PHF8在细胞成骨分化及骨修复过程中的作用研究

发布时间：2018-06-24 19:08

本文选题：PHF8 + 去甲基化　；参考：《山东大学》2014年博士论文

【摘要】：牙周病是口腔两大类主要疾病之一,是导致牙齿缺失的重要原因,在世界范围内有较高的患病率,在我国其患病率更居于龋病之上。牙周病是以牙周支持组织破坏为特征的慢性感染性疾病,可造成牙槽骨的吸收及牙周袋的形成,最终导致牙齿丧失。牙周病治疗的最终目的是重建因炎症过程而破坏的牙周组织,恢复牙周组织的结构和功能,即实现牙周组织再生。牙槽骨再生在牙周组织再生中具有重要的意义。骨组织的重建主要依赖于成骨细胞分泌骨基质,继而矿物质沉积。干细胞的增殖与分化在骨组织再生中发挥重要作用。干细胞是一类具有自我更新和分化潜能的细胞,其增殖和分化受多种内在机制和微环境因素影响。目前多采用生长因子刺激或改变基因组DNA等方法调控干细胞的成骨分化状态,但生长因子价格昂贵且效果不稳定,改变基因组DNA可能会造成不可逆的损伤。表观遗传学则是通过组蛋白修饰及DNA甲基化等的改变使DNA处于转录激活或者抑制状态,这种修饰不会造成基因组DNA的改变,因此可能导致的副作用较小。目前表观遗传学用于骨组织再生方面的研究较少。因此本实验探讨表观遗传学机制在干细胞成骨分化中的作用及作用机制,并进一步研究其在体内促进骨组织再生中的作用。这将有助于我们了解表观遗传学机制在干细胞成骨分化中的作用,为临床研发新的更有效的骨组织再生方法开辟新的思路。 PHD锌指蛋白8(PHD finger protein8, PHF8)是一种组蛋白去甲基化酶,可作用于多种组蛋白调节组蛋白单甲基化与双甲基化,进而从转录水平调控基因的表达。PHF8通过其PHD结构域结合到H3K4me3核小体并发挥对靶基因转录起始位点(TSS)组蛋白H3K9, H3K27和H4K20的去甲基化作用,进一步调控基因的转录活性。PHF8最初被证实与X染色体相关智力障碍有关。后续多项研究证实PHF8参与多种生物学过程,如调控细胞周期相关基因表达并影响肿瘤细胞迁移和侵袭；调控细胞黏附相关蛋白及细胞骨架蛋白如RhoA, Racl和GSK3β的表达；调节视黄酸对急性早幼粒细胞白血病的反应,对该类白血病的治疗有一定指导意义；通过调节组蛋白H3K9me1/2的去甲基化活性来调控rRNA的合成等。最近的研究发现PHF8在调控颅颌面发育中发挥重要作用。对斑马鱼的研究发现PHF8主要表达在头部及颌骨区域,注射zPHF8吗琳代能够导致颅颌面组织发育异常,而野生型PHF8治疗能够逆转由此引起的发育异常,而此种发育异常主要由于骨组织发育异常引起,因此该研究提示PHF8可能在骨组织发育再生中发挥重要作用。特殊富含AT序列结合蛋白2(Special AT-rich sequence-binding protein2, Satb2)是一种能够结合到特异的富含AT序列区域,调节染色体结构与基因表达的DNA结合蛋白。与PHF8相似,Satb2在鳃弓与成骨细胞系中也有表达。Satb2-/-的小鼠表现出成骨细胞的功能缺陷及分化异常,最终导致骨形成与矿化延迟。Satb2-/-胚胎表现出多种颅颌面异常,如下颌裂与腭裂。同时有报道证实Satb2基因变异的个体表现出广泛性骨质疏松及颅颌面发育畸形,包括腭裂,下颌发育不全等。上述多项研究已明确证实Satb2在成骨细胞分化及骨组织再生中发挥重要作用,因此Satb2可能成为用于促进颅颌面骨组织再生理想的转录因子。但是在骨髓间充质干细胞(Bone marrow stromal cells, BMSCs)向成骨细胞分化过程中表观遗传学因素与Satb2的相互作用机制尚未完全明了。以上的研究证实了PHF8与Satb2基因功能的相似性。其中任何一种基因的功能受到抑制都会导致严重的颅颌面发育异常,提示PHF8与Satb2在调控细胞成骨分化及骨组织再生方面可能存在某种联系。因此本实验的目的即探讨PHF8在BMSCs成骨分化及骨组织再生中的作用,并进一步研究PHF8调节细胞成骨分化是否通过其去甲基化酶活性调控Satb2基因的转录活性而发挥作用。材料和方法第一部分：PHF8在小鼠不同组织中的表达差异、细胞内定位及在BMSCs成骨分化中的表达变化。取8周龄C57BL/6J小鼠的不同组织制作组织切片,采用免疫组织化学的方法检测PHF8在不同组织中的表达。复苏冻存的MC3T3-E1细胞,传代培养后待细胞状态良好时,采用免疫荧光化学的方法检测PHF8的细胞内定位。