【作者】 陈明；

【导师】 吴补领； 陈婷；

【作者基本信息】 南方医科大学 ， 口腔医学， 2016， 博士

【摘要】 研究背景口腔内科中,牙体牙髓病的诊治一直以来都是依赖于根管治疗技术及其相关辅助设备的发展,而随着近年来再生医学和组织工程学的发展,牙体牙髓病治疗与研究的热点也逐渐聚焦到牙髓再生这个概念上,口腔医学界的科研工作者们希望能借助细胞组织工程学的发展来实现牙髓的再生,从而将一颗能重新行使功能的健康的牙齿还于患者。组织工程是指整合了医学、生命科学及工程学相关理论与技术实现组织修复或再生的技术,其中种子细胞、生物支架与细胞因子是组织工程得以实现的三个要素。人牙髓干细胞(human dental pulp stem cells, hDPSCs)是目前关注较多且较常应用的一种种子细胞。成牙本质细胞是牙齿发育最重要的细胞,它能分泌牙本质基质,但其作为有丝分裂终末期细胞,在体外难以分离培养。所以在牙髓再生研究中的第一步就是实现成牙本质细胞的再生,其中的一个关注热点就是探讨如何将这些种子细胞诱导分化为成牙本质细胞。而在牙本质的矿化过程中,细胞外基质起着重要的作用,其中牙本质涎磷蛋白(dentin sialophosphoprotein, DSPP)最为关键。多年来国内外学者的研究发现,DSPP与牙齿发育中上皮-间叶细胞的相互调控作用有关,它能促进生物矿化及牙髓创伤的修复,DSPP的突变会导致牙本质发育不全。DSPP的表达一直以来都是牙髓再生研究中的热点。数年来,国内外学者在种子细胞定向分化的调控机制方面做出了很多探索和进步,但是尚有许多问题仍然处在研究的初期阶段。近年来,学者们发现那些过往被忽视的非编码的小分子RNA在生物体的生命活动中同样起着重要的作用,对编码RNA的转录、翻译有着举足轻重的调控作用,从而引起了学界对非编码RNA的研究热潮。MicroRNA(miRNA)即是我们常见的小RNA之一。其长度一般在18-25nt之间,经过较长的初级转录物在一系列的核酸酶的剪切加工后形成,随后可以组装进入RNA诱导的沉默复合体,然后根据互补程度的大小来指导沉默复合体降解靶基因mRNA或者抑制相关mRNA的翻译,从而起到抑制目的基因表达的作用。miRNAs广泛存在于植物、线虫以及人类的细胞中,具有在翻译水平调控基因表达的功能。miRNA作为内源性的单链小分子RNA,对mRNA进行转录后的表达调控,miRNA与靶基因mRNA只需要部分的互补就可以起到作用。经过研究,学者们预测有30%的基因会受到miRNA的调控,而它们的作用特点是其中一个miRNA可以调控多个mRNA,而一个mRNA又可以受到多个miRNAs的调控。miRNAs作为一种新的调控因子广泛作用于靶基因的表达调控,并广泛参与细胞的各向分化,从而影响生物体细胞的增殖、分化、凋亡及个体发育等。本课题组Huang X等在前期实验中,已经通过软件及网站预测,后利用双荧光素酶报告基因系统验证了miR-32,miR-885-5p,miR-586可以靶向调控DSPP,最后验证了miR-586与DSPP的结合位点,证明了miR-586可以靶向调控DSPP的表达,抑制miR-586的表达,可导致DSPP表达上调,从而促进矿化诱导。此后本课题组Chen T等又将成血管相关因子SDF-1和VEGF的过表达慢病毒转染入内皮前体细胞(Endothelial progenitor cells, EPCs)中,构建了稳定过表达SDF-1和VEGF的EPCs,并将此稳定转染后的EPCs与DPSCs进行3D共培养,结果证明SDF-1和VEGF的联合运用对DPSCs/EPCs的3D共培养物的成牙本质向分化及血管形成均具有相应的促进作用。研究目的基于本课题组的前期研究结果,为了进一步寻找更适于作为牙髓再生研究的种子细胞,本课题将针对miR-586对于DSPP的调控作用做进一步研究。本实验将构建miR-586表达抑制的慢病毒,并将其转染入牙髓干细胞中且稳定表达。将此稳定转染的牙髓干细胞与内皮前体细胞进行共培养,分别对共培养物进行矿化诱导及成血管诱导培养,观察其各项矿化特性及血管形成特性,观察miR-586表达抑制处理后的此共培养物,是否能在不影响内皮前体细胞血管生成特性的基础上,同时促进共培养物向成牙本质细胞向分化。旨在让此miR-586表达抑制人牙髓干细胞和内皮前体细胞的共培养物既能向成牙本质细胞分化,又不影响其血管新生的能力,并将此细胞共培养模型作为牙髓再生后续实验的理想细胞组合。研究内容第一章人牙髓干细胞和内皮前体细胞的分离培养与鉴定一.方法1.本实验采用改良的组织块酶消化法分离和培养人牙髓干细胞,牙髓组织取材于成年人健康的第三磨牙或因正畸治疗需要而拔除的前磨牙,取出其牙髓组织进行培养,观察其形态及生长状态。2.采用多种方法对人牙髓干细胞的干性进行检查：有限稀释法纯化人牙髓干细胞及细胞克隆鉴定；采用流式细胞仪检测及分析干细胞表面标记物(CD29、CD44、CD90)；对培养的人牙髓干细胞进行多向分化潜能鉴定,包括成骨矿化诱导、成脂诱导及成软骨诱导,在经诱导21天后,分别进行茜素红染色、尼罗红染色及阿利新兰染色。3.本实验所采用的内皮前体细胞均为同行医师赠予。对赠予的内皮前体细胞进行复苏后常规培养。4.对传代培养的内皮前体细胞进行流式细胞检测,分析其内皮细胞表面标记物CD31、CD105、KDRCD105,造血细胞表面标记物CD45和CD14,干细胞表面标记物CD34和CD133。二.结果1.人牙髓干细胞的形态及生长状态的镜下观察：初代爬出的细胞呈长梭形,待细胞增殖至细胞培养瓶瓶底面积80%或多数牙髓组织块周围爬出的细胞已长至复层生长时,传代培养,传代后的细胞继续增殖,可呈旋涡状或放射状排列,此时可再次传代。细胞生长状态正常情况下,一般3-5天可传一代。2.流式细胞仪检测人牙髓干细胞结果显示：间充质干细胞表面标记物CD29、CD44、CD90均呈阳性表达,表达率分别为94.70%、100%和99.90%;造血干细胞表面标记物CD34、CD45阳性表达率分别为0.16%、0.47%。3.原代培养所得的人牙髓干细胞具有多向分化潜能。细胞经成骨矿化诱导后,茜素红染色可见大小不等的红褐色矿化结节形成；细胞经成脂诱导,尼罗红染色后可见细胞内红染；细胞经成软骨诱导,阿利新兰染色后可见蓝染。4. 内皮前体细胞生长形态：复苏所得细胞为第4代细胞,呈铺路石状排列,增殖速度快,2天传一代。5.流式细胞检测内皮前体细胞的表面标记物：内皮细胞表面标记物CD31、CD105、KDRCD105阳性表达率分别为99.82%、98.91%、92.11%；造血干细胞表面标记物CD45、CD14阳性表达率分别为0.06%、1.1%；间充质干细胞表面标记物CD34和CD133阳性表达率分别为7.51%和0%。第二章 miR-586表达抑制慢病毒稳定转染人牙髓干细胞一.