本文選題：KAT2A + 二氧化鈦��； 參考：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：研究背景生物醫(yī)學材料的性能強烈依賴于第一個發(fā)生相互作用時,材料表面接觸的生物環(huán)境[1];同時。在骨組織工程種子細胞發(fā)揮功能和分化過程中,由于骨頭本身最基本的結(jié)構(gòu)層次納米范圍內(nèi),而納米級別材料可以通過人體仿生來精確控制[2,3],因此,種子細胞與納米級別材料的研究受到人們研究和應(yīng)用的青睞。在口腔臨床治療應(yīng)用領(lǐng)域,種植體材料是最為常見的生物材料之一,用于牙齒缺失和骨組織缺損的修復(fù)[4];對于種植體的表面的處理技術(shù),研究所和企業(yè)也在不斷探索和改進。我們已知表觀遺傳學和微環(huán)境的改變有著密切的聯(lián)系[5],而種植體材料表面的形貌也為細胞的粘附、生長、增殖、分化提供了一個微環(huán)境,這兩者存在的聯(lián)系值得我們更為深入的研究。2012年葉玲等[6]研究顯示組蛋白去甲基化轉(zhuǎn)移酶KDM4B和KDM6B促進人間充質(zhì)干細胞成骨分化。2015年周永勝等[7]研究表明二氧化鈦納米管通過上調(diào)啟動子區(qū)域里成骨基因Runx2和OCN的組蛋白H3K4甲基化水平以及抑制去甲基化的RBP2表達,從而促進脂肪間充質(zhì)干細胞的成骨分化。然而對于表觀遺傳乙酰轉(zhuǎn)移酶在TiO_2納米管形貌與頜骨骨髓間充質(zhì)干細胞的成骨分化過程中發(fā)揮的作用還未做相關(guān)的報道。因此,明確表觀遺傳學在納米形貌與干細胞之間的相互作用有利于我們對口腔修復(fù)材料與細胞作用機制有著更加深入的認識,并為患者的臨床治療選擇提供新的思路。研究目的通過觀察體外實驗研究表觀遺傳酶對TiO_2納米管形貌與JMMSCs成骨分化作用的影響,探究表觀遺傳學在對種植體表面形態(tài)生物學性能是否起到作用,以及如何在種植體表面微觀形態(tài)與在頜骨骨髓干細胞成骨方向上作用的內(nèi)在機制,從而為種植體表面電化學處理技術(shù)提供相應(yīng)的理論支持,為改善種植體骨結(jié)合能力提供新的研究方向,同時為表觀遺傳酶在種植體修復(fù)中骨結(jié)合作用途徑提供一定的依據(jù)。方法1、JMMSCs分離、純化、培養(yǎng)及鑒定通過體外組織塊培養(yǎng)法和有限稀釋法培養(yǎng)頜骨來源骨髓間充質(zhì)干細胞(JMMSCs);流式細胞儀檢測細胞表面特定標記;噻唑藍(methylthiazolyl tetrazolium,MTT)檢測干細胞增殖能力;成骨、成脂分化誘導(dǎo)檢測干細胞多向分化能力。2、TiO_2納米管形貌對JMMSCs成骨分化能力的影響利用電化學陽極氧化的方法制備均勻有序的TiO_2納米管(NTs),通過掃描電子顯微鏡(SEM)進行材料表面形態(tài)的觀察,以確定納米級微觀形貌的獲取;電鏡下觀察TiO_2納米管上JMMSCs形態(tài),通過Cell count 8(CCK-8)法檢測與TiO_2納米管共培養(yǎng)的JMMSCs自我增殖能力;成骨誘導(dǎo)后通過茜素紅染色比較研究TiO_2納米管對JMMSCs成骨分化的作用。3、表觀遺傳酶在TiO_2納米管形貌對JMMSCs成骨分化方向上的影響先是通過NTs與JMMSCs的體外共培養(yǎng),利用qRT-PCR的方法在不同時間點下篩選出特異性表達的表觀遺傳酶,應(yīng)用小干擾RNA對篩選出來的特定基因的進行干擾,通過兩種方式:一種是應(yīng)用小干擾RNA后的與NTs共培養(yǎng)的JMMSCs進行常規(guī)成骨分化誘導(dǎo)7d、14d,對其進行堿性磷酸酶(ALP)的染色;一種是應(yīng)用小干擾RNA后與NTs共培養(yǎng)的JMMSCs進行常規(guī)成骨分化誘導(dǎo)14d后對細胞的成骨基因的表達進行qRT-PCR的檢測,探討特定基因干擾后JMMSCs成骨分化能力的變化。4、統(tǒng)計學分析所有數(shù)據(jù)采用SPSS 17.0軟件系統(tǒng)進行統(tǒng)計學分析。兩組間比較使用兩樣本t檢驗;多組間比較采用單因素方差分析。計量資料采用均數(shù)±標準差表示,以雙側(cè)P0.05為差異具有統(tǒng)計學意義。實驗結(jié)果1、所有樣本都能成功獲得原代JMMSCs;細胞在顯微鏡下呈長梭形;流式細胞儀檢測結(jié)果表明JMMSCs中間充質(zhì)干細胞表面特定標記(CD105、CD90)均為陽性表達,而造血干細胞表面特定標記(CD34、CD14、CD45)均為陰性表達;MTT實驗結(jié)果表明JMMSCs具有自我增殖能力;成骨分化誘導(dǎo)檢測可見到鈣化結(jié)節(jié)沉積,成脂分化誘導(dǎo)檢測可見脂滴形成。結(jié)果說明,JMMSCs具有干細胞多向分化的潛能。