从4周龄小鼠中取原代BMSCs,传代培养后第二代BMSCs施以成骨诱导液,分别于1,3,7,10,14,21天后提取总RNA,采用实时定量PCR (Real time PCR)方法检测各成骨相关因子及PHF8和Satb2在诱导成骨分化中的表达改变。第二部分：PHF8对小鼠BMSCs及前成骨细胞MC3T3-E1成骨分化的影响及作用机制。包装PHF8、PHF8shRNA及其相应对照空载体的慢病毒,运用慢病毒感染BMSCs及MC3T3-E1细胞,收集细胞并提取总RNA及总蛋白,用Real time PCR及western blot方法检测成骨相关因子mRNA及蛋白表达的变化；MC3T3-E1细胞成骨诱导7天及10天后,采用染色质免疫共沉淀(Chromatin Immunoprecipitation, ChIP)的方法检测PHF8是否能够结合到Satb2的启动子区域；用含有PHF8、PHF8shRNA及其相应对照空载体的慢病毒感染MC3T3-E1细胞,采用ChIP检测结合在Satb2基因上的单甲基化组蛋白H3K9mel的变化。第三部分：丝蛋白支架复合PHF8修饰的BMSCs对小鼠颅骨极量骨缺损修复的作用用含有PHF8、PHF8shRNA及其相应对照空载体的慢病毒感染BMSCs,将基因修饰的BMSCs与丝蛋白支架复合；在8周龄小鼠双侧颅骨制作直径4mm的缺损模型,将复合细胞的支架材料置于缺损区,对位缝合皮肤。5周后采用Micro CT,HE染色,免疫组织化学染色及Real Time PCR等方法检测PHF8修饰的BMSCs对小鼠颅骨极量骨缺损的修复作用。结果第一部分：PHF8在小鼠不同组织中的表达差异、细胞内定位及在骨髓基质细胞成骨分化中的表达变化对小鼠不同组织的组织切片进行免疫组织化学检测发现在长骨以及颅骨中有较多PHF8表达且PHF8阳性细胞主要集中于长骨的生长板及颅骨骨缝区域；而在心脏中有少量PHF8阳性细胞；肾,肝及肌肉组织中几乎看不到PHF8阳性细胞；细胞免疫荧光染色则证实PHF8主要表达于MC3T3-E1细胞的细胞核内。在BMSCs成骨诱导分化过程中,PHF8与Satb2mRNA的表达上升,且二者上升呈相同趋势。第二部分：PHF8促进小鼠BMSCs及前成骨细胞MC3T3-E1成骨分化及作用机制采用Real time PCR及western blot检测发现,在BMSCs及MC3T3-E1细胞中过表达PHF8能够上调成骨相关因子BSP, OC, Runx2, Satb2和OSX mRNA及蛋白的表达水平,而抑制PHF8表达能下调成骨相关因子mRNA及蛋白的表达水平；同时,本研究采用ChIP检测发现PHF8能够结合到Satb2的TSS区,且在成骨诱导分化过程中,PHF8与Satb2的结合能力增强；过表达PHF8能使结合在Satb2基因TSS区域的甲基化组蛋白H3K9mel减少,而用shRNA抑制PHF8能够增加甲基化组蛋白H3K9mel在Satb2基因TSS区域的结合。第三部分：丝蛋白支架复合PHF8修饰的BMSCs促进小鼠颅骨极量骨缺损的修复丝蛋白支架复合不同修饰的BMSCs用于修复小鼠颅骨极量骨缺损5周后,Micro CT结果证实PHF8修饰的BMSCs处理组新生骨体积(Bone volume, BV)较其他各组高,HE染色对骨缺损区域新生骨面积进行统计分析也得到同样的结果,表明PHF8修饰的BMSCs局部应用能够促进小鼠颅骨极量骨缺损的愈合；取新生组织提取RNA进行Real Time PCR结果表明PHF8修饰的BMSCs组骨缺损区域成骨相关基因的表达亦高于其他各组。结论 1.PHF8主要表达于长骨的生长板区域及颅骨骨缝区。 2.PHF8能够促进成骨相关因子mRNA及蛋白的表达,在BMSCs及MC3T3-E1成骨分化中发挥重要作用。3.PHF8发挥去甲基化酶活性,调节Satb2启动子区域组蛋白H3K9mel甲基化状态,进而调控Satb2基因的转录发挥成骨分化调节作用。 4.PHF8修饰的BMSCs能够促进小鼠颅骨极量骨缺损的愈合,因此可能成为用于骨组织再生的理想治疗分子。
