组织工程化口腔粘膜体外预构建
发布时间：2018-08-14 19:11
【摘要】：组织工程化口腔粘膜是近年来口腔医学领域的新热点,本文通过体外培养口腔粘膜上皮细胞和成纤维细胞,模拟组织工程化三维口腔粘膜的构建,探索组织工程化口腔粘膜体外培养的方法。本研究主要包括以下三方面内容：1.原代细胞培养目的：通过组织块法结合胰蛋白酶消化法培养、分离人口腔粘膜上皮细胞和成纤维细胞。材料与方法：10例第三磨牙拔除时切除的龈瓣,大小约1×0.5 cm2/块。将带有上皮层和固有层的组织块种植于培养瓶中,待组织块细胞增殖后,通过胰蛋白酶消化法分离获得原代上皮细胞和成纤维细胞。通过在不同培养基中传代培养,进一步纯化两种细胞。采用免疫荧光和HE染色进行细胞鉴定。结果：组织块植入后的第2d～3d可看到组织块周边出现新分裂增殖的细胞,约14 d细胞增殖范围达到0.5 cm～1cm后,进行初次传代。胰蛋白酶消化法可初步分离出上皮细胞和成纤维细胞。结论：组织块法结合胰蛋白酶消化法能快速高效的培养分离口腔粘膜上皮细胞和成纤维细胞,是一种实用的口腔粘膜细胞培养方法。2.支架筛选目的：筛选适合用于组织工程化口腔粘膜体外构建的支架材料。材料与方法：将5组上皮细胞和成纤维细胞分别种植于脱细胞异体真皮基质、PEG、PVA三种生物支架上,培养2 d后,采用CCK-8测定细胞活性,采用随机区组设计方差分析统计数据。将成纤维细胞种植于三种生物支架上,培养1周后再将上皮细胞种植于支架上,继续培养1周,HE染色及免疫组化观察细胞在支架中的生长状态。结果：统计分析显示,上皮细胞和成纤维细胞活性在三种生物材料中均无明显的差异(P0.05)。HE染色及免疫组化显示,三种生物支架材料中,脱细胞异体真皮基质中的细胞数目多,分布层次深,细胞标记蛋白表达较好。结论：三种生物支架中较适宜用于体外三维培养口腔粘膜上皮细胞及成纤维细胞的是脱细胞异体真皮基质。3.组织工程化口腔粘膜体外预构建 目的：通过体外三维培养口腔粘膜上皮细胞和成纤维细胞,模拟组织工程化口腔粘膜的构建,探索组织工程化口腔粘膜体外培养的方法。材料与方法：将成纤维细胞种植于脱细胞异体真皮基质上,培养1周后再将上皮细胞种植于支架上,继续培养1周后,将支架移至气-液平面培养5d。HE染色及免疫荧光观察口腔粘膜上皮细胞与成纤维细胞体外三维培养的情况。结果：免疫荧光及HE染色显示,经过2周的液下培养及5d的气-液培养后,细胞可以深入生长植入到支架全层,其中上皮细胞主要集中在支架表层及浅层,成纤维细胞主要分布在支架深层。结论：口腔粘膜上皮细胞及成纤维细胞体外三维培养后细胞的分布状态类似于口腔粘膜组织的基本分层,但细胞增殖数量少,细胞活性不佳,仍有待进一步研究改进实验方法。
[Abstract]:Tissue-engineered oral mucosa is a new hotspot in the field of stomatology in recent years. In this paper, oral epithelial cells and fibroblasts were cultured in vitro to simulate the construction of tissue-engineered three-dimensional oral mucosa, and the method of tissue-engineered oral mucosa culture in vitro was explored. Materials and Methods: Ten cases of gingival flaps resected during the extraction of the third molar were about 1 Primary epithelial cells and fibroblasts were isolated by trypsin digestion and further purified by subculture in different media. The cells were identified by immunofluorescence and HE staining. Conclusion: Tissue block method combined with trypsin digestion can isolate oral mucosal epithelial cells and fibroblasts quickly and efficiently. It is a practical method for oral mucosal cell culture. Material and Methods: Five groups of epithelial cells and fibroblasts were implanted on acellular allogenic dermal matrix, PEG and PVA scaffolds respectively. After 2 days of culture, CCK-8 was used to determine the cell activity and variance score was designed by randomized block design. Statistical data were analyzed. Fibroblasts were implanted on three kinds of biological scaffolds, then epithelial cells were implanted on the scaffolds after one week of culture, and then cultured for another week. HE staining and immunohistochemistry were used to observe the growth status of the cells in the scaffolds. Results: Statistical analysis showed that the activity of epithelial cells and fibroblasts was not obvious in the three kinds of biological materials. HE staining and immunohistochemistry showed that there were many cells in the acellular allogenic dermal matrix with deep distribution and good expression of cell marker protein in the three scaffolds. Skin matrix. 