本文選題：PHF8 + 去甲基化��； 參考：《山東大學》2014年博士論文

【摘要】：牙周病是口腔兩大類主要疾病之一,是導致牙齒缺失的重要原因,在世界范圍內(nèi)有較高的患病率,在我國其患病率更居于齲病之上。牙周病是以牙周支持組織破壞為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,最終導致牙齒喪失。牙周病治療的最終目的是重建因炎癥過程而破壞的牙周組織,恢復牙周組織的結(jié)構和功能,即實現(xiàn)牙周組織再生。牙槽骨再生在牙周組織再生中具有重要的意義。骨組織的重建主要依賴于成骨細胞分泌骨基質(zhì),繼而礦物質(zhì)沉積。干細胞的增殖與分化在骨組織再生中發(fā)揮重要作用。干細胞是一類具有自我更新和分化潛能的細胞,其增殖和分化受多種內(nèi)在機制和微環(huán)境因素影響。目前多采用生長因子刺激或改變基因組DNA等方法調(diào)控干細胞的成骨分化狀態(tài),但生長因子價格昂貴且效果不穩(wěn)定,改變基因組DNA可能會造成不可逆的損傷。表觀遺傳學則是通過組蛋白修飾及DNA甲基化等的改變使DNA處于轉(zhuǎn)錄激活或者抑制狀態(tài),這種修飾不會造成基因組DNA的改變,因此可能導致的副作用較小。目前表觀遺傳學用于骨組織再生方面的研究較少。因此本實驗探討表觀遺傳學機制在干細胞成骨分化中的作用及作用機制,并進一步研究其在體內(nèi)促進骨組織再生中的作用。這將有助于我們了解表觀遺傳學機制在干細胞成骨分化中的作用,為臨床研發(fā)新的更有效的骨組織再生方法開辟新的思路。 PHD鋅指蛋白8(PHD finger protein8, PHF8)是一種組蛋白去甲基化酶,可作用于多種組蛋白調(diào)節(jié)組蛋白單甲基化與雙甲基化,進而從轉(zhuǎn)錄水平調(diào)控基因的表達。PHF8通過其PHD結(jié)構域結(jié)合到H3K4me3核小體并發(fā)揮對靶基因轉(zhuǎn)錄起始位點(TSS)組蛋白H3K9, H3K27和H4K20的去甲基化作用,進一步調(diào)控基因的轉(zhuǎn)錄活性。PHF8最初被證實與X染色體相關智力障礙有關。后續(xù)多項研究證實PHF8參與多種生物學過程,如調(diào)控細胞周期相關基因表達并影響腫瘤細胞遷移和侵襲；調(diào)控細胞黏附相關蛋白及細胞骨架蛋白如RhoA, Racl和GSK3β的表達；調(diào)節(jié)視黃酸對急性早幼粒細胞白血病的反應,對該類白血病的治療有一定指導意義；通過調(diào)節(jié)組蛋白H3K9me1/2的去甲基化活性來調(diào)控rRNA的合成等。最近的研究發(fā)現(xiàn)PHF8在調(diào)控顱頜面發(fā)育中發(fā)揮重要作用。對斑馬魚的研究發(fā)現(xiàn)PHF8主要表達在頭部及頜骨區(qū)域,注射zPHF8嗎琳代能夠?qū)е嘛B頜面組織發(fā)育異常,而野生型PHF8治療能夠逆轉(zhuǎn)由此引起的發(fā)育異常,而此種發(fā)育異常主要由于骨組織發(fā)育異常引起,因此該研究提示PHF8可能在骨組織發(fā)育再生中發(fā)揮重要作用。 特殊富含AT序列結(jié)合蛋白2(Special AT-rich sequence-binding protein2, Satb2)是一種能夠結(jié)合到特異的富含AT序列區(qū)域,調(diào)節(jié)染色體結(jié)構與基因表達的DNA結(jié)合蛋白。與PHF8相似,Satb2在鰓弓與成骨細胞系中也有表達。Satb2-/-的小鼠表現(xiàn)出成骨細胞的功能缺陷及分化異常,最終導致骨形成與礦化延遲。Satb2-/-胚胎表現(xiàn)出多種顱頜面異常,如下頜裂與腭裂。同時有報道證實Satb2基因變異的個體表現(xiàn)出廣泛性骨質(zhì)疏松及顱頜面發(fā)育畸形,包括腭裂,下頜發(fā)育不全等。上述多項研究已明確證實Satb2在成骨細胞分化及骨組織再生中發(fā)揮重要作用,因此Satb2可能成為用于促進顱頜面骨組織再生理想的轉(zhuǎn)錄因子。但是在骨髓間充質(zhì)干細胞(Bone marrow stromal cells, BMSCs)向成骨細胞分化過程中表觀遺傳學因素與Satb2的相互作用機制尚未完全明了。 以上的研究證實了PHF8與Satb2基因功能的相似性。其中任何一種基因的功能受到抑制都會導致嚴重的顱頜面發(fā)育異常,提示PHF8與Satb2在調(diào)控細胞成骨分化及骨組織再生方面可能存在某種聯(lián)系。因此本實驗的目的即探討PHF8在BMSCs成骨分化及骨組織再生中的作用,并進一步研究PHF8調(diào)節(jié)細胞成骨分化是否通過其去甲基化酶活性調(diào)控Satb2基因的轉(zhuǎn)錄活性而發(fā)揮作用。 材料和方法 第一部分：PHF8在小鼠不同組織中的表達差異、細胞內(nèi)定位及在BMSCs成骨分化中的表達變化。 