[Abstract]:Periodontal disease is one of the two major diseases in the oral cavity. It is an important cause of tooth loss. There is a high prevalence in the world. In our country, the prevalence rate is above caries. Periodontitis is a chronic infectious disease characterized by periodontal support tissue damage, which can be made into alveolar bone and the formation of periodontal pocket. Finally, it leads to the formation of the periodontal pocket. Tooth loss. The ultimate aim of periodontal disease treatment is to reconstruct periodontal tissue that is damaged by the inflammatory process, restore the structure and function of periodontal tissue, that is to realize periodontal tissue regeneration. Alveolar bone regeneration is of great significance in periodontal tissue regeneration. The reconstruction of bone tissue is mainly dependent on the osteoblasts secreting the bone matrix and then minerals. The proliferation and differentiation of stem cells play an important role in bone tissue regeneration. Stem cells are a kind of cells with potential for self renewal and differentiation. The proliferation and differentiation of stem cells are affected by various internal mechanisms and microenvironmental factors. At present, the osteogenic differentiation of stem cells is regulated by the methods of stimulating or changing the genomic DNA by growth factors. However, the growth factor is expensive and the effect is unstable. The change of the genomic DNA may cause irreversible damage. Epigenetics is by the change of histone modification and DNA methylation to make DNA in the transcriptional activation or inhibition state, this modification does not cause the change of genomic DNA, thus may lead to less side effects. Epigenetics is seldom used in bone tissue regeneration. Therefore, this study explores the role and mechanism of epigenetic mechanism in osteogenic differentiation of stem cells, and further studies its role in promoting bone tissue regeneration in vivo. This will help us to understand the role of epigenetic mechanism in the osteogenesis of stem cells. It will open up new ideas for clinical research and development of new and more effective methods of bone tissue regeneration.
