本文選題：牙周膜干細(xì)胞 + 根端牙胚細(xì)胞�。� 參考：《福建醫(yī)科大學(xué)》2015年碩士論文

【摘要】：正畸治療過程中,常常發(fā)生患者口腔衛(wèi)生情況不佳導(dǎo)致的牙槽骨吸收,牙槽骨作為牙周組織,借助牙周膜纖維與牙齒緊密相連,在維持牙齒的穩(wěn)定和行使正常生理功能上發(fā)揮著重要作用。在臨床上發(fā)現(xiàn),由于牙周疾病或者正畸力量過大,往往導(dǎo)致牙槽骨吸收。人牙周韌帶中存在有牙周膜干細(xì)胞(periodontal ligament stem cells,PDLSCs),其具有自我增殖和多向分化潛能,可作為組織工程最直接可靠的種子細(xì)胞。研究表明,h PDLSCs在不同的培養(yǎng)環(huán)境和細(xì)胞因子的作用下,可不斷增殖分化成為成骨細(xì)胞、成牙骨質(zhì)細(xì)胞和成纖維細(xì)胞;其可修復(fù)牙周缺損,使牙周組織再生。Micro RNA在間充質(zhì)干細(xì)胞向成骨細(xì)胞分化過程中起著重要作用。h PDLSCs屬于間充質(zhì)干細(xì)胞,具有多向分化潛能,然而在h PDLSCs向成骨細(xì)胞方向分化過程中,hsa-mi R-146a是否起到了重要的調(diào)控作用尚未得知。目的為了研究在特定體外微環(huán)境中人牙周膜干細(xì)胞向成骨細(xì)胞方向分化過程中mi R46a-5p是否發(fā)揮調(diào)控作用,從而為牙槽骨的再生及發(fā)育機(jī)制提供基礎(chǔ)性研究。實(shí)驗(yàn)方法本實(shí)驗(yàn)首先構(gòu)建慢病毒特異性上/下調(diào)mi R146a-5p,進(jìn)而通過由根端牙胚細(xì)胞(Apical Tooth germ cells,APTGC)構(gòu)成的體外微環(huán)境誘導(dǎo)h PDLSCs向成骨細(xì)胞方向分化,對(duì)誘導(dǎo)前后的h PDLSCs進(jìn)行堿性磷酸酶(Alkaline Phosphatase,ALP)活性檢測(cè)以及成骨相關(guān)基因:(ALP、BSP及OCN)檢測(cè)。1.h PDLSCs的體外培養(yǎng)和篩選;2.APTG誘導(dǎo)培養(yǎng)基的制備;3.RT-QPCR驗(yàn)證mi R146a-5p表達(dá)差異;4.慢病毒轉(zhuǎn)染h PDLSCs;5.ALP活性檢測(cè);6.Real-time PCR檢測(cè)特異性上/下調(diào)mi R146a-5p后成骨相關(guān)基因:(ALP、BSP及OCN)的變化。實(shí)驗(yàn)結(jié)果1.通過免疫磁珠法篩選獲得h PDLSCs。2.RT-QPCR檢測(cè)驗(yàn)證mi R146a-5p在h PDLSCs向成骨細(xì)胞方向分化過程中表達(dá)增高。3.慢病毒特異性轉(zhuǎn)染效率檢測(cè)顯示LV-has-mi R146a-5p及LV-has-mi R146a-5pinhibitor導(dǎo)入人牙周膜干細(xì)胞后其mi R146a-5p特異性表達(dá)為上調(diào)及下調(diào)。4.ALP活性檢測(cè)顯示,LV-has-mi R146a-5p上調(diào)組細(xì)胞ALP活性較對(duì)照組有顯著升高,LV-has-mi R146a-5pinhibitor組細(xì)胞ALP活性較對(duì)照組有顯著降低。5.RT-PCR檢測(cè)顯示LV-has-mi R146a-5p上調(diào)組細(xì)胞成骨細(xì)胞相關(guān)基因(ALP、BSP及OCN)的轉(zhuǎn)錄水平表達(dá)量增加,LV-has-mi R146a-5pinhibitor組細(xì)胞成骨細(xì)胞相關(guān)基因(ALP、BSP及OCN)的轉(zhuǎn)錄水平表達(dá)量降低。結(jié)論mi R-146a-5p促進(jìn)人牙周膜干細(xì)胞向成骨細(xì)胞分化。
[Abstract]:During orthodontic treatment, alveolar bone resorption often occurs due to poor dental hygiene. Alveolar bone, as a periodontal tissue, is closely connected with teeth by periodontal ligament fibers. It plays an important role in maintaining dental stability and exercising normal physiological function. Clinically, alveolar bone resorption is often due to periodontal disease or excessive orthodontic force. There are periodontal ligament stem cells (PDLSCs) in human periodontal ligament, which have the potential of self-proliferation and multi-differentiation, and can be used as the most direct and reliable seed cells in tissue engineering. The results showed that PDLSCs could proliferate and differentiate into osteoblasts, cementoblasts and fibroblasts under different culture conditions and cytokines, and could repair periodontal defects. Making periodontal tissue regenerate. Micro RNA play an important role in the differentiation of mesenchymal stem cells into osteoblasts. H PDLSCs belongs to mesenchymal stem cells and has the potential of multiple differentiation. However, whether hsa-mi R-146a plays an important regulatory role in the differentiation of h PDLSCs into osteoblasts is not known. Objective to investigate whether mi R46a-5p plays a regulatory role in the differentiation of human periodontal ligament stem cells into osteoblasts in a specific microenvironment in vitro, so as to provide basic research for the regeneration and development of alveolar bone. Methods Lentivirus specific up-/ down-regulated mi R146a-5p was constructed firstly, and then h PDLSCs was induced to differentiate into osteoblast by microenvironment composed of apical Tooth germ cells (Apical Tooth germ cells). The alkaline phosphatase (ALP) activity of h PDLSCs before and after induction and the osteoblast-associated genes (BSP and OC) were detected by alkaline phosphatase and osteoblasts. 1. The culture and screening of 2. APTG induced culture medium were carried out. 3. RT-QPCR was used to verify the difference of mi R146a-5p expression. The activity of ALP in lentivirus transfected hPDLSCs5. Real-time PCR was used to detect the changes of osteoblast-associated genes (BSP and OCNs) after specific up-/ down-regulation of mi R146a-5p. Experimental results 1. H PDLSCs.2.RT-QPCR detection was performed by immunomagnetic bead assay to verify the increased expression of mi R146a-5p during the differentiation of h PDLSCs into osteoblasts. Detection of lentivirus-specific transfection efficiency showed that the specific expression of mi R146a-5p was up-regulated and down-regulated after the introduction of LV-has-mi R146a-5p and LV-has-mi R146a-5pinhibitor into human periodontal ligament stem cells. 4. Detection of ALP activity showed that the ALP activity of LV-has-mi R146a-5p upregulated group was significantly higher than that of control group. The activity of ALP in R146a-5pinhibitor group was significantly lower than that in control group. 5. RT-PCR assay showed that LV-has-mi R146a-5p up-regulated the expression of osteoblast-associated genes (ALPG-BSP and OCN-C) in LV-has-mi R146a-5pinhibitor group, and increased the expression level of ALPH-BSP and OCN-related genes in LV-has-mi R146a-5pinhibitor group. Conclusion mi R-146a-5p can promote the differentiation of human periodontal ligament stem cells into osteoblasts.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R783.5

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本文編號(hào)：1915689