方法1.miR-586表达抑制慢病毒载体的制备,载体使用的是吉凯基因公司的GV慢病毒载体系列、pHelper 1.0载体和pHelper 2.0载体三质粒组成。本实验所要构建的miR-586表达抑制慢病毒所采用的是GV-232载体。利用载体进行miR-586表达抑制慢病毒的包装以及对其进行滴度检测。2.慢病毒感染预实验：嘌呤霉素梯度筛选实验,找出嘌呤霉素用于人牙髓干细胞筛选的最优浓度,用于后续慢病毒稳定转染细胞株的筛选。3.慢病毒感染复数MOI的确认：设计慢病毒的梯度感染复数及不同的感染条件,找出miR-586表达抑制慢病毒感染人牙髓干细胞的最佳MOI及最佳感染条件。4.人牙髓干细胞稳定转染miR-586表达抑制慢病毒及转染后miR-586及其靶基因DSPP表达量的验证。实验分组：DPSCs(miR-586 inhibition)组,DPSCs(Empty Vector, EV)和DPSCs组。qRT-PCR检测稳定转染后miR-586的相对表达量,Western Blot检测稳定转染后人牙髓干细胞中DSPP的蛋白表达。二.结果1.获取miR-586表达抑制慢病毒,对阳性克隆的测序结果进行blast比对发现,设计的miR-586反义互补序列可以和成熟体miR-586的序列互补配对。miR-586表达抑制慢病毒的滴度为3E+8TU/ml。2.嘌呤霉素用于筛选人牙髓干细胞的最优浓度为2μg/ml,此浓度下的嘌呤霉素能在3天内杀灭所有人牙髓干细胞。3.miR-586表达抑制慢病毒感染人牙髓干细胞的MOI为50时能达到90%以上感染效率。感染条件是人牙髓干细胞在Complete Medium+5μg/ml polybrene下容易被感染。4.人牙髓干细胞转染miR-586表达抑制慢病毒后,经过嘌呤霉素筛选,可稳定表达,生长状态良好,经传代后用嘌呤霉素进行再次筛选仍有90%以上细胞存活,且细胞增殖能力良好。qRT-PCR结果显示,转染了miR-586表达抑制慢病毒的DPSCs(miR-586 inhibition)组的miR-586的表达量为对照组DPSCs组的(0.3952±0.0357)倍,表达水平显著降低,具有统计学意义(P=0.000,P<0.05)。DPSCs(miR-586 inhibition)组的miR-586的表达量为阴性对照病毒空载体组DPSCs (EV)组的(0.3817±0.0345)倍,表达水平显著降低,具有统计学意义(P=0.000,P<0.05)。DPSCs组与DPSCs (EV)组之间无统计学差异(P=0.400,P>0.05)。第三章miR-586表达抑制对牙髓干细胞/内皮前体细胞共培养中成牙本质向分化的影响一.方法1.将转染后的人牙髓干细胞与内皮前体细胞进行共培养,混合比例为1：1,培养液将含10%FBS的DMEM与10%FBS的EGM-2 MV混合,比例为1：1。实验分为5组,分别为：DPSCs组、DPSCs(miR-586 inhibition)组、DPSCs/EPCs组、DPSCs(Empty Vector,EV)/EPCs组、DPSCs(miR-586 inhibition)/EPCs组,后文叙述中5组细胞将简化为：DPSCs组,DPSCs(586)组,DPSCs/EPC组,DPSCs(EV)/EPCs组和DPSCs(586)/EPCs组。5组细胞均进行不同时间点(3天、7天、14天)的矿化诱导。2.经各时间点矿化诱导后的5组细胞,分别进行多项鉴定：(1)MTT检测miR-586表达抑制对于DPSCs增殖的影响；(2)茜素红染色；(3)qRT-PCR检测矿化相关因子DSPP、DMP-1、ALP和BSP的表达；(4)Western Blot检测5组细胞中DSPP的蛋白表达情况。二.结果1.MTT结果显示,DPSCs(586)组OD值在培养第3天时比DPSCs(EV)组显著升高(P<0.01),在培养第5和7天时,DPSCs(586)组OD值比DPSCs(EV)组显著升高(P<0.05)。2.茜素红染色结果显示：经矿化诱导14天后,DPSCs/EPCs组、DPSCs(586)/EPCs组、DPSCs(586)组、DPSCs(EV)/EPCs组形成的红褐色矿化结节均比对照组DPSCs组多且深,其中DPSCs(586)/EPCs组形成的矿化结节最多,颜色最深。3. qRT-PCR结果显示：矿化诱导3天时,DPSCs(586)组的DSPP表达量为对照组DPSCs组的(3.2172±0.2280)倍,表达量显著升高,具有统计学意义(P<0.05)。DPSCs(586)/EPCs组的DSPP表达量为对照组DPSCs组的(4.4134±0.2756)倍,表达量显著升高,具有统计学意义(P<0.05)。矿化诱导7天时,DPSCs(586)组的DSPP表达量为对照组DPSCs组的(5.0632±0.0537)倍,表达量显著升高,具有统计学意义(P<0.05)。DPSCs(586)/EPCs组的DSPP表达量为对照组DPSCs组的(9.1833±1.0052)倍,表达量显著升高,具有统计学意义(P<0.05)。矿化诱导14天时,DPSCs(586)组DSPP表达量为对照组DPSCs组的(4.1587±0.3162)倍,表达量显著升高,具有统计学意义(P<0.05)。DPSCs(586)/EPCs组DSPP表达量为对照组的(4.0150±0.5468)倍,表达量显著升高,具有统计学意义(P<0.05)。4. Western Blot结果显示,在矿化诱导3天的实验组中,DPSCs(586)/EPCs组和DPSCs(586)组的DSPP蛋白表达量均比其余三组高,其余三组未见明显的蛋白条带；在矿化7天的实验组中,DPSCs(586)/EPCs组和DPSCs(586)组的DSPP蛋白表达量同样均比其余三组高,其余三组亦出现了较明显的蛋白条带,其中,DPSCs(586)/EPCs组的DSPP蛋白条带最深；而在矿化诱导14天时,整体情况与前两个时间点相似,DPSCs(586)/EPCs组和DPSCs(586)组的DSPP蛋白表达量仍是最高,但其中DPSCs(586)组的条带比DPSCs(586)/EPCs组较深。第四章miR-586表达抑制对牙髓干细胞／内皮前体细胞共培养中成血管的影响一.方法1.将转染后的人牙髓干细胞与内皮前体细胞进行共培养,混合比例为1：1,培养液混合比例为1：1,共分为4组,分别为：EPCs组、DPSCs/EPCs组、DPSCs(EV)/EPCs组、DPSCs(586)/EPCs组。4组均进行7天的成血管诱导培养。2.7天后收集4组细胞,分别进行后续实验：(1)Tube formation assay; (2)qRT-PCR检测成血管相关因子CD34、VEGF.Flk-1和SDF-1的表达；(3) Western Blot检测5组细胞中VEGF的蛋白表达情况,加入了DPSCs组作为对照。