新型上皮细胞层片组织工程制备、冷冻保存及其应用于尿道修复的实验研究

Although tissue engineering has been thought of having great potential in urethroplasty, it is still limited by immune rejection of the scaffold materials, inflammation caused by fibrosis as well as cell proliferation within the stent material. Cell sheet technology (CST), by which cultured cells can be harvested as intact sheets, has much superiority over traditional tissue engineering. In our preliminary study, two kinds of tissue-engineered cell sheets (5-10cm in diameter) were successfully obtained on thermo-responsive culture dishes using cells harvested from a small piece of urethra buccal and foreskin of male New Zealand rabbit. The foreskin derived cell sheet showed better strength and thickness with shorter culture time, suggesting more suitable for urethral reconstruction. In this research project, firstly, we aim to develop a novel cell-sheet manipulation technology using temperature-responsive culture surfaces combined with serum-free culture medium; in which cell growth factors will be applied to substitute animal feeder cells, so as to meet the ethical request of clinical application in future. Secondly, we will try to establish an optimized technique for cryopreservation of tissue-engineered cell sheets for the purpose of cell-sheets tissue banking. As it is has been proved that human epidermal cells lose allogeneic T-cell activating ability, HLA-DR expression and Langerhans cell markers after growth in culture. Moreover, our previous study and other studies demonstrated that allograft immunogenicity can be reduced by cryopreservation procedure. Therefore, we deduce that cyropreserved tissue-engineered cell sheets might be tolerant in allogeneic recipient. So, thirdly, we will investigate the immunogenicity of both fresh and frozen-thawed tissue-engineered cell sheets after allotransplantation in urethral stricture animal model and its mechanism will be clarified. In conclusion, with CST, cultured autologous/allogeneic engineered epidermal cell sheets in vitro used as transplant sources are expected to overcome the problems of allogeneic immunological rejection as well as the shortage of substitute materials for urethroplasty.

复杂尿道下裂或长段尿道狭窄，因缺乏理想的尿道修复材料而疗效不佳。组织工程技术发展为尿道修复带来了新希望，然而支架材料因组织相容性问题及炎症反应限制了其临床应用。细胞层片技术不需要支架且保留了完整的细胞连接蛋白和细胞外基质，优势明显，显现出良好应用前景。我们前期研究发现：包皮来源的上皮细胞层片优于尿道粘膜上皮细胞层片，可能成为新的尿道修复材料。本课题组首次将细胞层片技术应用于尿道修复研究。首先，我们将进一步改进技术，利用条件培养皿创建无动物血清和动物喂养细胞的新型上皮细胞层片制备技术。其次，基于我们前期在低温生物学研究方面的优势，优化上皮细胞层片冷冻保存技术，并探究冷冻保存对上皮细胞层片免疫原性影响及其机制，论证上皮细胞层片组织库的建立及其应用于同种异体尿道修复的可行性。通过本项目研究，预期获得具有自主知识产权的新型上皮细胞层片制备及长期存储技术，并完成动物实验研究，为今后临床应用奠定基础。

尿道狭窄或尿道下裂是泌尿外科的常见疾病，本课题研究使用组织工程自体上皮细胞层片重建尿道，取得了优异的修复效果。本课题组通过优化培养技术，成功的利用条件培养皿创建出无需添加动物血清和采用动物饲养细胞的新型上皮细胞层片制备技术。研究发现和传统培养方法相比，收获后新型细胞层片无皱缩，形态完整紧凑、均一透明，细胞活性及细胞形态无明显差异，层片厚度则较薄，力学强度稍弱。随后，我们使用优化的冷冻保护剂，并采用微量泵灌流+程序化冷冻的方案，成功的对新型上皮细胞层片进行了长达3个月的冷冻保存。基于先前课题中保存哺乳动物卵巢和膀胱粘膜的冷冻保护剂，我们发现含有二甲基亚砜（DMSO）、果糖、抗坏血酸和Y22632组分的冷冻保护剂可有效提高上皮层片的细胞活性，从7%提高到50%以上。对上皮细胞层片的免疫原性进行流式检测后发现，冻存后的上皮细胞层片HLA-I阳性表达仍高于50%，相对于新鲜膜片约下降10%。最后，本课题在新西兰兔的尿道狭窄模型中对新鲜层片和冷冻层片分别进行了自体和异体的验证。结果发现在自体移植中，新鲜细胞层片和冻存细胞层片修复后的尿道，排尿通畅，尿道粘膜光滑，未观察到明显狭窄、尿瘘等并发症。而异体移植中，新鲜细胞层片和冻存细胞层片组都观察到了瘢痕狭窄、尿瘘等并发症。本研究还采用上述的培养技术及冷冻保存技术对人包皮上皮细胞层片的培养和冷冻进行了初步验证，并进行了完整性、活力、凋亡、免疫原性、力学性能和细胞功能等方面的评价。.综上，本研究所创建的新型上皮细胞层片培养技术和冷冻存储技术在新西兰兔的自体尿道修复实验中获得了良好的修复效果，为临床上尿道修复重建提供了一种新的自体生物材料来源，为上皮细胞层片组织库的建立奠定了基础。项目资助研究成果发表SCI论文7篇。培养博士2名，其中2名已取得博士学位。项目投入经费57万，支出25.6202万，各项支持基本与预算相符，剩余经费31.3798万，计划用于本项目后续研究。