發(fā)布時(shí)間：2018-08-14 19:11

【摘要】：組織工程化口腔粘膜是近年來(lái)口腔醫(yī)學(xué)領(lǐng)域的新熱點(diǎn),本文通過體外培養(yǎng)口腔粘膜上皮細(xì)胞和成纖維細(xì)胞,模擬組織工程化三維口腔粘膜的構(gòu)建,探索組織工程化口腔粘膜體外培養(yǎng)的方法。本研究主要包括以下三方面內(nèi)容：1.原代細(xì)胞培養(yǎng)目的：通過組織塊法結(jié)合胰蛋白酶消化法培養(yǎng)、分離人口腔粘膜上皮細(xì)胞和成纖維細(xì)胞。材料與方法：10例第三磨牙拔除時(shí)切除的齦瓣,大小約1×0.5 cm2/塊。將帶有上皮層和固有層的組織塊種植于培養(yǎng)瓶中,待組織塊細(xì)胞增殖后,通過胰蛋白酶消化法分離獲得原代上皮細(xì)胞和成纖維細(xì)胞。通過在不同培養(yǎng)基中傳代培養(yǎng),進(jìn)一步純化兩種細(xì)胞。采用免疫熒光和HE染色進(jìn)行細(xì)胞鑒定。結(jié)果：組織塊植入后的第2d～3d可看到組織塊周邊出現(xiàn)新分裂增殖的細(xì)胞,約14 d細(xì)胞增殖范圍達(dá)到0.5 cm～1cm后,進(jìn)行初次傳代。胰蛋白酶消化法可初步分離出上皮細(xì)胞和成纖維細(xì)胞。結(jié)論：組織塊法結(jié)合胰蛋白酶消化法能快速高效的培養(yǎng)分離口腔粘膜上皮細(xì)胞和成纖維細(xì)胞,是一種實(shí)用的口腔粘膜細(xì)胞培養(yǎng)方法。2.支架篩選目的：篩選適合用于組織工程化口腔粘膜體外構(gòu)建的支架材料。材料與方法：將5組上皮細(xì)胞和成纖維細(xì)胞分別種植于脫細(xì)胞異體真皮基質(zhì)、PEG、PVA三種生物支架上,培養(yǎng)2 d后,采用CCK-8測(cè)定細(xì)胞活性,采用隨機(jī)區(qū)組設(shè)計(jì)方差分析統(tǒng)計(jì)數(shù)據(jù)。將成纖維細(xì)胞種植于三種生物支架上,培養(yǎng)1周后再將上皮細(xì)胞種植于支架上,繼續(xù)培養(yǎng)1周,HE染色及免疫組化觀察細(xì)胞在支架中的生長(zhǎng)狀態(tài)。結(jié)果：統(tǒng)計(jì)分析顯示,上皮細(xì)胞和成纖維細(xì)胞活性在三種生物材料中均無(wú)明顯的差異(P0.05)。HE染色及免疫組化顯示,三種生物支架材料中,脫細(xì)胞異體真皮基質(zhì)中的細(xì)胞數(shù)目多,分布層次深,細(xì)胞標(biāo)記蛋白表達(dá)較好。結(jié)論：三種生物支架中較適宜用于體外三維培養(yǎng)口腔粘膜上皮細(xì)胞及成纖維細(xì)胞的是脫細(xì)胞異體真皮基質(zhì)。3.組織工程化口腔粘膜體外預(yù)構(gòu)建 目的：通過體外三維培養(yǎng)口腔粘膜上皮細(xì)胞和成纖維細(xì)胞,模擬組織工程化口腔粘膜的構(gòu)建,探索組織工程化口腔粘膜體外培養(yǎng)的方法。材料與方法：將成纖維細(xì)胞種植于脫細(xì)胞異體真皮基質(zhì)上,培養(yǎng)1周后再將上皮細(xì)胞種植于支架上,繼續(xù)培養(yǎng)1周后,將支架移至氣-液平面培養(yǎng)5d。HE染色及免疫熒光觀察口腔粘膜上皮細(xì)胞與成纖維細(xì)胞體外三維培養(yǎng)的情況。結(jié)果：免疫熒光及HE染色顯示,經(jīng)過2周的液下培養(yǎng)及5d的氣-液培養(yǎng)后,細(xì)胞可以深入生長(zhǎng)植入到支架全層,其中上皮細(xì)胞主要集中在支架表層及淺層,成纖維細(xì)胞主要分布在支架深層。結(jié)論：口腔粘膜上皮細(xì)胞及成纖維細(xì)胞體外三維培養(yǎng)后細(xì)胞的分布狀態(tài)類似于口腔粘膜組織的基本分層,但細(xì)胞增殖數(shù)量少,細(xì)胞活性不佳,仍有待進(jìn)一步研究改進(jìn)實(shí)驗(yàn)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R782
，

本文編號(hào)：2183845