应用组织工程口腔黏膜重建尿道的初步实验研究

A PRELIMINARY EXPERIMENTAL STUDY ON URETHRAL RECONSTRUCTION USING TISSUE ENGINEERED ORAL MUCOSA

目的探讨兔口腔黏膜细胞的体外培养方法,并以其为种子细胞与异体膀胱黏膜下脱细胞基质(bladder acellular matrix graft,BAMG)复合,构建组织工程尿道。方法18只10周龄雄性新西兰大耳白兔,体重0.3～0.5kg。取其中12只兔口腔黏膜组织,分离获得口腔黏膜细胞。将口腔黏膜细胞分别接种于有3T3细胞及无3T3细胞的培养皿进行培养,接种后第2天于倒置相差显微镜下观察细胞形态变化,绘制细胞生长曲线,观察生长增殖情况,并行免疫荧光组织化学染色鉴定。取余6只兔膀胱,经脱细胞处理制备BAMG,随机取1cm×1cm组织行HE染色,观察脱细胞效果。将接种于有3T3细胞培养皿培养获得的第2代口腔黏膜细胞种植于BAMG上,培养1周后,行HE染色及扫描电镜观察口腔黏膜细胞与BAMG的复合状况。结果倒置相差显微镜下观察,接种于有3T3细胞培养皿的口腔黏膜细胞可传至7～8代,其细胞形态均一,功能良好;无3T3细胞培养皿的口腔黏膜细胞形态多样,传至第2代时,即开始出现老化。细胞生长曲线显示,两种方法培养的口腔黏膜细胞均于第8天开始呈对数增长,第14天达峰值。接种于有3T3细胞培养皿的口腔黏膜细胞,培养至融合时所获得细胞数量明显多于接种于无3T3细胞培养皿的口腔黏膜细胞。两种方法培养的口腔黏膜细胞行免疫荧光染色,均呈绿色荧光。HE染色观察,BAMG经脱细胞处理后未见细胞,脱细胞效果良好。复合培养1周后,HE染色及扫描电镜观察口腔黏膜细胞与BAMG复合良好。结论兔口腔黏膜细胞可在体外成功培养、扩增,与同种异体BAMG复合后生长良好,为构建组织工程尿道提供了一种新的选择。

Objective To investigate the feasibility of replacing urinary epithelial cells with oral mucosa cell to reconstruct tissue engineered urethra by being seeded on bladder acellular matrix graft （BAMG）. Methods Eighteen male New Zealand rabbits, aged 10 weeks, weighing 0.3-0.5 kg, were used in this study. Oral mucosa cell of 12 rabbits were isolated and seeded onto a culture dish with a feeder layer of 3T3 and a culture dish without 3T3, respectively. The morphologic change and growth condition of oral mucosa cells were observed by inverted phase contrast microscope after 2 days of seeding. The quantity of oral mucosa cells was counted using cell counting meter; the cell growth curve was drawn and the immunofluorescence staining with broad-spectrum keratin antibody was carried out. The bladders taken from the rest 6 rabbits were decelluled to make BAMG and the tissue of 1 cm × 1 cm was randomly selected to observe the effect of acellularization. The second passage oral mucosa cells cultured with 3T3 were applied to sterilized BAMG to obtain a tissueengineered mucosa. The tissue-engineered mucosa was assessed using HE staining and scanning electron microscope after being cultured for 1 week. Results Oral mucosa cells seeded onto a feeder layer of 3T3 could be passaged for 7 or 8 generations with homogeneous forms and full function. Oral mucosa cells cultured without 3T3 could only be subcultured for 2 generations before aging and had multiple shapes and different sizes. Oral mucosa cells cultured by the two methods both started logarithmic growth on the 8th day and reached the peak value on the 14th day, which was indicated by the cell growth curve. However, more cells could be obtained through oral mucosa cells cultured with 3T3 than those cultured without 3T3. Oral mucosa cells manifestated green colour fluorescence cultured with or without 3T3. After the cells were removed, the BAMG presented as a porous membrane. The HE staining showed that the effect of acellularization was good and there were no cells at BAMG. The second passage oral mucosa cells cultured with 3T3 were expanded and seeded onto sterilized BAMG to obtain a tissue-engineered mucosa. Good compatibility of the compound graft was assessed using HE staining and scanning electron microscope. HE staining and scanning electron microscope showed that oral mucosa cells had good biocompatibility with BAMG after the tissue engineered mucosa was cultured for 1 week. Conclusion Oral mucosa cells of rabbit can be cultured in vitro and attain magnitude quantities. Oral mucosa cell also have good biocompatibility with BAMG and the compound graft could be a new material for urethral reconstruction.