二.结果1. Tube formation assay结果显示：DPSCs/EPCs组、DPSCs(EV)/EPCs组、DPSCs(586)/EPCs组形成的管腔结构比EPCs组的管腔结构连接处更为紧密和牢固,管腔连接处形成了特殊的复合体,面积更大,管腔总长度更长,平均管腔周长也更长,并且这三组所形成的管腔的持续时间能达到92小时,而EPCs组形成的管腔结构在48小时就出现了降解的情况。2. qRT-PCR结果显示：CD34、VEGF、Flk-1、SDF-1在DPSCs/EPCs组、DPSCs(EV)/EPCs组、DPSCs(586)/EPCs组这三个共培养组的表达量均显著高于EPCs单细胞组(P<0.05)。多重比较结果显示,3个共培养组之间无显著差异(P>0.05)。3. Western Blot结果显示,DPSCs/EPCs组、DPSCs(EV)/EPCs组、DPSCs(586)/EPCs组的VEGF表达量均高于EPCs组,其中DPSCs(586)/EPCSs组条带最深。将Western blot检测的VEGF蛋白条带进行灰度值比较,结果显示,DPSCs(586)/EPCSs的VEGF蛋白条带灰度值为EPCs组的(2.3178±0.1030)倍,显著升高(P<0.05)。多重比较结果显示,DPSCs(586)/EPCSs的VEGF蛋白条带灰度值同样显著高于DPSCs/EPCs组(P=0.001,P<0.05)以及DPSCs(EV)/EPCs组(P=0.001,P<0.05)。结论1.采用改良的组织块酶消化法分离和培养人牙髓干细胞,能保证较高的原代牙髓干细胞的游出率,培养出的原代细胞形态良好,呈长梭形,透明饱满,增殖速度快,具有克隆生长的特性,高表达间充质干细胞表面分子标记物,极低表达造血干细胞表面分子标记物,具有向成牙本质细胞、脂肪样细胞及软骨细胞分化的潜能,其特性与骨髓间充质干细胞类似,可定义其是一类牙髓组织来源的,具有多向分化潜能和干细胞特性的细胞群。受赠的内皮前体细胞复苏后经过鉴定,具有分化为成熟的内皮细胞的潜能。2.牙髓干细胞与内皮前体细胞共培养,在细胞数量为1：1,且培养液混合比例为1：1的条件下,共培养物生长状态良好,两种细胞均增殖较快。3.成功构建了miR-586表达抑制的慢病毒载体,并成功感染人牙髓干细胞。筛选出了稳定转染miR-586表达抑制慢病毒的人牙髓干细胞,成功构建的此稳转细胞模型可用于后续成牙本质特性及成血管特性的实验研究。4.成功构建了miR-586表达抑制人牙髓干细胞／内皮前体细胞的细胞共培养模型,可作为较好的种子细胞组合,用于牙髓再生研究。5. 内皮前体细胞共培养及miR-586表达抑制的同时应用,对于定向诱导人牙髓干细胞向成牙本质细胞分化具有协同促进作用。将内皮前体细胞与人牙髓干细胞进行直接接触共培养,能促进人牙髓干细胞的成牙本质向分化。6.miR-586表达抑制对于人牙髓干细胞／内皮前体细胞共培养物的成血管特性有间接促进作用。将人牙髓干细胞与内皮前体细胞进行直接接触共培养,能促进内皮前体细胞的血管新生。7. 人牙髓干细胞／内皮前体细胞共培养物形成的血管样结构的持续时间比单细胞培养的内皮前体细胞明显增加,并会在其管腔连接处形成更为稳固的复合体。更多还原

【Abstract】 BackgroundIn the endodontics clinic, the treatment of endodontic diseases are always depended on the development of the RCT (Root canal therapy) technique and the auxiliary equipment. As the rapid development of regenerative medicine and tissue engineering, the treatment of endodontic diseases are gradually transfer the focus onto a concept of pulp regeneration. The researchers desired to make this concept come true with the help of tissue engineering techniques. They hope to achieve a success that functional brand new teeth can be regenerated and replanted them back in patients’ mouths. Tissue engineering is a kind of technology that integrated with the theory and technologies of medicine, bioscience, life science and engineering. And there are three important elements in the theory of tissue engineering, which contains seed cells, biomaterial scaffold and growth factors.Dental pulp stem cells (DPSCs) is the most common one that used by researchers on the study of pulp regeneration. Odontoblast, a significant kind of cells in the process of tooth development, can secrete dentin matrix. Odontoblast, as a cell in the final stage of mitosis, is difficult to isolated and cultured in vitro. Therefore, the first step to achieve pulp regeneration is to figure out how to regenerate odontoblast. One of the hotspot in this field is to induced the seed cells differentiated into odontoblast. In the process of