【摘要】：誘導多能干細胞(iPS)技術(shù)作為一種新的醫(yī)療策略,在牙齒修復領(lǐng)域有廣闊的應(yīng)用前景。多種來源的細胞已被證實可以轉(zhuǎn)化為iPS細胞。同時,這些iPS細胞兼具有組織記憶性,使得之后的誘導過程更為簡易有效。課題組先前的實驗已成功將人源iPS細胞誘導分化為具有成牙潛能的上皮細胞,在與胎鼠口腔間充質(zhì)共培養(yǎng)之后移植到裸鼠中成功再生出牙齒。為了進一步發(fā)現(xiàn)該牙齒再生模型的可行性以及間充質(zhì)對牙齒再生的影響,我們進行了本課題的研究。本研究將人尿液來源的誘導多能干細胞誘導分化為上皮樣細胞,利用qPCR以及免疫熒光檢測其最適合的移植時期,從而選得上皮細胞。實驗將E48天的胎豬中的牙胚進行消化分離得到口腔間充質(zhì),將該間充質(zhì)以及消化上皮樣細胞得到的上皮細胞層在體外重新結(jié)合后共培養(yǎng)1-2天移植入裸鼠腎包膜上進行牙齒的再生。之后對比先前實驗中鼠間充質(zhì)來源的再生牙齒和豬間充質(zhì)來源的再生牙齒的大小以及承壓率,并對牙齒進行HE染色等實驗。結(jié)果顯示,通過對上皮細胞相關(guān)基因進行檢測發(fā)現(xiàn)人尿來源的iPS細胞誘導的上皮細胞層在第7天的時同間充質(zhì)的結(jié)合能力最為優(yōu)異。將該上皮細胞層同胎豬口腔間充質(zhì)重新結(jié)合后共培養(yǎng)的組織塊移植入腎包膜后成功再生出牙齒。統(tǒng)計發(fā)現(xiàn)豬牙胚再生的牙齒的成牙率為92.86%,豬間充質(zhì)共培養(yǎng)再生牙齒的成牙率為72.97%,遠高于已發(fā)表文章中鼠間充質(zhì)共培養(yǎng)再生牙齒(21.81%)的成牙率。同時,所有生成的類牙齒組織都大于鼠口腔間充質(zhì)來源的。實驗中移植的牙齒在20周時,其上皮細胞分化為成釉細胞,并伴有牙髓的生成。綜上所述,實驗表明,我們的人iPS再生牙齒模型有很好的適用性,同時口腔間充質(zhì)對牙齒的再生有很大的影響。這為我們之后使用人源iPS細胞再生出牙齒提供了良好的基礎(chǔ),加深了我們對口腔間充質(zhì)的認知,為之后人的牙齒再生應(yīng)用起一定的推動作用。
[Abstract]:As a new medical strategy, induced pluripotent stem cells (iPS) has broad application prospects in dental restoration. Cells from a variety of sources have been proved to be able to transform into iPS cells. At the same time, these iPS cells also have tissue memory, which makes the induction process easier and more effective. Previous experiments in our team have successfully induced human iPS cells to differentiate into epithelial cells with odontogenic potential, and successfully regenerated teeth in nude mice after co-culture with fetal rat oral mesenchymal cells. In order to find the feasibility of the tooth regeneration model and the effect of mesenchymal on tooth regeneration, we carried out this study. In this study, human urine-derived induced pluripotent stem cells were induced to differentiate into epithelial-like cells. QPCR and immunofluorescence were used to detect the most suitable period of transplantation to select epithelial cells. Oral mesenchymal tissue was obtained by digesting and separating tooth germs from E48-day-old pigs. The epithelial cell layer obtained from the mesenchymal and digestive epithelial-like cells was recombined in vitro and then co-cultured for 1 day and 2 days after transplantation into the renal capsule of nude mice for tooth regeneration. Then we compared the size and compression rate of the regenerated rat mesenchymal teeth and porcine mesenchymal regenerated teeth in previous experiments, and the teeth were stained with HE. The results showed that the epithelial cell layer induced by human urine-derived iPS cells had the best ability to bind to mesenchymal cells on the 7th day after the detection of epithelial cell-related genes. The epithelial cell layer was recombined with fetal porcine oral mesenchymal tissue and the co-cultured tissue was transplanted into the renal capsule to regenerate the teeth successfully. It was found that the rate of tooth formation was 92.86% for porcine tooth germ regeneration and 72.97% for porcine mesenchymal co-culture regeneration teeth, which was much higher than that of mouse mesenchymal co-culture regeneration teeth (21.81%) in published articles. At the same time, all the generated tooth-like tissues were larger than those derived from mouse oral mesenchymal tissue. At 20 weeks after transplantation, the epithelial cells of the transplanted teeth differentiated into ameloblasts, accompanied by pulp formation. In conclusion, the experiment shows that our human iPS dental regeneration model has good applicability, and oral mesenchymal has a great effect on tooth regeneration. This provides a good basis for us to regenerate teeth by using human iPS cells, deepen our understanding of oral mesenchymal tissue, and promote the application of human tooth regeneration.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R78;R318.08

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