MicroRNA对人毛囊神经嵴干细胞向Schwann细胞分化影响的实验研究

发布时间：2018-04-04 10:02

  本文选题：MicroRNA　切入点：神经嵴干细胞　出处：《吉林大学》2014年博士论文

【摘要】：近年来，随着口腔种植医学的迅猛发展和材料学的进步，种植体支持的义齿已逐渐成为广大牙列缺损和牙列缺失患者的首选修复方法。由于种植体周缺乏牙周膜结构，对负荷的定位能力及整体的感觉的敏感性比天然牙弱，因此，我们期望种植义齿能够更加灵敏的感知咬合力的大小和方向，种植义齿能够更接近天然牙的功能。这一目标可以通过神经再生方面的研究加以实现。 研究表明，Schwann细胞(SCs)能够促进周围神经组织的修复再生。SCs是周围神经系统特有的胶质细胞，周围神经损伤后，SCs吞噬变形的轴突与髓鞘碎屑，而且迅速增殖形成Bungner带，并且在分泌神经营养因子、细胞外基质和粘附分子，引导轴突生长方向，维持神经元活力，促进损伤区的周围神经再支配过程。神经嵴干细胞（NCSCs）起源于胚胎期的神经嵴结构，具有自我更新的能力及多分化潜能，毛囊来源的NCSCs取材简便，是SCs的前体细胞，是理想的SCs来源。MicroRNAs（miRNAs）是一组生物体内源性小RNA分子,是含有18-25个核苷酸的单链RNA分子，通过与mRNA的3’-UTR端结合抑制mRNA翻译或者使mRNA在转录后降解调控基因表达，，参与多种生理过程，如干细胞分化、肿瘤形成和转移、细胞凋亡、炎症以及胚胎发育。 本实验通过分离培养人毛囊神经嵴干细胞，并诱导其向Schwann细胞分化，应用Microarray技术，筛选并验证干细胞向Schwann细胞分化相关miRNAs，并且深入探讨miRNAs调控的相关功能及信号转导通路，明确miRNAs的作用机制，为未来miRNAs的临床应用打下理论基础。 1、诱导人毛囊神经嵴干细胞向Schwann细胞分化 目的：分离培养人毛囊神经嵴干细胞，诱导其向Schwann细胞分化，表达Schwann细胞相关标志物。 方法：取材新鲜皮肤组织，分离收集毛囊，消化得到的原代细胞加入含4ng/ml碱性成纤维细胞生长因子的HESC培养基培养。利用流式细胞术分选神经嵴干细胞标志物p75和HNK1双阳性细胞，得到纯化后的神经嵴干细胞。MesenPRO培养基加入20ng/ml神经调节蛋白1诱导NCSCs分化为SCs。流式细胞术检测SCs特征性标志物S100和GFAP表达情况。 结果：将毛囊种植于培养皿内可见隆突部位迁移出梭形细胞，流式分选出HNK1和p75双阳性细胞即NCSCs，约占所有细胞比例的10%左右。将NCSCs种于培养基5天左右时，形成典型的干细胞团块。在诱导第30天时，通过流式细胞术进行检测发现，约有63.8±3.7%的细胞表达S100，约有72.6±4.9%的细胞表达GFAP，说明在诱导因子神经调节蛋白1(Nrg1)的刺激下，NCSCs可分化为Schwann样细胞。 结论：本实验成功培养人毛囊神经嵴干细胞，并改良干细胞纯化方法，得到良好纯度干细胞，形成较为典型的NCSCs团块。NCSCs在Nrg1的刺激下可成功分化为Schwann细胞，表达特异性标志物S100和GFAP。 2、人毛囊神经嵴干细胞向Schwann细胞分化中的microRNA筛选和验证 目的：筛选参与NCSCs向SCs分化的miRNAs，选择特异性miRNA验证其表达，观察其对于分化过程的影响。 方法：应用microarray技术，将S16细胞与未分化NCSCs对比，NCSCs诱导40天与诱导前对比，筛选表达出现变化的miRNAs。选择miR-21，qRT-PCR方法验证其在诱导0、10、20、30、40天时间点表达变化。在诱导NCSCs向SCs分化过程中加入miR-21激动剂agomir-21和阻断剂antagomir-21，调控miR-21水平，通过流式细胞术和qRT-PCR检测S100和GFAP表达水平。 结果：通过microarray检测发现，诱导分化后干细胞相对于未分化干细胞中约有76种miRNAs水平下调4倍以上，89种miRNAs上调4倍以上。在诱导过程中在不同的时间点通过qRT-PCR检测了miR-21的表达， miR-21在诱导分化SCs的第10天上升约3倍(p0.05)，并且随着诱导时间的推移不断上升，40天时达到约6倍。此结果表明miR-21在NCSCs向SCs分化中可能起到了潜在性的调节作用。 转染agomir-21的NCSCs在诱导后约有75.3±5.6%的细胞同时表达S100和GFAP，明显高于其阴性对照agomir-NC组（64.2±4.1%，p0.05）；antagomir-21组的NCSCs在诱导后约有41.1±3.2%的细胞同时表达S100和GFAP，明显低于其阴性对照antagomir-NC组（62.0±2.7%，p0.01）。 qRT-PCR对各组细胞中的S100和GFAP mRNA进行了检测（以对照组S100和GFAP mRNA表达为1），结果发现转染agomir-21的NCSCs在诱导后S100mRNA表达量为2.13±0.20，明显高于其阴性对照agomir-NC组（1.03±0.21，p0.05）；antagomir-21组的NCSCs在诱导后S100mRNA表达量为0.50±0.06，明显低于其阴性对照antagomir-NC组（0.95±0.13，p0.05）。agomir-21组和antagomir-21组NCSCs在诱导后GFAP mRNA表达量分别为3.27±0.44和0.53±0.08，分别高于/低于其阴性对照agomir-NC组（0.88±0.15，p0.01）和antagomir-NC组（1.17±0.19，p0.05）。