【作者】 黄欣；

【导师】 吴补领； 高杰；

【作者基本信息】 南方医科大学 ， 外科学， 2011， 博士

【摘要】 牙齿是特殊的矿化器官,由釉质、牙本质、牙骨质和牙髓组成,其中牙本质构成牙齿的主体。牙本质起源于牙源性上皮和外胚间充质的相互作用,进而诱导成牙本质细胞分化成熟,合成和分泌细胞外有机基质,然后羟基磷灰石晶体沉积,矿化开始形成。在矿化过程中,细胞外基质起着非常重要的作用。其中最关键的是牙本质涎蛋白(dentin sialoprotein,DSP)和牙本质磷蛋白(dentin phosphoprotein,DPP),它们来源于同一个编码基因——牙本质涎磷蛋白基因(dentin sialophosphoprotein,DSPP)。长期以来,国内外学者对DSPP基因及其编码蛋白做了大量研究,DSPP基因的表达调控一直是研究的焦点。近年来,研究人员发现那些过去不被重视的非编码RNA在生物体的生命活动中起着不可忽视的作用,对编码RNA的转录、翻译有着巨大的调控作用,掀起了非编码RNA研究的热潮。1nicroRNA(miRNA)即是非编码RNA大家族中的一贝。miRNAs是一类长约22个核苷酸(nt)的内源性的、非编码的单链RNA分子,它们广泛存在于从植物、线虫到人类的细胞中。迄今为止,在动植物中已经发现了数千个miRNA基因,目前认为在不同物种中,编码miRNA的基因约占蛋白编码基因的1%左右,它们通过与所靶向基因的mRNA的3’UTR区互补结合,实现对靶向mRNA的降解或者翻译抑制,进而作为一类重要的调控分子,参与对生物体的生长、发育、衰老和死亡的调控。在正常生理状态下,生物体内的miRNAs表达呈严格的组织及发育阶段特异性,不同的组织和细胞在发育的不同阶段,miRNA的表达水平有显著差异。在口腔颌面部发育研究领域,Jevnaker等最先运用芯片研究miRNAs在大鼠牙胚和涎腺中的表达,发现在牙胚发育的不同阶段miRNAs表达是动态调控的,一些miRNAs的表达呈组织特异性,并通过生物信息学预测这些miRNAs是牙齿发育,乃至口腔颌面部组织器官发育调控的必需因子。基于前人的研究成果,为了更全面、深入的探讨miRNA对DSPP表达的调控作用,及其对成牙本质细胞分化的作用,本课题利用生物信息学软件对可能靶向DSPP基因的miRNAs进行预测,并结合双荧光素酶报告基因系统和实时定量PCR的方法筛选和验证能够靶向调控DSPP基因的miRNAs,然后选取其中一个miRNA——mir586做进一步的研究。同样用生物信息学方法预测mir586与DSPP的结合位点,并通过双荧光素酶报告基因系统进行验证。最后,构建过表达mir586和mir586表达抑制的慢病毒表达载体,转染入牙髓细胞,观察在牙髓细胞向成牙本质细胞分化过程中,mir586对细胞功能的影响。本论文主要包括以下三章内容：第一章：靶向DSPP基因的microRNAs的筛选和验证首先利用Targetscan、mirBase、microRNA.org和mirGen-miRanda生物学软件和数据库对可能靶向DSPP基因的miRNAs进行预测,分别得到了17种、15种、10种、10种miRNAs。然后取有两个以上交集的靶位点的miRNA作为研究对象,得到10种miRNAs。为了进一步筛选和验证在成牙本质细胞中能够靶向调节DSPP基因的miRNAs,本研究采用了双荧光素酶报告基因系统和定量PCR的方法。研究结果显示mir885-5p、mir32、mir586能够抑制荧光素酶的活性,即能够对DSPP基因起作用。在牙髓细胞向成牙本质细胞分化过程中,当DSPP基因的表达增多趋于稳定时,mir885-5p、mir32和mir586在10-14 d的表达量都有所下降。结合文献查阅,以及本章所得结果,我们选择抑制荧光素酶活性最强、表达量变化更显著的has-mir586作为进一步研究对象,探讨其与DSPP基因之间的靶向关系,及其对成牙本质细胞分化的作用。第二章：mir586靶向调节DSPP基因的结合位点分析首先利用Targetscan和MicroRNA.org软件预测mir586靶向调节DSPP基因的可能结合位点,两者显示了同一结合位点(AAUGCAUG)。为了进一步证实mir586通过此位点对DSPP基因起作用,将结合位点序列突变为UUACGUA,分别构建正常的DSPP 3’UTR表达载体和突变型DSPP 3’UTR表达载体,再通过双荧光素酶报告基因系统,检测荧光素酶活性。结果显示mir586能够与DSPP3’UTR结合,从而抑制荧光素酶活性；而mir586不能与突变型DSPP 3’UTR结合,证明mir586通过与DSPP 3’UTR上190-196碱基序列(AAUGCAUG)结合起作用。同时,进一步证实mir586与DSPP基因以这种“短种子”序列结合,可能起到抑制DSPP mRNA翻译的作用。第三章：mir586的功能研究本章主要包括两方面的内容：1.获取mir586过表达和表达抑制的慢病毒首先提取正常人全血中的基因组DNA。分别设计mir586过表达序列和表达抑制序列(anti-mir586):从NCBI中查找到has-mir-586的初始序列,选取包含has-mir-586成熟序列及其两端各约200个碱基的序列作为mir586过表达序列；取mir586互补序列进行3次串联,并根据文献设计anti-mir586序列。然后,以提取的正常基因组DNA为模板,PCR扩增mir586基因片段；以anti-mir586聚合引物序列为模板,使之形成双链anti-mir586基因片段。两个基因片段分别与慢病毒表达载体pLVX-shRNA1相连后,转化到感受态DH5α菌中,挑取阳性克隆,提取质粒并进行鉴定。采用Lenti-XTM HTX Packaging System,将构建好的两个慢病毒过表达载体和包装载体Lenti-XTM HTX Packaging Mix,及转染试剂Xfect transfection,转染293T细胞,收集病毒上清,过滤,浓缩,分装,测定病毒滴度约为5×106IFU/ml。最后,慢病毒感染人牙髓细胞,选择合适浓度的G418筛选稳定转染慢病毒的细胞,扩大培养。应用实时荧光定量PCR的方法分别测定mir586过表达、anti-mir586和阴性对照组感染牙髓细胞后的mir586表达情况,结果显示mir586过表达组mir586的表达量比阴性对照组增加了约6倍,而anti-mir586组的表达量减少了约4倍,mir586的表达非常微弱。2.研究在矿化液诱导牙髓细胞向成牙本质样细胞分化过程中,mir586对细胞增殖和分化的影响选取矿化液诱导的0、3、7、14和21 d作为测定各项指标的时间点。采用MTT法评估细胞的增殖情况,发现正常牙髓细胞在矿化诱导分化开始后,随着细胞数目的增多,细胞生长加快,第7 d,细胞逐渐分化、增殖,呈复层生长,矿化结节增多,14 d后细胞密度过高,出现细胞死亡,生长变缓；anti-mir586组细胞在初期时生长较缓慢,第3 d后生长速度较对照组明显加快,到第10 d,生长速度即开始减慢；mir586过表达组细胞数目不断增多,但细胞增长速度较对照组有所减慢。总的来说,在分化过程中,mir586对细胞增殖的影响不大,mir586表达抑制时,可以在一定程度上促进细胞的生长。通过检测碱性磷酸酶活性评估牙髓细胞的分化情况：正常牙髓细胞矿化诱导下,碱性磷酸酶活性随着分化的进行逐渐增强。mir586过表达组细胞的ALP活性变化不大,后期有上升趋势。而anti-mir586组细胞的ALP活性从第7 d开始显著上升,在矿化诱导14 d达到顶峰。DMP1和DSPP两个基因编码的蛋白是牙齿发育过程中非常重要的非胶原基质蛋白,被认为是成牙本质细胞的特异性因子。牙髓细胞在矿化液诱导下,可形成成牙本质样细胞,DMP1的表达早于DSPP基因,在诱导分化的第7 d即有大量DMP1表达,14 d达到顶峰,随着分化的进行,DSPP的表达在诱导分化7d后逐渐增多。当mir586过表达时,DSPP的表达速度减慢,表达量减少；DMP1的表达也有所降低。而当mir586受到抑制时,从诱导第7 d开始,DSPP的表达量明显多于对照组；DMP1的表达水平则与对照组没有明显差异。用Western blotting检测DSP蛋白的表达情况时发现：对照组牙髓细胞在矿化诱导第10 d可以看到清晰的DSP蛋白条带,而anti-mir586组在诱导第7 d即可以看到蛋白条带；与对照组相比较,mir586过表达组在诱导第10 d时,蛋白条带明显减弱,表明DSP蛋白被降解,致使表达量减少。总的来说,mir586表达抑制时,能够促进牙髓细胞向成牙本质样细胞的分化；当mir586过表达时,牙本质特异性基因DSPP、DMP1表达减少,DSP的表达延迟,且表达量明显减少,大大减缓了牙髓细胞分化的进程。综上所述,本研究通过生物信息学方法预测到多个能作用于牙本质涎磷蛋白基因的miRNAs,通过双荧光素酶报告基因系统和实时荧光定量PCR的方法,重点研究了mir586靶向调控牙本质涎磷蛋白基因的结合位点,并通过构建mir586过表达和表达抑制慢病毒,稳定转染人牙髓细胞,检测矿化液诱导牙髓细胞分化过程中,mir586对细胞增殖和分化的影响。初步研究了miRNAs在成牙本质细胞分化过程中的功能,以及miRNAs对牙本质特异性因子——牙本质涎磷蛋白及其编码基因的调控作用,有利于扩大研究多种miRNAs在牙本质形成中的机制作用,发现新的调控因子和调节途径。更多还原

