【作者】 任大鹏；

【导师】 王春玲； 袁晓； Aimin Peng；

【作者基本信息】 山东大学 ， 口腔临床医学， 2016， 博士

【摘要】 背景和目的正畸治疗过程中,牙齿在正畸力的作用下发生移动,其生物学基础在于牙周组织在应力刺激下发生改建。正畸力经牙齿传递到牙周组织,牙周膜在这一过程中发挥了桥梁作用。牙周膜成纤维细胞(periodontal ligament fibroblasts, PDLFs)是牙周膜的主要构成细胞,在应力刺激下能合成细胞因子并通过胞内信号传递系统影响细胞行为(增殖,分化,凋亡等),从而介导牙周组织的改建,修复和再生。研究应力刺激在PDLF胞内的信号转导以及对PDLF行为的影响,对于阐明正畸力作用下牙周组织改建的分子机理,发现有利于促使牙齿移动的力学环境都具有重要意义。成骨细胞是促进骨基质分泌和矿化的主要细胞。核心结合因子a1 (core binding factor al, Cbfal),又称Runt相关转录因子2(]runt related transcription factor 2, Runx2),是学者们公认的最重要的成骨细胞特异性转录因子之一。Runx2蛋白能特异性结合许多成骨基因启动子上的顺式作用元件,并促进成骨靶基因的转录表达。虽然Runx2基因和蛋白的表达水平在矿化组织和成骨细胞系中远远高于一些非骨组织来源的细胞,但是目前越来越多的研究表明其基因和蛋白水平与成骨细胞的功能和分化并不是直接线性相关,而Runx2蛋白的活化状态也是其发挥功能的重要影响因素。目前,学者们一致认为Runx2是联系胞外成骨诱导因素和胞内影响成骨细胞功能分化的信号通路的枢纽。细胞外信号调节激酶1/2(extracellular signal regulated kinase 1/2, ERK1/2)是最先被发现并且最具有代表性的有丝分裂原活化蛋白激酶(mitogen-activatedprotein kinase,MAPK)家族的成员。经胞外刺激因素活化后的ERK1/2可以进一步影响其下游转录因子的表达和活化。有研究表明Runx2是 ERK1/2众多下游靶基因中的一员,但是ERK1/2-Runx2通路是否可以在应力刺激下的PDLF中被激活还未知；如果其确实参与了应力刺激下PDLF的成骨分化,其具体的分子机理也有待深入研究。本研究首先通过携带Runx2基因的慢病毒载体转染原代培养的PDLF,探究Runx2对PDLF成骨分化的作用,然后构建了PDLF体外培养一应力刺激模型,观察Runx2和其他相关成骨基因在应力刺激下的表达变化,以及ERK1/2通路在应力刺激下的激活情况；最后通过比较对正常PDLF 和P Runx2过表达PDLF实施应力刺激,以及特异性阻断ERK1/2通路对应力刺激PDLF成骨分化的影响,研究ERK1/2 和 Runx2在介导应力刺激促进PDLF成骨分化过程中发挥的作用,对应力刺激,ERK1/2, Runx2,以及PDLF的成骨基因表达这几个因素之间的关系进行阐述,以明确ERK1/2-Runx2通路介导应力刺激调控PDLF成骨分化的分子机理,为临床正畸矫治提供新的分子生物学基础。主要实验方法及结果1牙周膜成纤维细胞的分离培养及鉴定采用组织块联合酶消化法对牙周膜成纤维细胞进行分离培养,并绘制细胞生长曲线。免疫细胞化学染色结果显示角蛋白染色为阴性,波形丝蛋白染色为阳性,HE染色胞浆伊红,胞核嗜碱性。成纤维细胞特异性表面蛋白1(FSP1)免疫荧光染色阳性。2稳定过表达Runx2基因的PDLF的构建及Runx2对PDLF的成骨诱导作用利用Ⅱ型Runx2基因以及LV5慢病毒载体构建LV5-Runx2慢病毒载体。有限稀释法测定病毒液滴度为6x105TU/ml。通过靶细胞侵染预实验对MOI值以及Puromycin筛选浓度进行摸索,最终获取稳定过表达Ⅱ型Runx2的牙周膜成纤维细胞。荧光定量PCR以及Western Blot证实稳定转染LV5-Runx2的牙周膜成纤维细胞中Runx2的nRNA和蛋白水平都升高显著。荧光定量PCR结果显示Ⅱ型Runx2过表达可以有效促进PDLF中成骨转录因子SP7,骨钙素(OCN)和骨涎蛋白(BSP)的转录表达,但对1型胶原(COL-1)和碱性磷酸酶(ALP)的表达无影响,并且轻微抑制转录激活因子4(ATF4)的表达。茜素红染色实验证实Ⅱ型Runx2过表达的PDL F体外培养3周后可以观察到钙结节。3周期性张应力刺激体外培养的牙周膜成纤维细胞模型构建使用多通道细胞体外牵张应力加载系统,对体外培养的牙周膜成纤维细胞施加振幅10%,频率0.5HZ的周期性牵张力,加力作用时间为1h,3h,6h,12h,18h,24h,以不加力组作为对照组,采用荧光定量PCR测定并绘制成骨基因Runx2, SP7, OCN, BSP和ATF4的mRNA随加力时间延长而变化的表达曲线；利用Western Blot和免疫沉淀法测定Runx2蛋白水平在不同加力时间点的表达,以及Runx2 和 ERK1/2在不同加力时间的激活情况。结果证实了周期性张应力可以促使牙周膜成纤维细胞中上述成骨基因的表达,并且在这一过程中同时伴随Runx2和ERK1/2通路的磷酸化。4ERK1/2-Runx2通路介导了周期性张应力刺激下牙周膜成纤维细胞的成骨分化对正常PDLF和过表达Runx2的PDLF分别进行应力刺激3h,荧光定量PCR结果显示周期性张应力刺激以及Lunx2过表达对牙周膜成纤维细胞中成骨基因SP7, OCN和BSP的mRNA表达有协同促进作用,即对Runx2过表达的PDLF同时施加应力刺激比不加力的Runx2过表达PDLF或者加力的正常PDLF对上述成骨基因的转录刺激作用更强。对加力组PDLF同时应用ERK1/2通路的特异性抑制剂U0126后,上述成骨基因的转录表达水平较单纯加力组有所下降。通过对正常PDLF和过表达Runx2的PDLF中Runx2的mRNA和蛋白水平的测定,发现应力刺激都可以诱导Runx2基因的转录和翻译,但ERK1/2通路被抑制后,Runx2的mRNA水平显著下降,而蛋白水平无明显变化,说明应力刺激对Runx2的转录表达可以通过ERK1/2介导,但ERK1/2对Runx2蛋白的翻译过程影响不大。通过测定Runx2的蛋白磷酸化水平(p-Runx2),发现p-Runx2的表达趋势与上述成骨基因的表达趋势高度一致,从而推测ERK1/2-Runx2通路介导应力刺激对牙周膜成纤维细胞的成骨分化的机制在于提高Runx2蛋白表达水平的同时对其进行磷酸化修饰以提高Runx2蛋白的转录激活功能。进一步研究发现ERK1/2蛋白主要存在于胞浆中,而Runx2蛋白则存在于胞核中；应力刺激激活ERK1/2后促使p-ERK1/2进入胞核并与Runx2形成蛋白复合体,推测这为p-ERK1/2对Runx2的磷酸化修饰提供了条件；U0126抑制了应力刺激下ERK1/2的活化,从而阻止p-ERK1/2进入细胞核并激活Runx2。结论1. Runx2对牙周膜成纤维细胞的成骨分化具有促进作用,可提高下游成骨靶基因SP7, OCN和BSP的转录水平的表达。2.周期性张应力刺激可以促进牙周膜成纤维细胞成骨基因SP7, OC N和BSP的转录表达；Runx2基因的mRNA和蛋白表达水平也同时升高,并伴随Runx2蛋白和ERK1/2通路的磷酸化。3. ERK1/2-Runx2通路介导周期性张应力刺激促进牙周膜成纤维细胞成骨分化的机制在于提高胞内Runx2蛋白的磷酸化水平(p-Runx2),从而促进Runx2下游成骨基因的转录表达；Runx2总蛋白水平与下游成骨基因转录表达无相关性。应力刺激激活ERK1/2进入细胞核中与Runx2形成蛋白复合体,猜测这为p-ERK1/2对Runx2的磷酸化提供了条件；阻断ERK1/2通路影响了应力刺激对ERK1/2的激活以及入核,从而影响了Runx2蛋白的磷酸化修饰和成骨基因的转录表达。创新和意义本课题从细胞分子水平证实了ERK1/2-Runx2通路参与了周期性张应力刺激下牙周膜成纤维细胞的成骨分化,并进一步探讨了其具体的机制,认为Runx2的磷酸化水平的而非总蛋白水平在调控下游成骨靶基因转录表达方面起到至关重要的作用；而应力刺激下活化的ERK1/2则在转录水平提高Runx2的表达并同时进入细胞核与RLunx2蛋白结合以促使Runx2蛋白的磷酸化。以上观点为临床正畸治疗过程中牙周组织的骨改建提供新的分子靶点和思路。更多还原

