本文選題：MicroRNA　切入點(diǎn)：神經(jīng)嵴干細(xì)胞　出處：《吉林大學(xué)》2014年博士論文

【摘要】：近年來，隨著口腔種植醫(yī)學(xué)的迅猛發(fā)展和材料學(xué)的進(jìn)步，種植體支持的義齒已逐漸成為廣大牙列缺損和牙列缺失患者的首選修復(fù)方法。由于種植體周缺乏牙周膜結(jié)構(gòu)，對負(fù)荷的定位能力及整體的感覺的敏感性比天然牙弱，因此，我們期望種植義齒能夠更加靈敏的感知咬合力的大小和方向，種植義齒能夠更接近天然牙的功能。這一目標(biāo)可以通過神經(jīng)再生方面的研究加以實(shí)現(xiàn)。 研究表明，Schwann細(xì)胞(SCs)能夠促進(jìn)周圍神經(jīng)組織的修復(fù)再生。SCs是周圍神經(jīng)系統(tǒng)特有的膠質(zhì)細(xì)胞，周圍神經(jīng)損傷后，SCs吞噬變形的軸突與髓鞘碎屑，而且迅速增殖形成Bungner帶，并且在分泌神經(jīng)營養(yǎng)因子、細(xì)胞外基質(zhì)和粘附分子，引導(dǎo)軸突生長方向，維持神經(jīng)元活力，促進(jìn)損傷區(qū)的周圍神經(jīng)再支配過程。神經(jīng)嵴干細(xì)胞（NCSCs）起源于胚胎期的神經(jīng)嵴結(jié)構(gòu)，具有自我更新的能力及多分化潛能，毛囊來源的NCSCs取材簡便，是SCs的前體細(xì)胞，是理想的SCs來源。MicroRNAs（miRNAs）是一組生物體內(nèi)源性小RNA分子,是含有18-25個(gè)核苷酸的單鏈RNA分子，通過與mRNA的3’-UTR端結(jié)合抑制mRNA翻譯或者使mRNA在轉(zhuǎn)錄后降解調(diào)控基因表達(dá)，，參與多種生理過程，如干細(xì)胞分化、腫瘤形成和轉(zhuǎn)移、細(xì)胞凋亡、炎癥以及胚胎發(fā)育。 本實(shí)驗(yàn)通過分離培養(yǎng)人毛囊神經(jīng)嵴干細(xì)胞，并誘導(dǎo)其向Schwann細(xì)胞分化，應(yīng)用Microarray技術(shù)，篩選并驗(yàn)證干細(xì)胞向Schwann細(xì)胞分化相關(guān)miRNAs，并且深入探討miRNAs調(diào)控的相關(guān)功能及信號轉(zhuǎn)導(dǎo)通路，明確miRNAs的作用機(jī)制，為未來miRNAs的臨床應(yīng)用打下理論基礎(chǔ)。 1、誘導(dǎo)人毛囊神經(jīng)嵴干細(xì)胞向Schwann細(xì)胞分化 目的：分離培養(yǎng)人毛囊神經(jīng)嵴干細(xì)胞，誘導(dǎo)其向Schwann細(xì)胞分化，表達(dá)Schwann細(xì)胞相關(guān)標(biāo)志物。 方法：取材新鮮皮膚組織，分離收集毛囊，消化得到的原代細(xì)胞加入含4ng/ml堿性成纖維細(xì)胞生長因子的HESC培養(yǎng)基培養(yǎng)。利用流式細(xì)胞術(shù)分選神經(jīng)嵴干細(xì)胞標(biāo)志物p75和HNK1雙陽性細(xì)胞，得到純化后的神經(jīng)嵴干細(xì)胞。MesenPRO培養(yǎng)基加入20ng/ml神經(jīng)調(diào)節(jié)蛋白1誘導(dǎo)NCSCs分化為SCs。流式細(xì)胞術(shù)檢測SCs特征性標(biāo)志物S100和GFAP表達(dá)情況。 結(jié)果：將毛囊種植于培養(yǎng)皿內(nèi)可見隆突部位遷移出梭形細(xì)胞，流式分選出HNK1和p75雙陽性細(xì)胞即NCSCs，約占所有細(xì)胞比例的10%左右。將NCSCs種于培養(yǎng)基5天左右時(shí)，形成典型的干細(xì)胞團(tuán)塊。在誘導(dǎo)第30天時(shí)，通過流式細(xì)胞術(shù)進(jìn)行檢測發(fā)現(xiàn)，約有63.8±3.7%的細(xì)胞表達(dá)S100，約有72.6±4.9%的細(xì)胞表達(dá)GFAP，說明在誘導(dǎo)因子神經(jīng)調(diào)節(jié)蛋白1(Nrg1)的刺激下，NCSCs可分化為Schwann樣細(xì)胞。 結(jié)論：本實(shí)驗(yàn)成功培養(yǎng)人毛囊神經(jīng)嵴干細(xì)胞，并改良干細(xì)胞純化方法，得到良好純度干細(xì)胞，形成較為典型的NCSCs團(tuán)塊。NCSCs在Nrg1的刺激下可成功分化為Schwann細(xì)胞，表達(dá)特異性標(biāo)志物S100和GFAP。 