mineralization, extracellular matrix plays a vital role and dentin sialophosphoprotein (DSPP) can be defined as a key factor. Studies found that DSPP can repairs the pulp injury and promote the process of mineralization. The mutation or knock down of DSPP will induce tooth hypoplasia. The study of DSPP has been the hot area of research in the field of pulp regeneration for a long time.Over the years, a great progress and exploration has been made on the aspect of regulatory mechanism in seed cells differentiation by researchers worldwide. However, many issues still remain to us become an obstacle to the study of pulp regeneration. In recent years, researchers realized that the non-coding small RNA, which has been overlooked in the past, plays a significant role in the life of organism. The non-coding RNAs can regulate the transcription and translation of coding RNAs. A new upsurge of research on these non-coding RNAs has been made among the scientists. MicroRNA (miRNA) is one of these non-coding RNAs. The length of miRNA is between 18 and 25nt. After a long process of shearing by nuclease, miRNA can be assembled into RISCs. According to the complementary way between miRNA and the 3’UTR of the target gene, the RISCs will degrade or suppress the translation of the target gene’s mRNA. As a result, the miRNA regulate the target gene. miRNAs widely exist in the cells of plants, nematodes and humans and have the capacity to regulate the gene expression on the translation level. This regulation ability of miRNA can be process even the complementation is partial. Researchers also found that there’re nearly 30% of genes can be regulated by miRNAs. A single miRNA may regulate multiple genes and also the mRNA of gene can be regulated by multiple miRNAs. miRNA, as a new found regulatory factor, widely regulated the expression of target genes and widely participated in the differentiation, proliferation and apoptosis of cells.A serious previous experiments have been done by the members of our research group. First, they made a prediction on the relation between miRNAs and their target genes in the process of mineralization in DPSCs by software and the website database. According to the result of prediction, they operated a dual-luciferase reporter gene assay to verify the binding of miRNAs and DSPP. Finally, they confirmed that miR-586 can bind with the 3’UTR of DSPP and the binding site has also been verified. The inhibition of miR-586 will increased the expression level of DSPP and eventually promote the induction of mineralization. Second, they constructed a lentivirus that carried VEGF and SDF-1 overexpress gene and transfected the lentivirus into EPCs successfully. The VEGF and SDF-1 overexpress EPCs was 3D-cocultured with DPSCs. Results indicated that the combined utilization of VEGF and SDF-1 can promote the odontogenic and