本文關(guān)鍵詞：Periostin對(duì)牙周膜干細(xì)胞定向分化為神經(jīng)細(xì)胞作用的影響　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：研究背景神經(jīng)的構(gòu)建在生理性牙周膜重建的過程中是不可或缺的重要環(huán)節(jié),對(duì)于維持牙周膜的完整性以及牙周膜功能的行使具有一定意義。因此,牙周膜內(nèi)神經(jīng)的重建對(duì)于種植體周圍生理性牙周膜的構(gòu)建有重要意義。既往研究發(fā)現(xiàn),牙周膜干細(xì)胞有向神經(jīng)細(xì)胞分化的潛能,為生理性牙周膜的重建提供了重要的理論依據(jù)。因此,如何有效地促進(jìn)牙周膜干細(xì)胞分化為神經(jīng)細(xì)胞是本課題的核心內(nèi)容。實(shí)驗(yàn)?zāi)康臄M通過研究Periostin對(duì)牙周膜干細(xì)胞(hPDLSCs)定向分化為神經(jīng)細(xì)胞的作用及調(diào)控機(jī)制,進(jìn)而探索重建生理性牙周膜的可能,為種植體周圍生理性牙周膜的構(gòu)建提供重要理論依據(jù)。實(shí)驗(yàn)方法第一部分:人牙周膜干細(xì)胞的培養(yǎng)及誘導(dǎo)分化1.1人牙周膜干細(xì)胞(hPDLSCs)的誘導(dǎo)培養(yǎng)對(duì)照組:將hPDLSCs接種于六孔板,加入bFGF預(yù)誘導(dǎo)液,誘導(dǎo)液中bFGF的濃度為10ng/mL,在誘導(dǎo)24h后換液,用PBS輕輕洗滌三次后再加入誘導(dǎo)液,繼續(xù)進(jìn)行誘導(dǎo)培養(yǎng),隔天換液并拍照。實(shí)驗(yàn)組:加入誘導(dǎo)液同時(shí)添加100 ng/mL的Periostin外源性刺激的牙周膜干細(xì)胞繼續(xù)誘導(dǎo)培養(yǎng),隔天換液并拍照。1.2 Western blot、免疫熒光檢測(cè)牙周膜干細(xì)胞成神經(jīng)相關(guān)蛋白(β-tubulinⅢ、GFAP、NSE)的表達(dá)。第二部分:沉默牙周膜干細(xì)胞Periostin的表達(dá)對(duì)hPDLSCs定向分化為神經(jīng)細(xì)胞的影響2.1構(gòu)建Periostin基因干擾慢病毒,RT-PCR法檢測(cè)慢病毒LVX-PERIOSTIN-RNAi 下調(diào) PERIOSTIN 基因表達(dá)效率2.2 Periostin基因的干擾慢病毒感染hPDLSCs細(xì)胞,WB、免疫熒光檢測(cè)牙周膜干細(xì)胞成神經(jīng)相關(guān)蛋白(β-tubulin Ⅲ、GFAP、NSE)的表達(dá)。實(shí)驗(yàn)結(jié)果1.培養(yǎng)的hPDLSCs細(xì)胞呈現(xiàn)多角形、不規(guī)則或者是梭型。經(jīng)過Periostin刺激誘導(dǎo)分化后,細(xì)胞逐漸變?yōu)殚L(zhǎng)梭形,呈現(xiàn)出神經(jīng)細(xì)胞的特征性形態(tài)。2.WB及免疫熒光染色顯示,在hPDLSCs細(xì)胞中,β-tubulin Ⅲ、GFAP、NSE均呈低表達(dá),而經(jīng)成神經(jīng)誘導(dǎo)培養(yǎng)后,其β-tubulin Ⅲ、GFAP、NSE表達(dá)水平顯著升高,在此基礎(chǔ)上,使用外源性Periostin刺激后,β-tubulin Ⅲ、GFAP、NSE表達(dá)水平進(jìn)一步升高。WB結(jié)果顯示,在培養(yǎng)第4天時(shí),β-tubulinⅢ、GFAP、NSE表達(dá)水平達(dá)到一個(gè)相對(duì)的高峰,在誘導(dǎo)培養(yǎng)第7天時(shí),β-tubulin Ⅲ、GFAP、NSE表達(dá)水平較第4天時(shí)有所下降。3.我們成功構(gòu)建了 LVX-PERIOSTIN-RNAi,并使用其感染hPDLSCs細(xì)胞。進(jìn)一步采用western blot、免疫熒光法檢測(cè)了 LVX-PERIOSTIN-RNAi感染后神經(jīng)細(xì)胞特異性相關(guān)蛋白的表達(dá)。結(jié)果顯示,在感染空載體后,外源性Periostin能夠顯著上調(diào)神經(jīng)細(xì)胞特異性相關(guān)蛋白β-tubulin Ⅲ、GFAP、NSE 的表達(dá)。而在感染 LVX-PERIOSTIN-RNAi 后,β-tubulin Ⅲ、GFAP、NSE的表達(dá)水平較空載體組顯著下降。結(jié)論1.外源性Periostin能夠促進(jìn)牙周膜干細(xì)胞定向分化為神經(jīng)細(xì)胞;2.慢病毒沉默牙周膜干細(xì)胞Periostin的表達(dá),能夠顯著抑制牙周膜干細(xì)胞定向分化為神經(jīng)細(xì)胞。
[Abstract]:Background the construction of nerve is an indispensable link in the process of physiological periodontal ligament reconstruction, and it is of certain significance to maintain the integrity of periodontal ligament and the function of periodontal ligament. The reconstruction of periodontal ligament nerve plays an important role in the construction of physiological periodontal ligament around implants. Previous studies have found that periodontal ligament stem cells have the potential to differentiate into neural cells. It provides an important theoretical basis for the reconstruction of physiological periodontal ligament. How to effectively promote the differentiation of periodontal ligament stem cells into nerve cells is the core of this study. The role and regulatory mechanism of directional differentiation into nerve cells. To explore the possibility of reconstruction of physiological periodontal membrane. An important theoretical basis for the construction of physiological periodontal ligament around implants. Part one: culture and differentiation of Human periodontal ligament Stem cells (hPDLSCs). HPDLSCs was inoculated on the six-hole plate. The concentration of bFGF in the induction solution was 10ng / mL by adding bFGF pre-induction solution. After 24 hours of induction, the solution was changed, then gently washed with PBS for three times, and then added to the inducer solution, and then the induction culture was carried out. The experiment group: the periodontal ligament stem cells stimulated by 100 ng/mL Periostin were added to the solution and cultured continuously. The next day we changed the fluid and photographed. 1.2 Western blot.The immunofluorescence was used to detect the expression of 尾 -tubulin 鈪，

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