【作者】 林恒；

【导师】 陈智；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2013， 博士

【摘要】 一、它莫西芬诱导型可逆永生化小鼠牙乳头细胞系的建立和鉴定研究目的：成牙本质细胞是一种可分泌矿化基质的终末分化细胞,在牙髓牙本质复合体中发挥重要作用。小鼠牙乳头细胞(mDPCs)在体外诱导条件下可分化成成牙本质细胞样细胞。但是由于繁琐的原代培养过程和细胞有限的寿命,在实验过程中不易获得稳定的细胞来源。为解决该问题,本研究将传统的基于Cre/LoxP的可逆永生化系统与它莫西芬诱导的Cre重组酶系统相结合,试图建立一个它莫西芬诱导型可逆永生化小鼠牙乳头细胞系,并对其进行鉴定。研究方法：首先使用慢病毒介导的方法将已插入LoxP位点的SV40Tag-TK表达载体转染入原代培养的mDPCs。经过GCV筛选和单克隆筛选获得一个稳定表达SV40Tag并持续增值的克隆-mDPC6T,进而在mDPC6T过表达含有雌激素受体的ERT2CreERT2重组酶。经单克隆筛选SV40Tag和Cre阳性细胞,获得细胞系mDPCET。 mDPCET经过4-羟基它莫西芬(4-OHT)处理后获得逆永生化细胞。通过EdU掺入实验和PDLs计数法检测原代mDPCs、mDPCET和逆永生化细胞的细胞增殖动力学,通过免疫荧光、Real-time RT-PCR和Western Blot检测成牙本质相关标志物,茜素红染色检测细胞在诱导条件下矿化结节的形成。研究结果：与原代细胞相比,mDPCET增殖能力显著上调,寿命延长,但是同时保持了原代mDPCs的大部分生化和功能特性,当mDPCET细胞在4-OHT处理条件下,ERT2Cre ERT2从细胞质转移到细胞核,从而敲除已转入的SV40Tag-TK,使mDPCET发生逆永生化。与mDPCET相比,逆永生细胞的成牙本质相关基因及矿化结节形成能力显著上调,并重新获得可发生复制性衰老的能力。结论：建立并鉴定了一个它莫西芬诱导型可逆永生化小鼠牙乳头细胞系mDPCET。经4-OHT处理后,mDPCET可发生逆永生化,逆永生化细胞呈现低增殖高分化的状态,并重获可衰老的能力,这种状态更加类似分化的成牙本质细胞。二、Klf4通过调控Dmp1促进小鼠牙乳头细胞向成牙本质细胞样细胞分化研究目的：成牙本质细胞来源于牙乳头,是一种具有分泌基质功能的终未分化细胞。在成牙本质细胞分化过程中多种转录因子参与了调控。本课题组过去的研究发现,Kruppel-like factor4(Klf4)特异性表达在极化和延伸期的成牙本质细胞中,但是Klf4在成牙本质细胞分化过程中的作用尚不明确。本研究试图探索Klf4在成牙本质细胞分化过程中的功能,并分析其调控成牙本质细胞分化的分子机制。研究方法：在本研究中,使用矿化诱导液在体外诱导小鼠原代牙乳头细胞(mDPCs)和小鼠牙乳头细胞系即mDPC6T向成牙本质细胞样细胞分化作为研究模型。检测诱导过程中Klf4的mRNA和蛋白质表达水平、碱性磷酸酶活性和成牙本质细胞分化相关基因(Dspp、Dmp1和Alp)的表达：同时通过EdU掺入法检测细胞的增殖能力。在Klf4过表达和抑制条件下检测成牙本质细胞分化相关基因(Dspp、Dmp1和Alp)的表达和碱性磷酸酶活性,同时通过EdU掺入法检测细胞的增殖能力,茜素红染色检测矿化结节的形成。生物信息学预测Klf4潜在的下游基因Dmp1,并通过染色体免疫共沉淀(ChIP)、凝胶电泳迁移实验(EMSA)、双荧光素酶报告实验(DLA)检测Klf4对Dmp1的转录调控。在Klf4抑制条件下过表达Dmp1,检测Dmp1与Klf4间的调控关系。研究结果：Klf4在mDPCs和mDPC6T向成牙本质细胞样细胞分化过程中表达显著上调。过表达Klf4显著上调成牙本质相关基因,如Dmp1、Dspp和Alp,并可促进矿化结节的形成。抑制Klf4表达可下调Dmp1、Dspp和Alp的表达,并抑制矿化结节形成。生物信息学预测Klf4潜在的下游基因Dmp1。通过ChIP、EMSA和DLA的进一步分析,表明Klf4特异性结合于Dmp1启动子区,并转录激活其表达。Dmp1过表达可部分挽救由抑制Klf4表达对成牙本质分化的阻碍作用。结论：Klf4通过结合于Dmp1启动子上特定区域,转录上调Dmp1表达,从而促进成牙本质细胞分化。三、间充质中条件性敲除Klf4影响牙本质的矿化研究目的：Klf4(Kruppel-like factor4)又称为GKlf或Ezf,是Klf4锌指蛋白转录因子家族中的一员,在器官发育、干细胞干性维持和肿瘤发生发展中发挥重要作用。过去的研究发现Klf4特异性表达在极化和延伸期的成牙本质细胞中。在实验二中,通过体外研究探索了Klf4在成牙本质细胞分化中的作用。但是,体内环境下Klf4对成牙本质细胞分化和牙本质形成的调控作用还未可知。本研究试图探索在牙胚间充质中条件性敲除Klf4对成牙本质细胞分化和牙本质形成的影响。研究方法：将Wnt1-Cre:Klf4f/+小鼠与Klf4f/f小鼠交配就可以得到Wnt1-Cre；Klf4f/f,即在神经嵴来源的细胞中特异的失活Klf4的小鼠。Wnt1-Cre与R26R-LacZ(?)小鼠交配获得Wnt1-cre;R26R-LacZ即神经嵴来源的细胞呈x-gal染色阳性的小鼠。胎龄计算时以查到孕栓的当天中午计为胚胎E0.5天,小鼠出生当天中午计为出生后PN0.5天。按胎龄或出生后日龄获得的小鼠,分离尾鼠织,提取基因组DNA进行基因型鉴定。同一窝小鼠中基因型为Klf4f/+小鼠设为对照组小鼠,将基因型为Wnt1-cre；Klf4f/f小鼠设为实验组小鼠,即条件性敲除小鼠。分离小鼠下颌骨,体式显微镜照相,X-ray检测矿化组织密度,另外一部分组织经过固定,脱水,透明,石蜡包埋,切片用来进行组织学染色分析和免疫组化分析实验。研究结果：Wnt1-cre;R26R-LacZ经X-gal染色确认Cre重组酶在牙胚间充质细胞中被激活。免疫组织化学检测结果显示,在对照组(Klf4f/+)中,KLF4表达在E18.5与PN0.5的牙尖顶端分化的成牙本质细胞和成釉细胞中,在实验组中(Wnt1-cre;Klf4f/f) KLF4仅表达在PN0.5的牙尖顶端分化的成釉细胞中,成牙本质细胞不表达KLF4。HE染色结果显示在PN1W和PN2W的Klf4条件性敲除小鼠标本中,前期牙本质显著增厚。而Azan染色结果也发现Klf4条件性敲除小鼠的牙本质出现淡染。高分辨X-ray显示Klf4条件性敲除小鼠牙本质灰度显著下降,而牙釉质未发现显著的灰度改变。更有意思的是在PN3M时,2/4的Klf4条件性敲除小鼠出现龋损,并累及所有下颌磨牙,而对照小鼠未见龋损发生。结论：牙胚间充质中条件性敲除Klf4影响牙本质的矿化。更多还原