2、人毛囊神經(jīng)嵴干細(xì)胞向Schwann細(xì)胞分化中的microRNA篩選和驗(yàn)證 目的：篩選參與NCSCs向SCs分化的miRNAs，選擇特異性miRNA驗(yàn)證其表達(dá)，觀察其對于分化過程的影響。 方法：應(yīng)用microarray技術(shù)，將S16細(xì)胞與未分化NCSCs對比，NCSCs誘導(dǎo)40天與誘導(dǎo)前對比，篩選表達(dá)出現(xiàn)變化的miRNAs。選擇miR-21，qRT-PCR方法驗(yàn)證其在誘導(dǎo)0、10、20、30、40天時(shí)間點(diǎn)表達(dá)變化。在誘導(dǎo)NCSCs向SCs分化過程中加入miR-21激動(dòng)劑agomir-21和阻斷劑antagomir-21，調(diào)控miR-21水平，通過流式細(xì)胞術(shù)和qRT-PCR檢測S100和GFAP表達(dá)水平。 結(jié)果：通過microarray檢測發(fā)現(xiàn)，誘導(dǎo)分化后干細(xì)胞相對于未分化干細(xì)胞中約有76種miRNAs水平下調(diào)4倍以上，89種miRNAs上調(diào)4倍以上。在誘導(dǎo)過程中在不同的時(shí)間點(diǎn)通過qRT-PCR檢測了miR-21的表達(dá)， miR-21在誘導(dǎo)分化SCs的第10天上升約3倍(p0.05)，并且隨著誘導(dǎo)時(shí)間的推移不斷上升，40天時(shí)達(dá)到約6倍。此結(jié)果表明miR-21在NCSCs向SCs分化中可能起到了潛在性的調(diào)節(jié)作用。 轉(zhuǎn)染agomir-21的NCSCs在誘導(dǎo)后約有75.3±5.6%的細(xì)胞同時(shí)表達(dá)S100和GFAP，明顯高于其陰性對照agomir-NC組（64.2±4.1%，p0.05）；antagomir-21組的NCSCs在誘導(dǎo)后約有41.1±3.2%的細(xì)胞同時(shí)表達(dá)S100和GFAP，明顯低于其陰性對照antagomir-NC組（62.0±2.7%，p0.01）。 qRT-PCR對各組細(xì)胞中的S100和GFAP mRNA進(jìn)行了檢測（以對照組S100和GFAP mRNA表達(dá)為1），結(jié)果發(fā)現(xiàn)轉(zhuǎn)染agomir-21的NCSCs在誘導(dǎo)后S100mRNA表達(dá)量為2.13±0.20，明顯高于其陰性對照agomir-NC組（1.03±0.21，p0.05）；antagomir-21組的NCSCs在誘導(dǎo)后S100mRNA表達(dá)量為0.50±0.06，明顯低于其陰性對照antagomir-NC組（0.95±0.13，p0.05）。agomir-21組和antagomir-21組NCSCs在誘導(dǎo)后GFAP mRNA表達(dá)量分別為3.27±0.44和0.53±0.08，分別高于/低于其陰性對照agomir-NC組（0.88±0.15，p0.01）和antagomir-NC組（1.17±0.19，p0.05）。此結(jié)果表明miR-21具有促進(jìn)NCSCs分化為SCs的作用。 結(jié)論：本實(shí)驗(yàn)通過microarray實(shí)驗(yàn)篩選參與NCSCs向SCs分化的miRNAs，發(fā)現(xiàn)miR-21的表達(dá)上調(diào)，同時(shí)進(jìn)一步通過qRT-PCR驗(yàn)證了miR-21在NCSCs分化為SCs后表達(dá)升高。并且通過人為控制miR-21表達(dá)，證實(shí)miR-21能夠促進(jìn)NCSCs向SCs的分化。 3、miR-21通過抑制SOX2表達(dá)調(diào)控毛囊NCSCs向Schwann細(xì)胞分化 目的：尋找miR-21調(diào)控NCSCs分化的機(jī)制，明確miR-21的作用靶點(diǎn)和SOX2與miR-21的調(diào)控分化的關(guān)系。 方法：通過數(shù)據(jù)庫查詢miR-21的作用靶點(diǎn)，將SOX2的3’UTR端和突變后的片段分別構(gòu)建入luciferase報(bào)告系統(tǒng)，驗(yàn)證miR-21是否與SOX2的3’UTR端結(jié)合。通過轉(zhuǎn)染miR-21激動(dòng)劑agomiR-21和阻斷劑antagomiR-21，qRT-PCR和western blot法檢驗(yàn)SOX2表達(dá)水平，觀察miR-21對SOX2的mRNA和蛋白水平的影響。