此结果表明miR-21具有促进NCSCs分化为SCs的作用。 结论：本实验通过microarray实验筛选参与NCSCs向SCs分化的miRNAs，发现miR-21的表达上调，同时进一步通过qRT-PCR验证了miR-21在NCSCs分化为SCs后表达升高。并且通过人为控制miR-21表达，证实miR-21能够促进NCSCs向SCs的分化。 3、miR-21通过抑制SOX2表达调控毛囊NCSCs向Schwann细胞分化 目的：寻找miR-21调控NCSCs分化的机制，明确miR-21的作用靶点和SOX2与miR-21的调控分化的关系。 方法：通过数据库查询miR-21的作用靶点，将SOX2的3’UTR端和突变后的片段分别构建入luciferase报告系统，验证miR-21是否与SOX2的3’UTR端结合。通过转染miR-21激动剂agomiR-21和阻断剂antagomiR-21，qRT-PCR和western blot法检验SOX2表达水平，观察miR-21对SOX2的mRNA和蛋白水平的影响。为检测SOX2是否参与miR-21促干细胞分化，将NCSCs的SOX2基因敲除后，qRT-PCR和流式细胞术观察antagomir-21对干细胞分化的影响。 结果：数据库查询结果发现SOX2基因的3’UTR端包含一个7mer大小的序列与miR-21相匹配。Luciferase报告实验显示在miR-21作用下SOX2-3’UTR-wt组（野生型组）荧光强度明显低于其阴性对照组（miR-con），而SOX2-3’UTR-mut组（突变组）或空质粒组荧光强度与其阴性对照无明显差异。此结果表明，miR-21可以通过与SOX2mRNA3’UTR端结合而调节其翻译。 Western blot结果发现agomiR-21可显著降低NCSCs细胞中SOX2水平（p0.05），而antagomiR-21可显著升高NCSCs细胞中SOX2水平（p0.05）。但无论是agomiR-21还是antagomiR-21，均不影响SOX2mRNA表达。这些结果表明，miR-21可以通过与SOX2mRNA3’UTR端结合在转录后水平调节其蛋白表达水平。 我们通过慢病毒shRNA敲除NCSCs中的SOX2，并将此细胞（SOX2-KD-NCSCs）与普通NCSCs（Control）一起用于研究SOX2在miR-21调节干细胞分化中所扮演的角色。通过流式细胞术和qRT-PCR对S100和GFAP蛋白及mRNA检测发现，转染antagomiR-21的NCSCs分化能力明显低于转染antagomiR-NC的NCSCs（p0.01），而转染antagomiR-21的SOX2-KD-NCSCs分化能力与转染antagomiR-NC的SOX2-KD-NCSCs相比并无明显变化。此结果表明敲除SOX2可阻断antagomir-21的抗SCs分化能力，证明SOX2是miR-21调节NCSCs分化的下游靶点之一。 结论：本实验通过Luciferase assay方法验证了miR-21可以通过与SOX2mRNA3’UTR端结合而调节其翻译。并且，miR-21不影响SOX2的mRNA表达，通过与SOX2mRNA3’UTR端结合在转录后水平调节其蛋白表达。敲除SOX2可阻断antagomir-21的抗SCs分化能力，明确SOX2是miR-21调节NCSCs分化的下游靶点之一。
[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 卤 5.6 % , which was significantly higher than that of the negative control agomir - NC group ( 64.2 卤 4.1 % , p0.05 ) .
After induction , the NCSCs expressed S100 and GFAP at about 41.1 卤 3.2 % , which was significantly lower than that of control group ( 62.0 卤 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 卤 0.20 , which was significantly higher than that of the negative control agomir - NC group ( 1.03 卤 0.21 , p0.05 ) .
The expression of GFAP mRNA after induction of NCSCs was 0 . 50 卤 0 . 06 , 0 . 95 卤 0 . 13 and 0 . 53 卤 0 . 08 , respectively , and the expression of GFAP mRNA was 3.27 卤 0 . 44 and 0.53 卤 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 .

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R783.6

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