angiogenic differentiation.ObjectivesAccording to the results of previous study by our research group, the objectives of this study is to construct a miR-586 inhibition lentivirus and stably transfected DPSCs with it. The stably transfected DPSCs, which have been screening by puromycin, was cocultured with Endothelial progenitor cells (EPCs). DPSCs/EPCs will be induce towards odontogenic and angiogenic differentiation, respectively. The cells will be harvested for detecting the odontogenic and angiogenic relative factors to observe whether miR-586 inhibition will promote the odontogenic differentiation of DPSCs/EPCs without losing or suppressing the angiogenesis capacity of EPCs. We hope this DPSCs (miR-586 inhibition)/EPCs model can be utilized in the further study of pulp regeneration.Chapter One The isolation and identification of DPSCs and EPCs1. Methodsa. We used modified tissue explant collagenase method to isolated and cultured DPSCs. The pulp tissue was obtain from the patients whose third molar or premolar have an extraction or orthodontic treatment need. Pulp tissue was pull out of the tooth and cultured in vitro.b. The characteristics of stem cells were tested by different kind of methods. First, limiting dilution analysis was utilized to purified DPSCs. Second, the stem cells markers(CD29,CD4,CD90) were detected by flow cytometry. Third, the multiple differentiation potential were also tested.c. The EPCs were gift from others. EPCs were recovered from liquid nitrogen and cultured regularly.d. The EPCs were also tested for the endothelial cells markers(CD31,CD105,KDRCD105), hematopoietic cell markers(CD45,CD14) and stem cells markers(CD34,CD133) by flow cytometry after subcultured.2. Resultsa. The morphology and the growth status of DPSCs were observe under microscope. The cells was spindle shape. The cells can be subcultured until cell confluence was approximately 80%. The passage cells were proliferated continually which will finally showing a tendency of multi-layer growth. Generally, DPSCs can be subcultured every 3 days.b. The flow cytometry results showed that the positive expression rate of mesenchymal stem cells markers CD29,CD44 and CD90 was 94.70%,100% and 99.90%, respectively, while the hemopoietic stem cells markers CD34 and CD45 was 0.16% and 0.47%, respectively in DPSCs.c. The primary cells cultured from the pulp tissue in our study have the multiple differentiation potential. The mineralized nodules can be found in DPSCs after induction by Alizarin Red staining. And DPSCs can also be inducted differentiated into adipose cells and cartilage cell.d. The passage 4 EPCs, which proliferated fast, were obtained after recovery and the showing a cobblestone-like appearance. EPCs can be subcultured every 2 days.e. The flow cytometry results showed that the positive expression rate of endothelial cells