PHD zinc finger protein 8 (PHD finger protein8, PHF8) is a histone demethylation enzyme that can act on the monomethylation and dimethylation of a variety of histone regulating histone, and then from the transcriptional level regulating gene expression.PHF8 through its PHD domain to H3K4me3 nucleosome and to play the target gene transcription starting site (TSS) histone H3K9, The demethylation of H3K27 and H4K20, which further regulates the transcriptional activity of.PHF8, is initially linked to the mental disorders associated with the X chromosome. Subsequent studies have confirmed that PHF8 is involved in a variety of biological processes, such as regulating the expression of cell cycle related genes and affecting the migration and invasion of tumor cells, regulating cell adhesion related proteins and fine-tuning. The expression of cytoskeleton, such as RhoA, Racl and GSK3 beta, regulates the response of retinoic acid to acute promyelocytic leukemia, and has certain guiding significance for the treatment of this type of leukemia. The synthesis of rRNA is regulated by modulating the demethylation activity of histone H3K9me1/2. Recent studies have found that PHF8 plays an important role in the regulation of craniofacial development. The study of zebrafish found that PHF8 is mainly expressed in the head and jaw region, and the injection of zPHF8 can lead to abnormal development of craniofacial tissue, and the wild type PHF8 therapy can reverse the resulting dysplasia, which is mainly due to the abnormal development of bone tissue. Therefore, this study suggests that PHF8 may be in the bone group. The fabric plays an important role in the development and regeneration of the fabric.
Special AT sequence binding protein 2 (Special AT-rich sequence-binding protein2, Satb2) is a DNA binding protein capable of combining specific AT sequence regions to regulate chromosome structure and gene expression. Similar to PHF8, Satb2 also expresses the work of.Satb2-/- mice in the branchial and osteoblast lines expressing osteoblasts. Defects and abnormal differentiation, resulting in bone formation and mineralization delayed.Satb2-/- embryos showing a variety of craniofacial abnormalities, as follows: maxillary cleft and cleft palate. Meanwhile, it has been reported that individuals with Satb2 gene mutation show extensive osteoporosis and craniofacial malformation, including cleft palate and lower jaw development. A number of studies have clearly confirmed that Satb2 plays an important role in osteoblast differentiation and bone tissue regeneration, so Satb2 may become an ideal transcription factor for promoting the regeneration of craniofacial tissue. But the interaction mechanism of epigenetic factors with Satb2 in the differentiation of bone marrow mesenchymal stem cells (Bone marrow stromal cells, BMSCs) to osteoblasts It's not completely clear.
These studies have confirmed the similarity between PHF8 and Satb2 gene function. The inhibition of any one of these genes may lead to severe craniofacial dysplasia, suggesting that PHF8 and Satb2 may have some connection in the regulation of osteogenic differentiation and bone tissue regeneration. Therefore, the purpose of this experiment is to explore the PHF8 osteogenesis in BMSCs. The role of PHF8 in the regeneration of bone tissue and to further study whether the osteogenic differentiation of cells can regulate the transcriptional activity of the Satb2 gene by its demethylation activity.
Materials and methods
Part one: the differential expression of PHF8 in different tissues of mice, the intracellular localization and the expression changes of BMSCs in osteogenic differentiation.
Tissue sections were made from different tissues of 8 weeks old C57BL/6J mice. The expression of PHF8 in different tissues was detected by immunohistochemical method. When the frozen MC3T3-E1 cells were resuscitation, the intracellular location of PHF8 was detected by immunofluorescence chemical method. The original BMSCs was obtained from 4 weeks old mice. Second generations of BMSCs were treated with osteogenic inducer, and the total RNA was extracted after 1,3,7,10,14,21 days. The expression of bone related factors and the expression of PHF8 and Satb2 in the induction of osteogenic differentiation were detected by real-time quantitative PCR (Real time PCR).
The second part: the effect and mechanism of PHF8 on the osteogenic differentiation of BMSCs and osteoblast MC3T3-E1 in mice.