【Abstract】 1. Tamoxifen-mediated reversible immortalization of mouse dental papilla cell lineObjectives:Odontoblasts are a kind of non-proliferating and terminally differentiated cells which play an important part in pulpo-dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited lifespan. To solve this problem, we combined the traditional strategy of "Cre/LoxP-based reversible immortalization" with tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line.Methods:mDPCs were sequentially transduced with a floxed SV40Tag-TK and an ERT2CreERT2expressing plasmid. Clonal isolated SV40Tag and Cre positive cells were designated as mDPCET. The reverted cells were acquired by4-hydroxytamoxifen treatment. Primary mDPCs, mDPCET and the reverted cells were examined by investigation of cell proliferation kinetics, expression of odontoblastic-related makers and mineralization ability.Results:mDPCET showed upregulated growth rate and significantly extended lifespan, but retained most of the biochemical and functional characteristics of primary mDPCs. When mDPCET cells were treated with4-hydroxy tamoxifen, ERT2CreERT2was translocated from cytoplasm to nucleus which caused the excision of the SV40Tag-TK and led to the reversion of mDPCET. After the immortalization was reversed, cells underwent replicative senescence, showed higher expression level of odontoblastic-related genes and increased mineral deposition rate.Conclusions:Tamoxifen-mediated reversible immortalization, therefore, allows the expansion of primary mDPCs, leads to production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent as a safe and ready-to-use method due to its simple manipulation. 2. Klf4promoted odontoblastic differentiation of mouse dental papilla cells via DmplObjectives:Odontoblast cells, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of odontoblasts. We have recently found that Kriippel-like factor4(Klf4) was expressed in the polarizing and elongating odontoblasts, but the function of Klf4in the differentiation of odontoblasts is still unclear. We hypothesized Klf4promoted the differentiation of odontoblasts by up-regulating some odontoblast-related genes.Methods&Results:In this study, we found that the expression of Klf4increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4significantly up-regulated odontoblast-related genes, such as