采用温敏性基材培养并收获无损伤细胞片层的技术在无支架材料的组织重建方面具有良好应用前景。考虑到临床应用时种子细胞来源限制及治疗的时效性，在进行细胞片培养时，提高细胞贴附率以及加快细胞增殖和细胞片分离尤为关键。通过引入细胞粘附肽RGDS和生长因子胰岛素可分别促进细胞贴附和增殖，却存在如物理吸附方式不牢固、共价结合不利于细胞分离的缺点。针对这一问题，本研究拟采用分子印迹纳米粒子的表面涂层技术，构建对RGDS和胰岛素均具有温度响应性识别能力的双分子印迹表面，旨在通过特异性识别作用结合RGDS和胰岛素，有效促进细胞贴附和增殖，并能在降低温度时将其释放，提高细胞片层的分离效率，最终达到减少种子细胞需求、提高细胞利用率、缩短细胞片层收获周期的目的。研究中将探讨温度响应性双分子印迹表面的制备技术及其在细胞片培养和分离过程中的重要作用，有望为生物活性分子的引入方式提供新的思路，推进细胞片层技术的临床应用。

Harvesting intact cell sheets by using thermo-responsive culture substrates holds great promise in scaffold-free tissue reconstruction. In view of the limitation of cell source and the timeliness of treatment in clinical applications, it is very important to promote cell adhesion, accelerate cell proliferation and cell sheet detachment during cell sheet culture. Previous studies have shown that introducing a cell adhesive peptide RGDS or a growth factor insulin into the thermo-responsive culture substrates can markedly improve cell adhesion or proliferation, respectively. However, drawbacks exist with such approaches, including instability of physical absorption or covalent bond decelerating cell detachment. The proposed project aims to address these concerns by fabricating a thermo-responsive, bimolecularly imprinted culture surface through surface coating of a mixture of molecularly imprinted nanoparticles towards RGDS and insulin respectively. In this approach, the bioactive molecules will promote cell adhesion and proliferation during cell culture, while they can be released from surface by lowering temperature, which facilitates the detachment of matured cell sheet. Therefore, this technique has advantages by improving the efficiency of cell sheet detachment, reducing the need of cell dosage, and shortening the harvesting time of cell sheets. In the study, we will work out a technical route for preparation of thermo-responsive bimolecularly imprinted surfaces and investigate the role of such surfaces in cell sheet culture. We expect that the proposed work will lead to a novel approach to introducing bioactive molecules on cell culture substrates and promote the clinical applications of cell sheet engineering.