【Abstract】 Tooth is a special mineralized organ composed of the enamel, dentin, cementum and pulp, of which dentin is the main part of the teeth. Dentin originated in dental epithelial -mesenchymal interactions, which induced odontoblast differentiation and maturation, the organic synthesis and secretion of extracellular matrix, and hydroxyapatite crystal deposition, mineralization begins to form. In the mineralization process, extracellular matrix plays a very important role. One of the most critical is the dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), which comes from the same gene-dentin sialophosphoprotein gene (DSPP). Over the years, scholars have made a lot of research on DSPP gene and its encoded protein. The regulation of DSPP gene has always been the focus of research.In recent years, researchers found that those non-coding RNA which were not taken seriously in the past played a significant role in the organisms life activities. They have a huge role in regulating transcription and translation of coding RNA, setting off a wave of non-coding RNA research. microRNA (miRNA) that is a kind of non-coding RNA. miRNAs are a class of about 22 nucleotides (nt) endogenous, non-coding single-stranded RNA molecules, they are widely present in from plants, nematodes to human cells. To date, plants and animals have been found thousands of miRNAs. Currently in different species genes encoded miRNA account for about 1% of genes encoded protein. MiRNAs lead to the targeted mRNA degradation or translation inhibition by targeting mRNA with the 3’UTR, and thus as an important class of regulatory molecules involved in regulating the growth, development, aging and death of the organism. Under normal physiological conditions, expression of miRNAs in vivo have a strict organizational and developmental stage-specific. MiRNA expression of different tissues and cells in different stages of development were significantly different. In the field of oral and maxillofacial development, Jevnaker and colleagues were the first to use microarray studying miRNAs expression in rat teeth and salivary gland, and found miRNAs expression were changing in different stages of tooth development,some of which was tissue-specific. At the same time, bioinformatics analysis suggested these miRNAs were necessary for tooth development and even oral and maxillofacial tissues and organs developmental.Based on previous research, to study more comprehensivly, in-depth of miRNA regulating DSPP, and its role in odontoblast differentiation, our research uses bioinformatics software to predict miRNAs targeting DSPP gene, combined with dual luciferase reporter gene system and real-time quantitative PCR method to select and validat miRNAs targeting DSPP gene, and then select one of the miRNAs——mir586 to do further research. The same methods are bioinformatics to predict binding sites between mir586 and DSPP and the dual luciferase reporter gene system for validation. Finally, lentiviral vector of mir586 overexpression and mir586 suppression were transfected into human dental pulp cells to discover the effect of mir586 on odontoblast differentiation.The paper includes the following three parts:PartⅠ:selection and validation of microRNAs