【Abstract】 Background and objectiveDuring the orthodontic treatment, the teeth move towards expected direction because of the reconstruction of peridontal tissue. Periodontal ligament plays an important role in the transmission of orthodontical force from tooth to periodontal tissue. Periodontal ligament is dense connective tissue locating between tooth root and alveolar bone, which is consisted by cells and matrix. Periodontal ligament fibroblasts (PDLFs) is the predominant cell in consisting periodontal ligament, and is able to synthetise various kinds of cytokine to mediate its own cellular behavior such as proliferation, differentiation and apoptosis through signal transduction system, which contribute to the remolding and regeneration of periodontal system. The investigation of how does the orthodontic force transduce inside the PDLFs, as well as how does the orthodontic force affect the PDLFs cellular behavior is helpful in understanding the mechanism of periodontal remolding promoted by orthodontic force and in searching the most suitable mechanical environment that could sustain the periodontal stability.Osteoblasts play an important role in promoting the bone matrix secretion and mineralization. Core binding factor a 1 (Cbfal), also called runt related transcription factor 2 (Runx2), is accepted as one of the most important osteogenic transcription factors. The protein could bind to the cis-acting element that exist on a serious of osteogenic genes, thus promoting the transcription of the target genes. Even as a specific osteogenic transcription factor, the expression of Runx2 is highly confined in the mineral tissue and osteoblast cell line, but more and more studies have proved that the expression level of Runx2 is not linear correlated to the function and differentiation of osteoblasts, because the activation status of Runx2 also affect its roles potentially. Overall, Runx2 is greatly accepted as a hub in connecting extracellular factors that could induce osteodifferentiation and intracellular signalings that control osteoblast function and differentiation.Extracellular signal regulated kinase 1/2 (ERK1/2) is the most typical and the earliest to be found among the Mitogen-activated protein kinase (MAPK) family members. Activated by upstream kinases, ERK1/2 could then transmit the signal further toward nuclear and affect the expression or activation of downstream factors. Runx2 has been proved to be one of those downstream targets of ERK1/2 in osteoblasts, but it is still unknown whether the ERK1/2-Runx2 pathway can be activated in mastication stimulated PDLFs, and if it does involve in the osteodifferentiation of PDLFs after mastication stimulation, the molecular mechanism of which is further needed to be explored.Our present study first prove the ability of Runx2 to promote the osteodifferentiation of PDLFs through transfecting the primary cultured PDLFs with lentivirus vector containing type Ⅱ Runx2 gene. Then we simulate the orthodontic force by constructing the in vitro mechanical tension application model and demonstrate the expressions of Runx2 and other related osteogenes, as well as ERK1/2 pathway could all be activated in PDLFs by cyclic stretch application. Finally, we apply the stretch on both normal PDLFs and Runx2 overexpressed PDLFs, and inhibit the ERK1/2 pathway using specific inhibitor at the same time, in order to intervene both