取8周齡C57BL/6J小鼠的不同組織制作組織切片,采用免疫組織化學的方法檢測PHF8在不同組織中的表達。復蘇凍存的MC3T3-E1細胞,傳代培養(yǎng)后待細胞狀態(tài)良好時,采用免疫熒光化學的方法檢測PHF8的細胞內(nèi)定位。從4周齡小鼠中取原代BMSCs,傳代培養(yǎng)后第二代BMSCs施以成骨誘導液,分別于1,3,7,10,14,21天后提取總RNA,采用實時定量PCR (Real time PCR)方法檢測各成骨相關因子及PHF8和Satb2在誘導成骨分化中的表達改變。 第二部分：PHF8對小鼠BMSCs及前成骨細胞MC3T3-E1成骨分化的影響及作用機制。 包裝PHF8、PHF8shRNA及其相應對照空載體的慢病毒,運用慢病毒感染BMSCs及MC3T3-E1細胞,收集細胞并提取總RNA及總蛋白,用Real time PCR及western blot方法檢測成骨相關因子mRNA及蛋白表達的變化；MC3T3-E1細胞成骨誘導7天及10天后,采用染色質(zhì)免疫共沉淀(Chromatin Immunoprecipitation, ChIP)的方法檢測PHF8是否能夠結(jié)合到Satb2的啟動子區(qū)域；用含有PHF8、PHF8shRNA及其相應對照空載體的慢病毒感染MC3T3-E1細胞,采用ChIP檢測結(jié)合在Satb2基因上的單甲基化組蛋白H3K9mel的變化。 第三部分：絲蛋白支架復合PHF8修飾的BMSCs對小鼠顱骨極量骨缺損修復的作用 用含有PHF8、PHF8shRNA及其相應對照空載體的慢病毒感染BMSCs,將基因修飾的BMSCs與絲蛋白支架復合；在8周齡小鼠雙側(cè)顱骨制作直徑4mm的缺損模型,將復合細胞的支架材料置于缺損區(qū),對位縫合皮膚。5周后采用Micro CT,HE染色,免疫組織化學染色及Real Time PCR等方法檢測PHF8修飾的BMSCs對小鼠顱骨極量骨缺損的修復作用。 結(jié)果 第一部分：PHF8在小鼠不同組織中的表達差異、細胞內(nèi)定位及在骨髓基質(zhì)細胞成骨分化中的表達變化 對小鼠不同組織的組織切片進行免疫組織化學檢測發(fā)現(xiàn)在長骨以及顱骨中有較多PHF8表達且PHF8陽性細胞主要集中于長骨的生長板及顱骨骨縫區(qū)域；而在心臟中有少量PHF8陽性細胞；腎,肝及肌肉組織中幾乎看不到PHF8陽性細胞；細胞免疫熒光染色則證實PHF8主要表達于MC3T3-E1細胞的細胞核內(nèi)。在BMSCs成骨誘導分化過程中,PHF8與Satb2mRNA的表達上升,且二者上升呈相同趨勢。 第二部分：PHF8促進小鼠BMSCs及前成骨細胞MC3T3-E1成骨分化及作用機制 采用Real time PCR及western blot檢測發(fā)現(xiàn),在BMSCs及MC3T3-E1細胞中過表達PHF8能夠上調(diào)成骨相關因子BSP, OC, Runx2, Satb2和OSX mRNA及蛋白的表達水平,而抑制PHF8表達能下調(diào)成骨相關因子mRNA及蛋白的表達水平；同時,本研究采用ChIP檢測發(fā)現(xiàn)PHF8能夠結(jié)合到Satb2的TSS區(qū),且在成骨誘導分化過程中,PHF8與Satb2的結(jié)合能力增強；過表達PHF8能使結(jié)合在Satb2基因TSS區(qū)域的甲基化組蛋白H3K9mel減少,而用shRNA抑制PHF8能夠增加甲基化組蛋白H3K9mel在Satb2基因TSS區(qū)域的結(jié)合。 第三部分：絲蛋白支架復合PHF8修飾的BMSCs促進小鼠顱骨極量骨缺損的修復 絲蛋白支架復合不同修飾的BMSCs用于修復小鼠顱骨極量骨缺損5周后,Micro CT結(jié)果證實PHF8修飾的BMSCs處理組新生骨體積(Bone volume, BV)較其他各組高,HE染色對骨缺損區(qū)域新生骨面積進行統(tǒng)計分析也得到同樣的結(jié)果,表明PHF8修飾的BMSCs局部應用能夠促進小鼠顱骨極量骨缺損的愈合；取新生組織提取RNA進行Real Time PCR結(jié)果表明PHF8修飾的BMSCs組骨缺損區(qū)域成骨相關基因的表達亦高于其他各組。 結(jié)論 1.PHF8主要表達于長骨的生長板區(qū)域及顱骨骨縫區(qū)。 2.PHF8能夠促進成骨相關因子mRNA及蛋白的表達,在BMSCs及MC3T3-E1成骨分化中發(fā)揮重要作用。3.PHF8發(fā)揮去甲基化酶活性,調(diào)節(jié)Satb2啟動子區(qū)域組蛋白H3K9mel甲基化 狀態(tài),進而調(diào)控Satb2基因的轉(zhuǎn)錄發(fā)揮成骨分化調(diào)節(jié)作用。 4.PHF8修飾的BMSCs能夠促進小鼠顱骨極量骨缺損的愈合,因此可能成為用于骨組織再生的理想治療分子。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R781.4

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