Targeting DSPP geneFirstly, we use Targetscan, mirBase, microRNA.org and mirGen-miRanda four different bioinformatics software to predict miRNAs targeting DSPP gene, respectively, got 17,15,10,10 miRNAs. Then ten miRNAs were chosen based upon their targeted prediction by more than one program. In order to further select and validat these miRNAs regulating DSPP gene in the odontoblast, the study used a dual luciferase reporter gene system and quantitative PCR methods. The results showed mir885-5p, mir32, mir586 can inhibit the activity of luciferase, which can regulate DSPP gene. During dental pulp cells differentiation to odontoblast, when the DSPP gene expression increased, mir885-5p, mir32 and mir586 expression on day 10-14 have decreased. Combination of literature and the results of this chapter, we chose has-mir586 that strongestly inhibite luciferase activity and express more significantly as a further study to investigate the relationship with DSPP gene, and its effect on odontoblast differentiation.PartⅡ:analysis of the binding sites between mir586 and DSPP geneFirstly, Targetscan and MicroRNA.org were used to predict the binding sites between mir586 and DSPP gene. Both show the same binding site (AAUGCAUG). To further confirm the mir586 regulating DSPP gene through this binding site, mutations of the binding site were constructed. Normal DSPP 3 ’UTR expression vector and mutant DSPP 3’UTR expression vector were used in the dual luciferase reporter gene system to detect luciferase activity. The results showed that mir586 regulate DSPP 3 ’UTR through this binding site, thereby inhibiting the luciferase activity.But mir586 can not combine to mutant DSPP 3’ UTR, which proved mir586 combined to sequence of 190～196 of DSPP 3 ’UTR (AAUGCAUG).PartⅢ:the function of mir586This part mainly includes two aspects:1. Get mir586 overexpression lentivirus and mir586 inhibition lentivirus.First, genomic DNA was extracted from normal human whole blood. Sequences were designed for mir586 over-expression and mir586 suppression (anti-mir586).Sequence of has-mir-586 were got from NCBI, and designed mir586 overexpression sequence contains the has-mir-586 mature sequence and 200 base pairs at each end. According to the literature, take mir586 sequence 3 times in series as anti-mir586 sequence. Then, the extracted genomic DNA as template normal, mir586 gene fragment was amplified by PCR. Anti-mir586 primers as a template polymer to make it a double-stranded anti-mir586 gene. Two gene fragments were connected with pLVX-shRNA1 respectively and then transferred into competent DH5 a bacteria. Positive clones were picked out and then plasmids were extracted and testified. With Lenti-X TM HTX Packaging System, two lentiviral vector and packaging vector Lenti-X TM HTX Packaging Mix, and the transfection reagent Xfect transfection were transfected into 293T cells. Then viral supernatant was collected, filtered, concentrate, packaging. Titer of virus were about 5×106 IFU/ml. Finally, lentivirus transfected into human dental pulp cells and use the appropriate concentration of G418 to select stably transfected cells. Real time quantitative PCR method were determined the expression of mir586 among the mir586 overexpression group, anti-mir586 group and negative control group, The result showed that expression of mir586 in mir586 overexpression is as 6 times as that of the negative control group and anti-mir586 group decreased by about 4 times, so expression of mir586 in anti-mir586 group is very weak.2. During human dental pulp cells cultured in a mineralizing medium differentiation to the