The lentivirus of PHF8, PHF8shRNA and its corresponding control space carrier, using slow virus infection of BMSCs and MC3T3-E1 cells, collecting cells and extracting total RNA and total protein, using Real time PCR and Western blot to detect the changes of mRNA and protein expression of bone related factors; 7 days and 10 days after induction of MC3T3-E1 fine cell osteogenesis, immunization with chromatin Chromatin Immunoprecipitation (ChIP) method was used to detect whether PHF8 could bind to the promoter region of Satb2; MC3T3-E1 cells with PHF8, PHF8shRNA and their corresponding control space carriers were infected with MC3T3-E1 cells, and ChIP detection combined with the H3K9mel change of mono methylation histone on the Satb2 gene.
The third part: the effect of silk protein scaffold combined with PHF8 modified BMSCs on the repair of cranial bone defects in mice.
The gene modified BMSCs was combined with the silk protein scaffold with PHF8, PHF8shRNA and its corresponding control space carrier. The defect model of 4mm in the diameter of 8 weeks old mice was made in 8 weeks old mice. The scaffold materials of the composite cells were placed in the defect area. After.5 weeks, Micro CT, HE staining, and immunohistochemistry were used. Staining and Real Time PCR were used to detect the repair effect of PHF8 modified BMSCs on cranial bone defect in mice.
Result
Part one: the differential expression of PHF8 in different tissues of mice, intracellular localization and expression changes in osteogenic differentiation of bone marrow stromal cells.
The immunohistochemical detection of tissue sections of different tissues of mice showed that there were more PHF8 expressions in the long bone and the skull, and the PHF8 positive cells were mainly concentrated in the growth plate of the long bone and the area of the cranial seams; and there were a few PHF8 positive cells in the heart; the cells in the kidney, liver and muscle group almost did not see the positive cells of the PHF8; Immunofluorescence staining showed that PHF8 was mainly expressed in the nucleus of MC3T3-E1 cells. The expression of PHF8 and Satb2mRNA increased in the process of BMSCs osteogenesis induced differentiation, and the two increased in the same trend.
The second part: PHF8 promotes the osteogenic differentiation and mechanism of BMSCs and osteoblast MC3T3-E1 in mice.
Real time PCR and Western blot detected that the overexpression of PHF8 in BMSCs and MC3T3-E1 cells could up regulate the expression level of BSP, OC, Runx2, Satb2 and protein. In the TSS region of Satb2, the binding ability of PHF8 to Satb2 is enhanced during the osteogenic differentiation, and overexpression of PHF8 can reduce the methylation histone H3K9mel in the TSS region of the Satb2 gene, and the shRNA inhibition PHF8 can increase the binding of the methylated histone H3K9mel in the Satb2 base.
The third part: silk fibroin scaffold combined with PHF8 modified BMSCs promotes the repair of cranial bone defects in mice.
The combination of silk protein scaffold and different modified BMSCs was used to repair the extreme bone defect of the skull in mice for 5 weeks. The results of Micro CT confirmed that the new bone volume (Bone volume, BV) in the PHF8 modified BMSCs treatment group was higher than that of the other groups. The statistical analysis of the new bone area in the bone defect area by HE staining also obtained the same results, indicating the BMSCs Bureau of PHF8 modification. The application can promote the healing of the extreme bone defect of the skull in mice, and the results of Real Time PCR in the extraction of RNA from the newborn tissues indicate that the expression of the bone related genes in the region of the PHF8 modified BMSCs group is also higher than that of the other groups.
conclusion
1.PHF8 is mainly expressed in the growth plate area of the long bone and the suture area of the skull.
2.PHF8 can promote the expression of mRNA and protein in bone related factors. It plays an important role in BMSCs and MC3T3-E1 osteogenesis,.3.PHF8 plays the activity of demethase and regulates the H3K9mel methylation of the Satb2 promoter region histone
In order to regulate the transcription of Satb2 gene, it can regulate osteogenic differentiation.
4.PHF8 modified BMSCs can promote the healing of cranial bone defects in rats, so it may become an ideal therapeutic molecule for bone tissue regeneration.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R781.4

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