Dmpl, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4down-regulated expression of Dmpl, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4-Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4was able to specifically bind to the Dmpl promoter and transactivate its expression. Furthermore, forced expression of Dmpl in the Klf4knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Conclusions:Our data confirmed our hypothesis that Klf4promotes the differentiation of odontoblasts via the up-regulation of Dmpl. 3. Conditional knockout Klf4in dental mesenchyme affected dentin formation.Objectives:Kriippel-like factor4(Klf4) is a member of Klf zink finger transcriptional factor family. The Klf4plays a pivotal role in cellular differentiation. Previously, we have found that Klf4was expressed in the polarizing and elongating odontoblasts. In Part2, we applied in vitro study and showed that Klf4transcriptional activate the expression of Dmpl and promoted odontoblastic differentiation of mouse dental papilla cells. In this study, we hypothesized conditional knockout of Klf4in odontoblasts may affect differentiation of odontoblasts and dentin formation.Methods:Mouse containing inactivated Klf4in their neural crest cells (Wnt1-Cre; Klf4f/f) were obtained by crossing Wntl-Cre; Klf4f/+mice with Klf4mf/f line. Mouse containing activated LacZ in their neural crest cells was obtained by crossing Wntl-Cre mice with R26R-LacZ line. Mouse tail was dissected and the genome DNA was isolated for genotyping. Embryonic head samples were dissected and fixed individually in4%paraformaldehyde (PFA) overnight at4℃, and processed for paraffin section for histological and immunostaining or for X-ray scan.Results:Wntl-cre;R26R-LacZ mouse showed positive X-gal staining in dental mesenchyme. Immunostaining showed that, for the control mice (Klf4f/+), KLF4was expressed in the differentiated odontoblasts and ameloblasts in the cusp of E18.5and PN0.5molar germ. But in conditional knockout mice, expression of Klf4was detected only in the ameloblasts but not the odontoblasts. HE staining showed thicken predentin layer in the conditional knockout mice but not the control mice. Besides conditional knockout mice also showed weaker blue in the dentin for the azan staining. By high resolution X-ray, we have found decrease of the grey scale in the dentin but not the enamel in the conditional knockout mice. More interestingly, we observed caries involve all the mandibular molar in2/4of the3month old conditional knockout mice but none in the control mice.Conclusions:Klf4is essential in the odontoblasts for the dentin mineralization.更多还原