3. Prefabrication of tissue-engineered oral mucosa in vitro Objective: To explore the method of tissue-engineered oral mucosa culture in vitro by three-dimensional culture of oral epithelial cells and fibroblasts, and to simulate the construction of tissue-engineered oral mucosa. Essentially, epithelial cells were implanted on the scaffold after 1 week of culture, and then cultured on the gas-liquid plane for 5 days. HE staining and immunofluorescence were used to observe the three-dimensional culture of oral mucosal epithelial cells and fibroblasts in vitro. The epithelial cells were mainly concentrated in the surface and superficial layers of the scaffold, while fibroblasts were mainly distributed in the deep layers of the scaffold. A small number of cells and poor cell activity still need further research and improvement of experimental methods.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R782
，
本文编号：2183845
[Abstract]:Tissue-engineered oral mucosa is a new hotspot in the field of stomatology in recent years. In this paper, oral epithelial cells and fibroblasts were cultured in vitro to simulate the construction of tissue-engineered three-dimensional oral mucosa, and the method of tissue-engineered oral mucosa culture in vitro was explored. Materials and Methods: Ten cases of gingival flaps resected during the extraction of the third molar were about 1 Primary epithelial cells and fibroblasts were isolated by trypsin digestion and further purified by subculture in different media. The cells were identified by immunofluorescence and HE staining. Conclusion: Tissue block method combined with trypsin digestion can isolate oral mucosal epithelial cells and fibroblasts quickly and efficiently. It is a practical method for oral mucosal cell culture. Material and Methods: Five groups of epithelial cells and fibroblasts were implanted on acellular allogenic dermal matrix, PEG and PVA scaffolds respectively. After 2 days of culture, CCK-8 was used to determine the cell activity and variance score was designed by randomized block design. Statistical data were analyzed. Fibroblasts were implanted on three kinds of biological scaffolds, then epithelial cells were implanted on the scaffolds after one week of culture, and then cultured for another week. HE staining and immunohistochemistry were used to observe the growth status of the cells in the scaffolds. Results: Statistical analysis showed that the activity of epithelial cells and fibroblasts was not obvious in the three kinds of biological materials. HE staining and immunohistochemistry showed that there were many cells in the acellular allogenic dermal matrix with deep distribution and good expression of cell marker protein in the three scaffolds. Skin matrix. 3. Prefabrication of tissue-engineered oral mucosa in vitro Objective: To explore the method of tissue-engineered oral mucosa culture in vitro by three-dimensional culture of oral epithelial cells and fibroblasts, and to simulate the construction of tissue-engineered oral mucosa. Essentially, epithelial cells were implanted on the scaffold after 1 week of culture, and then cultured on the gas-liquid plane for 5 days. HE staining and immunofluorescence were used to observe the three-dimensional culture of oral mucosal epithelial cells and fibroblasts in vitro. The epithelial cells were mainly concentrated in the surface and superficial layers of the scaffold, while fibroblasts were mainly distributed in the deep layers of the scaffold. A small number of cells and poor cell activity still need further research and improvement of experimental methods.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R782
，
本文编号：2183845
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