odontoblast-like cell, the effect of mir586 on cell proliferation and differentiationSamples were harvested at 0,3,7,10,14 or 21 days of differentiation to detect indicators.Cell proliferation was assessed by MTT method and found that the normal dental pulp cells cultured in a mineralizing medium grew quickly with the increase of cells. On day 7, cells gradually differentiation to odontoblast-like cells grew logarithmicly, so that cells were multiple layer growth and mineralized nodules increased. After 14 days the cell density was too high, cell death occurred. The cells of anti-mir586 group grew slowly at the beginning, after 3 days the growth rate were markedly accelerated, to 10 days, the growth rate began to slow down,perhaps because of excessive growth. The number of cells in mir586 overexpression group was increasing,while the growth rate was slower compared to control group. Overall, in the differentiation process, mir586 little effect on cell proliferation. When mir586 expression inhibited, to a certain extent, promote cell growth.The differentiation of dental pulp cells were evaluated by alkaline phosphatase activity. In normal group, alkaline phosphatase activity were gradually increased with differentiation. ALP activity of cells in mir586 overexpression group changed little, while ALP activity of cells in anti-mir586 group increased significantly from day 7 and to 14 days in mineralization it induced peak. DMP1 and DSPP proteins encoded by two genes is a very important non-collagen matrix proteins during tooth development, which is considered the odontoblast specific factors. Dental pulp cells cultured in the mineralizing medium can differentiate to odontoblast-like cells. DMP1 gene expresses earlier than DSPP, and with the differentiation DSPP expression increased gradually. From day 7 there were a large number of DMP1 expression and on day 14 it reached peak.When mir586 was overexpressed, DSPP and DMP1 expression were reduced. When mir586 expression was inhibited, DSPP expression was significantly more than the control group from day 7, but DMP1 expression is no significant difference compare to the control group. The expression of DSP was detected by Western blotting. DSP protein bands can be seen clearly in the control group on day 10, which appeared in the anti-mir586 group on day 7.Compared with the control group, protein bands was significantly reduced in mir586 overexpression group from day 10, which indicate that the DSP protein is degraded, resulting in reduced expression.In general, when mir586 expression is suppressed, mir586 can promote the dental pulp cells differentiating to odontoblast-like cells. When mir586 is overexpressed, the expression of dentin-specific DSPP gene decreased, DSP expression is delayed and decreased, which greatly slowed the process of differentiation.In summary, this study predicted some miRNAs targeting DSPP by bioinformatics methods. Applying to the dual luciferase reporter gene system and real-time fluorescence quantitative PCR method, we focus on the binding site between mir586 and DSPP. Mir586 overexpression lentivirus and mir586 suppression lentivirus were used to stably transfect to human pulp cells and to detect the effect of mir586 on cell proliferation and differentiation during the differentiation of pulp cells. This is a preliminary study of the miRNAs effect on the process of odontoblast differentiation, and on specific factors of dentin-DSPP and its proteins, which is conducive to extend research of variety of miRNAs in dentinogenesis and to discover new regulatory factors and regulatory pathways.更多还原