為檢測SOX2是否參與miR-21促干細(xì)胞分化，將NCSCs的SOX2基因敲除后，qRT-PCR和流式細(xì)胞術(shù)觀察antagomir-21對干細(xì)胞分化的影響。 結(jié)果：數(shù)據(jù)庫查詢結(jié)果發(fā)現(xiàn)SOX2基因的3’UTR端包含一個(gè)7mer大小的序列與miR-21相匹配。Luciferase報(bào)告實(shí)驗(yàn)顯示在miR-21作用下SOX2-3’UTR-wt組（野生型組）熒光強(qiáng)度明顯低于其陰性對照組（miR-con），而SOX2-3’UTR-mut組（突變組）或空質(zhì)粒組熒光強(qiáng)度與其陰性對照無明顯差異。此結(jié)果表明，miR-21可以通過與SOX2mRNA3’UTR端結(jié)合而調(diào)節(jié)其翻譯。 Western blot結(jié)果發(fā)現(xiàn)agomiR-21可顯著降低NCSCs細(xì)胞中SOX2水平（p0.05），而antagomiR-21可顯著升高NCSCs細(xì)胞中SOX2水平（p0.05）。但無論是agomiR-21還是antagomiR-21，均不影響SOX2mRNA表達(dá)。這些結(jié)果表明，miR-21可以通過與SOX2mRNA3’UTR端結(jié)合在轉(zhuǎn)錄后水平調(diào)節(jié)其蛋白表達(dá)水平。 我們通過慢病毒shRNA敲除NCSCs中的SOX2，并將此細(xì)胞（SOX2-KD-NCSCs）與普通NCSCs（Control）一起用于研究SOX2在miR-21調(diào)節(jié)干細(xì)胞分化中所扮演的角色。通過流式細(xì)胞術(shù)和qRT-PCR對S100和GFAP蛋白及mRNA檢測發(fā)現(xiàn)，轉(zhuǎn)染antagomiR-21的NCSCs分化能力明顯低于轉(zhuǎn)染antagomiR-NC的NCSCs（p0.01），而轉(zhuǎn)染antagomiR-21的SOX2-KD-NCSCs分化能力與轉(zhuǎn)染antagomiR-NC的SOX2-KD-NCSCs相比并無明顯變化。此結(jié)果表明敲除SOX2可阻斷antagomir-21的抗SCs分化能力，證明SOX2是miR-21調(diào)節(jié)NCSCs分化的下游靶點(diǎn)之一。 結(jié)論：本實(shí)驗(yàn)通過Luciferase assay方法驗(yàn)證了miR-21可以通過與SOX2mRNA3’UTR端結(jié)合而調(diào)節(jié)其翻譯。并且，miR-21不影響SOX2的mRNA表達(dá)，通過與SOX2mRNA3’UTR端結(jié)合在轉(zhuǎn)錄后水平調(diào)節(jié)其蛋白表達(dá)。敲除SOX2可阻斷antagomir-21的抗SCs分化能力，明確SOX2是miR-21調(diào)節(jié)NCSCs分化的下游靶點(diǎn)之一。
[Abstract]:In recent years , with the rapid development of dental implant medicine and the progress of material science , the artificial tooth supported by implant has become the preferred repair method for the patients with missing teeth and missing teeth . Because of the lack of periodontal membrane structure around the implant , the sensitivity of the implant to the positioning ability and the whole feeling of the implant is weaker than that of the natural tooth . Therefore , it is expected that the implant tooth can sense the size and direction of the bite force more sensitively , and the implant tooth can be closer to the function of the natural tooth . This goal can be realized through the research of nerve regeneration .