markers CD31, CD105 and KDRCD105 was 99.82%,98.91% and 92.11%, respectively, while the hemopoietic stem cells markers CD45 and CD 14 was 0.06% and 1.1%, respectively in EPCs. The positive expression rate of mesenchymal stem cells markers CD34 and CD133 was 7.51% and 0%, respectively in EPCs.Chapter Two DPSCs stably transfected with miR-586 inhibition lentivirus1. Methodsa. The miR-586 inhibition lentivirus vector were obtain from the Genechem corporation. The vector system were constructed by 3 kind of vectors which contains pHelper 1.0 vector, pHelper 2.0 vector and GV vector system. The GV vector system contains HIV basic and auxiliary elements. pHelper 1.0 vector contain gag. gene from HIV. pHelper 2.0 vector contains VSV-G gene. All these vectors were participant in the lentivirus packaging. And the lentivirus titer were also identified.b. Preliminary experiment of transfection by lentivirus. The gradient screening experiment with puromycin. The suitable concentration of puromycin were tested for the further screening of study.c. Confirm of multiplicity of infection (MOI). A gradient MOI and different condition of infection were designed to tested for the best MOI and condition of infection.d. The detection of miR-586 and its target gene after stably transfected by miR-586 inhibition lentivirus by qRT-PCR. Furthermore, western blot was also operated to tested for the expression level of DSPP.2. Resultsa. The harvest of miR-586 inhibition lentivirus was done and the first generation DNA sequencing technology was utilized to test the positive cloning sequence of the lentivirus. The results showed that the antisense oligonucleotides sequence can be paired with the sequence of the mature miR-586. The miR-586 inhibition lentivirus titer is 3E+8TU/ml.b. The suitable screening concentration of puromycin for DPSCs is 2μg/ml. DPSCs can be eliminated in 3 days.c. DPSCs can be transfected easily under the condition of Complete Medium +5μg/ml polybrene by miR-586 inhibition lentivirus. Transfection efficiency may over 90% when MOI is 50.d. DPSCs have a good status and morphology after transfection and screening. The proliferation of DPSCs are still fast after screening. The subcultured DPSCs were screening by puromycin again and the cells condition remain good. The qRT-PCR results showed that the miR-586 relative expression level in DPSCs(miR-586 inhibition) group was (0.3952±0.0357) times to DPSCs group. Compared to DPSCs group, the miR-586 expression level is significantly suppressed in DPSCs(miR-586 inhibition) group(P<0.05).And the miR-586 expression level is also significantly suppressed in DPSCs(miR-586 inhibition) group when compared with DPSCs (EV) group(P<0.05).Chapter Three The role of miR-586 inhibition in the odontogenic differentiation of DPSCs/EPCs1. Methodsa. miR-586 inhibition DPSCs cocultured with EPCs at