扩增.对照组兔则出现排尿困难、尿道狭窄,光镜下发现黏膜及黏膜下存在严重的炎症反应.结论 兔口腔黏膜细胞与同种异体BAMG复合后,可成功用于尿道缺损的修复,构建组织工程化尿道.

Urethral reconstruction using oral keratinocytes-seeded bladder acellular matrix graft

Objective To investigate the feasibility of replacing urinary epithelial cells with oral keratinocytes by being seeded on bladder acellular matrix graft(BAMG). Methods Twenty-four male rabbits were randomly divided into 2 groups:experimental group and control group.A length of 2.0 cm and width of 0.8 cm penile urethral mucosal defect was induced in the anterior urethra 2.0 cm awav from the urinary meatus in all the rabbits.Oral keratinocytes were isolated from a small buecal mucosa(1.0 cm×0.4 cm)of the 12 rabbits in experimental group and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin(i3T3).Passage 2 oraI keratinocytes cuItured with i3T3 were expanded and seeded onto sterilized BAMG(2.2 cm×1.0 cm)to repair the de-fects of the urethra.Urethroplasty was performed with BAMG with no cell seeding in the controlgroup.Catheter examination and retrograde urethI ography was done in 1,2 and 6 months after graft-ing.The urethral grafts were harvested and analyzed by histological staining. Results Oral kerati-nocytes seeded onto a feeder layer of i3T3 had better morphous and amplification capability.The corn-patibility of the compound graft was assessed by HE staining and scanning electron microscope.Twelve rabbits in the experimental group could void without difficulty.Catheter examination and ret-rograde urethrogram revealed that a wide urethral caliber was maintained with no sign of strictures and fistula formation.Histological examination showed the grafted urethra was covered with smooth mu- cosa with no strictures at 1,2 and 6 months.The oral keratinocytes of the graft still existed even 6 months after grafting.On contrast,gross and urethrogram demonstrated urethral stricture in the con-trol group.And inflammatory reactions could be found in the area of the stricture through light micro-scope. Conclusions Oral keratinocytes have good compatibility with BAMG and the compound graft could be used for urethral reconstruction in the animal model.