The research shows that Schwann cells ( SCs ) can promote the repair and regeneration of peripheral nerve tissue . SCs are glial cells which are characteristic of peripheral nervous system . After peripheral nerve injury , SCs phagocytose and deform axons and myelin debris , and they rapidly proliferate to form Bungner bands . The NCSCs are a group of endogenous small RNA molecules . It is an ideal source of SCs . MicroRNAs ( NCSCs ) are a group of endogenous small RNA molecules . They are involved in various physiological processes , such as stem cell differentiation , tumor formation and metastasis , apoptosis , inflammation and embryonic development .

By separating and culturing the neural crest stem cells of human hair follicle and inducing them to differentiate into Schwann cells , we apply the technique of biotechnology to screen and verify the differentiation of stem cells into Schwann cells .

1 . Induction of differentiation of human hair follicle neural crest stem cells into Schwann cells

Objective : To isolate cultured human hair follicle neural crest stem cells and induce them to differentiate into Schwann cells and express Schwann cell related markers .

Methods : Fresh skin tissue was isolated from fresh skin , and the primary cells obtained from digestion were cultured in HESC culture medium containing 4ng / ml basic fibroblast growth factor . The neural crest stem cell marker p75 and HNK1 double positive cells were sorted by flow cytometry to obtain purified neural crest stem cells . The cultured neural crest stem cells were obtained by adding 20ng / ml neuromodulation protein 1 to induce NCSCs differentiation into SCs . Flow cytometry was used to detect the expression of the characteristic markers S100 and GFAP in SCs .

Results : It was found that NCSCs could be differentiated into Schwann cells under the stimulation of induction factor neuromodulation protein 1 ( Nrg1 ) .

Conclusion : This experiment successfully cultured human hair follicle neural crest stem cells and improved the method of stem cell purification to obtain good purity stem cells . The NCSCs can be successfully differentiated into Schwann cells under the stimulation of Nrg1 and express specific markers S100 and GFAP .

2 . microRNA screening and verification of human hair follicle neural crest stem cells into Schwann cell differentiation

Objective : To screen the expression of NCSCs in the differentiation of SCs and to observe its effect on the differentiation process .

Methods : The expression of miR - 21 and qRT - PCR was used to compare the expression of miR - 21 agonist agomir - 21 and blocking agent , and control the expression of miR - 21 by flow cytometry and qRT - PCR . The expression of miR - 21 was detected by flow cytometry and qRT - PCR .

Results : The expression of miR - 21 was detected by qRT - PCR and miR - 21 increased about 3 times ( p . 05 ) on the 10th day of induction and differentiation of SCs by qRT - PCR , and the expression of miR - 21 was detected by qRT - PCR at different time points . The results showed that miR - 21 could play a potential role in the differentiation of SCs into SCs .

After induction , the NCSCs transfected with agomir - 21 expressed S100 and GFAP at 75.3 鹵 5.6 % , which was significantly higher than that of the negative control agomir - NC group ( 64.2 鹵 4.1 % , p0.05 ) .
After induction , the NCSCs expressed S100 and GFAP at about 41.1 鹵 3.2 % , which was significantly lower than that of control group ( 62.0 鹵 2.7 % , p0.01 ) .