ratio 1:1. The culture medium is also mix with 10% FBS DMEM and 10% FBS EGM-2 MV at ratio 1:1. Experiment group was designed into 5:DPSCs group, DPSCs(miR-586 inhibition) group, DPSCs/EPCs group, DPSCs(Empty Vector, EV)/EPCs group and DPSCs(miR-586 inhibition)/EPCs group which will abbreviated as DPSCs, DPSCs(586), DPSCs/EPCs, DPSCs(EV)/EPCs and DPSCs(586)/EPCs in the following description. These 5 groups will be induce towards odontogenic differentiation at different time points (3d,7d,14d).b. Some analysis will be run to detect the cells in these 5 groups after induction at different time points. First, MTT for decteting on proliferation of DPSCs. Second, Alizarin red staining. Then, the expression level of DSPP, DMP-1, ALP and BSP will be detected by qRT-PCR. At last, the protein expression level of DSPP will be detected with western blot.2. Resultsa. MTT showed that the OD value of DPSCs(miR-586 inhibition) group were statitcally higher than DPSCs (EV) group on day 3,5 and 7.b. Alizarin ren staining showed that the number and color of mineralized nodules in the three cocultured groups are more and darker than that in DPSCs group. The quantity of mineralized nodules in DPSCs(586)/EPCs are the most in 5 groups.c. qRT-PCR showed that the expression level of DSPP in DPSCs(586) was (3.2172±0.2280) times to DPSCs group(P<0.05) at day 3 after induction. The expression level of DSPP in DPSCs(586)/EPCs group was (4.4134±0.2756) times to DPSCs group(P<0.05) at day 3 after induction. The expression level of DSPP in DPSCs(586) group was (5.0632±0.0537) times to DPSCs group(P< 0.05), while the expression level of DSPP in DPSCs(586)/EPCs group was (9.1833±1.0052) times to DPSCs group(P<0.05) at day 7 after induction. The expression level of DSPP in DPSCs(586) group was (4.1587±0.3162) times to DPSCs group(P<0.05), while the expression level of DSPP in DPSCs(586)/EPCs group was (4.0150±0.5468) times to DPSCs group(P<0.05) at day 14 after induction.d. Western blot:Results showed that the protein expression level of DSPP in DPSCs(586)/EPCs and DPSCs(586) are higher than the other three groups at day 3 after induction. The protein expression level of DSPP in DPSCs(586)/EPCs and DPSCs(586) are also higher than the other three groups and the DPSCs(586)/EPCs group was the highest at day 7 after induction. The other three groups show a distinct protein band. At day 14 after induction, the situation was almost the same as that at day 7. DPSCs(586)/EPCs and DPSCs(586) groups were still the highest two among 5 groups. However, the protein band of DPSCs(586) was higher than that of DPSCs(586)/EPCs.Chapter Four The role of miR-586 inhibition in the angiogenic differentiation of DPSCs/EPCs1. Methodsa. miR-586 inhibition DPSCs cocultured with EPCs at ratio 1:1. The culture medium is also mix with 10% FBS DMEM and 10% FBS EGM-2 MV at ratio 