组织工程舌黏膜构建的研究

目的 探讨舌黏膜细胞的体外培养方法,及其作为种子细胞与同种异体膀胱脱细胞移植物(BAMG)复合构建组织工程化黏膜的方法.方法 分离并获取雄性新西兰大耳白兔舌黏膜细胞进行培养,定期观察细胞形态变化及生长增殖情况.对体外培养的舌黏膜细胞进行广谱角蛋白抗体(AE1/AE3)免疫荧光染色鉴定;流式细胞仪检测第2代培养细胞的AE1/AE3阳性细胞百分比;细胞计数及噻唑蓝(MTT)比色法测定以判断细胞增殖能力.取同种异体兔膀胱经脱细胞处理制成BAMG,随机取小块组织行HE和Masson染色观察脱细胞效果,然后将第2代体外培养的舌黏膜细胞种植于BAMG上,通过HE染色及扫描电镜观察口腔黏膜细胞与BAMG的复合情况.结果 舌黏膜细胞形态均一有较强的增殖能力可传代至4～5代,传至第4代时开始出现老化,细胞贴壁及增殖能力均明显减弱.免疫荧光染色显示近100%体外培养的舌黏膜细胞AE1/AE3免疫荧光染色阳性.流式细胞仪检测结果表明第2代舌黏膜细胞AE1/AE3阳性细胞百分比大于96%.舌黏膜细胞传至第3代时可获得细胞数量在2×107以上.HE和扫描电镜观察均显示舌黏膜细胞和BAMG复合良好.结论 兔舌黏膜细胞可在体外成功培养、扩增;兔舌黏膜细胞与同种异体BAMG复合后生长良好.

Preliminary study on tissue-engineered lingual mucosa reconstruction

Objective To investigate the feasibility of tissue-engineered mucosa reconstruction u-sing lingual mucosa cells seeded on bladder acellular matrix graft (BAMG). Methods Lingual mucosa ceils in rabbits were isulated and seeded onto a culture dish. Lingual mucosa cells were observed under an inverted phase contrast microscope from the second day after seeding for morphologic change and growth condition. The number of lingual mucosa cells was counted using cell counting meter and the cell growth curve was drawn and immunofluorescence staining with broad-spectrum keratin antibody A1/A3 was car-ried out. The bladder taken from variant male rabbits of homogeneity was decelluled, made into BAMG and HE staining was carried out randomly to observe the effect of acellularization. Passage 2 lingual mucosa cells were applied to the sterilized BAMG to obtain a tissue-engineered mucosa. The tissue-engineered mu-cosa was assessed using HE staining and scanning electron microscope. Results Lingual mucosa cells could be passaged for 4 or 5 generations. Lingual mucosa ceils started logarithmic growth at 8th day and reached the peak value at 14th day. Most of lingual mucosa cells manifested green color fluorescence. After the cells were removed,the BAMG we got presented as a porous membrane. The acellularization was fine.Passage 2 lingual mucosa ceils were expanded and seeded onto the sterilized BAMG to obtain a tissue-engi-neered mucosa. Fine biocompatibility of the compound graft was assessed using HE staining and scanning electron microscope. Conclusion Lingual mucosa cells of rabbits can be cultured in vitro and attain mag-nitude quantity. Lingual mucosa ceils also have fine biocompatibility with BAMG.