of the factors in ERK1/2-Runx2 pathway. Our study interpreted the relationship among those factors:orthodontic force stimulation, Runx2, ERK1/2 and osteogene expression, clarified the mechanism of the PDLFs osteodifferentiation through ERKl/2-Runx2 pathway by cyclic tension loading. Our study proposed the possible molecular mechanism through which the orthodontic force stimulation could promote the PDLF osteodifferentiation and further the entire periodontal tisuue osteogenic remodeling, aiming to provide some new idea in clinical orthodontical treatment.Major methods and results1. Isolation and identification of PDLFsPrimary PDLFs were isolated through tissue combined with enzyme digestion methord. PDLFs growth curve were made by counting cell number. The endochylema was proved to be eosinophilic and the nuclear was proved to be basophilous according to HE staining. Keratin positive and vimentin negative results proved that the cultured cells derived from mesenchymal tissue. The result of immunofluorescence staining of fibroblast specific protein 1 (FSP1) was positive.2. Construction of the stably transfected PDLFs with type II Runx2 and effect of Runx2 in inducing the osteodifferentiation of PDLFs.We construct the LV5-Runx2 vector using type II Runx2 gene segment and lentivirus vector LV5. The virus stock solution was collected, and the concentration measured by limiting dilution assay was 6x105TU/ml. After the preliminary experiment was conducted for the optimal selecting concentration of Puromycin, as well as the optimal MOI value, we finally got the cloned PDLF overexpressing Runx2. Realtime PCR and Western blot were performed to confrim that mRNA and protein level of Runx2 were all elevated in stably transfected PDLFs. Realtime PCR results demonstrated that mRNA levels of osteogenes SP7, OCN and BSP were all promoted in Runx2 overexpressed PDLFs, COL-1 and ALP mRNA levels were unaffected, and ATF4 mRNA level reduced slightly in Runx2 overexpressed PDLFs. Alizarin red stain exprement found that 3 weeks culture of Runx2 overexpressed PDLFs led to the apperance of calcium nodule. Those results above proved that Runx2 overexpression alone could promote the osteodifferentiation of PDLFs.3. Construction of the cyclic stretch stimulated PDLFs modelWe applied the cyclic stretch of 10% amplitude and 0.5HZ frequency on PDLFs in vitro by using the multichannel cellular mechanical loading system, in order to simulate the biological orthodontical force loading onto the PDLFs. The loading time was 1h,3h,6h,12h,18h and 24h and unloaded PDLFs served as control group. Realtime PCR was conducted to detect the dynamic mRNA levels of Runx2, SP7, OCN, BSP and ATF4 in different loading time. Western blot and immunoprecipitation were conducted to detect the protein level of Runx2, as well as the activation of Runx2 and ERK1/2 pathway in different loading time. Those results above confirmed that current stretch loding system could effectively induce the osteogenes transcriptional expressions, and also activate the phosphorylation of Runx2 protein and ERK1/2 pathway during mechanical loading.4. ERK1/2-Runx2 pathway mediate the osteodifferentiation of PDLFs induced by cyclic stretchBoth the normal PDLFs and Runx2 overexpressed PDLFs were subject to cyclic stretch for 3h, and the mRNA level of ATF4, SP7, OCN and BSP were detected by realtime PCR. The results of realtime PCR showed that the mechanical stimualtion and Runx2 overexpression