1:1. Experiment group was designed into 4:EPCs, DPSCs/EPCs, DPSCs(EV)/EPCs and DPSCs(586)/EPCs group.b. Different analysis will be run to detect the angiogenic properties. First, Tube formation assay. Second, the expression level of CD34、VEGF、Flk-1 and SDF-1 will be detected by qRT-PCR. Third, the protein expression level of VEGF will be detected with western blot.2. Resultsa. Tube foemation assay:The vessel-like structure constructed in DPSCs/EPCs, DPSCs(EV)/EPCs and DPSCs(586)/EPCs groups are more stable than that in EPCs group. The junctional area of the vessel-like structure in the three coculture groups are much larger than that in the EPCs group. The Total tube length and the mean loop perimeter of the three cocultured groups are longer than that in EPCs group.b. qRT-PCR showed that the expression level of CD34, VEGF, Flk-1 and SDF-1 in the three cocultured groups are significantly higher than that in EPCs group(P< 0.05). There is no significant difference between the three cocultured groups on the expression level of CD34, VEGF, Flk-1 and SDF-1(P>0.05).c. Western blot:Results showed that the protein expression level of VEGF in three cocultured groups are higher than that in EPCs group. The protein bands of three cocultured groups did not show a distinct difference and the DPSCs group did not show a distinct band.Conclusions1. There will be a high success rate of isolation and cultivation of DPSCs when using the modified tissue explant collagenase method. The DPSCs in this study show a high expression rate of mesenchymal stem cells markers and show a low expression rate of the hemopoietic stem cells markers. EPCs used in this study have the potential to differentiate into mature endothelial cells after identified.2. DPSCs cocultured with EPCs are well-growth at ratio 1:1 and also with a 1:1 mixture medium with DMEM and EGM-2 MV.3. miR-586 inhibition lentivirus vector was successfully constructed. And the stably transfected miR-586 inhibition DPSCs was also successfully constructed. The miR-586 inhibition DPSCs model can be used for further study of odontogenic and angiogenic differentiation.4. miR-586 inhibition DPSCs/EPCs model was successfully constructed. This model can be used for the pulp regeneration research as a well-combined seed cells.5. The combination of miR-586 inhibition and EPCs can synergistically facilitate the odontogenic differentiation on DPSCs. EPCs cocultured with DPSCs can promote the odontogenic differentiation on DPSCs.6. miR-586 inhibition has indirectly facilitate the angiogenesis differentiation on DPSCs/EPCs. DPSCs cocultured with EPCs can promote the angiogenic differentiation on EPCs.7. The duration of vessel-like structure constructed in DPSCs/EPCs are longer than that in EPCs. And the junctional area of the vessel-like structure will formed a special complex in DPSCs/EPCs.更多还原