The expression of S100 and GFAP in each group was detected by qRT - PCR ( the expression of S100 and GFAP mRNA in the control group was 1 ) . The results showed that the expression of NCSCs transfected with agomir - 21 was 2.13 鹵 0.20 , which was significantly higher than that of the negative control agomir - NC group ( 1.03 鹵 0.21 , p0.05 ) .
The expression of GFAP mRNA after induction of NCSCs was 0 . 50 鹵 0 . 06 , 0 . 95 鹵 0 . 13 and 0 . 53 鹵 0 . 08 , respectively , and the expression of GFAP mRNA was 3.27 鹵 0 . 44 and 0.53 鹵 0.08 , respectively . The results show that miR - 21 has the function of promoting the differentiation of NCSCs into SCs .

Conclusion : The expression of miR - 21 in NCSCs can be increased and the expression of miR - 21 is increased through qRT - PCR , and miR - 21 can promote the differentiation of NCSCs to SCs by artificially controlling miR - 21 expression .

3 . miR - 21 regulates the differentiation of hair follicle NCSCs to Schwann cells by inhibiting SOX2 expression

Objective : To find the mechanism of miR - 21 to regulate and regulate the differentiation of NCSCs , and clarify the relationship between the action target of miR - 21 and the regulation and differentiation of SOX2 and miR - 21 .

Methods : The effects of miR - 21 on the mRNA and protein levels of SOX2 were investigated by querying the target of miR - 21 by database . The effects of miR - 21 on the mRNA and protein levels of SOX2 were investigated . The effects of miR - 21 on the mRNA and protein levels of SOX2 were observed .

Results : The results of database query found that the 3 ' - terminal of SOX2 gene contained a 7 mer - size sequence matched with miR - 21 . The results of Lucifer ' s report showed that the fluorescence intensity of SOX2 - 3 ' UU- wt group ( wild - type group ) was significantly lower than that of the negative control group ( miR - con ) under the action of miR - 21 . The results showed that miR - 21 could be translated by binding to the SOX2 - 3 ' - terminal .

Western blot analysis showed that the expression of SOX2 mRNA in NCSCs cells was significantly lower than that in NCSCs ( P < 0.05 ) .

We found that the ability of SOX2 - KD - NCSCs to differentiate into NCSCs was significantly lower than that of NCSCs transfected with NCSCs ( p0.01 ) , but SOX2 - KD - NCSCs was transfected into NCSCs by flow cytometry and qRT - PCR .

Conclusion : Through Lucifer assay , miR - 21 can be translated by binding to SOX2 mRNA3 ' - terminal . Furthermore , miR - 21 does not affect the mRNA expression of SOX2 , and regulates its protein expression at post - transcriptional level by binding to the SOX2 mRNA3 ' terminal . The knock - out SOX2 can block the differentiation of the SCs , and clarify that SOX2 is one of the downstream targets of miR - 21 regulation of NCSCs differentiation .

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R783.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 張韶輝;魏廣和;劉立新;;MicroRNA研究的基本方法及流程[J];濟(jì)寧醫(yī)學(xué)院學(xué)報(bào);2013年02期

2 喬士沖;呂曉飛;莊龍飛;姜煥煥;張志勇;賴紅昌;;牙種植體周圍骨內(nèi)神經(jīng)感受功能的初步研究[J];上�？谇会t(yī)學(xué);2011年02期

3 張凱;王毅;邢國勝;;間充質(zhì)干細(xì)胞的生物學(xué)特性及多向分化潛能[J];中國組織工程研究與臨床康復(fù);2008年03期

4 顧丹丹;張沛云;;許旺細(xì)胞對周圍神經(jīng)再生的促進(jìn)效應(yīng)[J];中國組織工程研究與臨床康復(fù);2008年25期

5 張殿英,姜保國,傅忠國,閆家智,唐杞衡;周圍神經(jīng)損傷后S-100蛋白的分布和變化研究[J];中國矯形外科雜志;2002年04期

6 林野;陳琰;;種植義齒的骨感知現(xiàn)象及研究[J];中華口腔醫(yī)學(xué)研究雜志(電子版);2008年04期

本文編號：1709471