had a cooperative role in inducing the transcription of those genes above. Subsequently, inhibiting ERK1/2 pathway by U0126 on both normal PDLFs and Ruxn2(+)PDLFs while conducting mechanical stimulation significantly reduced the transcripts of those genes above, comparing to the mechanical stimulation without blocking ERK1/2 pathway. Further, by studing the mRNA level and protein level of Runx2 in the cells, we found that cyclic stretch could promote the transcriptional and translational expression of Runx2 in PDLFs; when ERK1/2 pathway was inhibited, the mRNA level of Runx2 could decrease, while Ruxn2 protein level didn’t reduce evidently. These results proved that the mechanical stimulation could induce the Runx2 transcriptional expression through ERK1/2 pathway, but ERK1/2 has little effect on Runx2 translational regulation. By studying the phosphorylation level of Runx2 (p-Ruxn2), we found that the expression ofp-Runx2 is highly related to the mRNA levels of those target genes of Runx2. Our conclusion is that the mechanism of ERK1/2-Runx2 in mediating the mechanical stretch stimulated PDLFs osteodifferentiation is promoting the phosphorylated protein level of Runx2, which could elevate the transactivation function of Runx2 in order to increasing the target genes transcription. Further analysis of both Runx2 and ERK1/2 protein revealed that ERK1/2 exist in cytoplasm in resting cells, while Runx2 stays in nuclear; mechanical stimulation activate the ERK1/2 and promote them to translocate into unclear where they could form protein complex with Runx2, which could provide the condition for Runx2 to be phosphorylated; specific inhibition of ERK1/2 by U0126 prevented the stretch-induced activation of ERK1/2 as well as its nuclear translocation, thus abate its effect on Runx2 phosphorylation.Conclusion1. Runx2 could promote the osteodifferentiation of PDLFs by inducing the transcription of its target genes such as SP7, OCN and BSP2. Orthodontic force simulated by cyclic stretch could elevate the mRNA expressions of SP7, OCN and BSP, as well as both the mRNA and protein level of Runx2. Besides, the ERKl/2 and Runx2 could be activated during the cyclic stretch stimulation3. The ERK1/2-Runx2 pathway participates in mechanical stretch-stimulated PDLFs osteodifferentiation through increased level of p-Runx2 protein, thus promote transcriptional expressions of its target osteogene; total Runx2 protein level is not correlated with the transcription level of its target osteogene. Stretch activated and promote the nuclear translocation of ERK1/2, which complex with Runx2, which presumably create condition for Runx2 phosphorylation. Inhibition of ERK1/2 by U0126 prohibit the effect of stretch on ERK1/2 and thus the phosphorylation of Runx2, which affect the osteogenes expressions in stretch loaded PDLFs.Innovation and significanceThis present study confirmed that the ERK1/2-Runx2 pathway involves in the cyclic stretch induced PDLFs osteodifferentiation from cellular and molecular level. Our study proposed the idea that activated Ruxn2 rather than total Runx2 is the predominant factor in regulating the transcriptions of its target genes, while activated ERK1/2 could lead to increase in Ruxn2 mRNA level, as well as further complex and phosphorylate Runx2 protein after nuclear translocation. Those opinions above could provide some new fresh ideas regarding the periodontal tissue remolding during clinical orthodontic treatment.更多还原