2、利用陽極氧化法在一定濃度的電解溶液的條件下,掃描電子顯微鏡(SEM)觀察材料成功制備出TiO_2納米管形貌,通過CCK8試劑盒檢測發(fā)現(xiàn)TiO_2納米管對JMMSCs的增殖能力有一定的促進作用,成骨誘導(dǎo)28d以后茜素紅結(jié)果顯示NTs組的對JMMSCs的成骨分化能力促進作用更顯著。結(jié)果說明,TiO_2納米管形貌具有促進JMMSCs的成骨分化的作用。3、根據(jù)qRT-PCR在3d、7d檢測乙�；D(zhuǎn)移酶基因表達結(jié)果,綜合對比分析得出,在乙酰基轉(zhuǎn)移酶家族中KAT2A在TiO_2納米管與JMMSCs共培養(yǎng)過程中特異性的表達;應(yīng)用小干擾RNA后,相比于未干擾組,干擾組NTs上的JMMSCs的成骨基因ALP、COL-1、OCN表達相應(yīng)的出現(xiàn)下調(diào);同時堿性磷酸酶染色結(jié)果表示,干擾后與NTs共培養(yǎng)的JMMSCs相比不干擾組成骨分化能力下降。結(jié)果說明,KAT2A在納米管形貌促進JMMSCs成骨分化能力起著關(guān)鍵作用。結(jié)論1、體外培養(yǎng)獲取JMMSCs具有干細胞的特性,可以應(yīng)用于體外實驗。2、TiO_2納米管形貌促進JMMSCs的成骨分化。3、KAT2A在TiO_2納米管促進細胞成骨分化的過程中起著促進作用。
[Abstract]:Background the performance of biomedical materials is strongly dependent on the first biological environment, [1], on the surface of the material when the first interaction occurs. At the same time, in the process of function and differentiation of the bone tissue engineering seed cells, the nanoscale material can be refined through the bionics of the human body because of the most basic structural level of the bone itself. In the field of oral clinical treatment, implant materials are one of the most common biomaterials, which are used in dental defects and bone tissue defects to repair [4], and for the treatment of implant surfaces, research institutes and enterprises, in the field of oral clinical treatment. We have been exploring and improving. We have known that epigenetics and microenvironment changes have a close relationship with [5], and the surface morphology of the implant material also provides a microenvironment for cell adhesion, growth, proliferation and differentiation. The relationship between the two is worthy of further study in the study of [6] and other [6] studies in.2012. Methylation transferase KDM4B and KDM6B promote osteogenic differentiation of human mesenchymal stem cells for.2015 Zhou Yongsheng and other [7] studies, suggesting that TiO2 nanotubes promote osteogenic differentiation of adipose mesenchymal stem cells by up regulation of the level of histone H3K4 methylation and the inhibition of demethylation of RBP2 expression in the promoter region of the promoter region of Runx2 and OCN. However, the role of epigenetic acetyltransferase in the osteogenesis of TiO_2 nanotube and bone marrow mesenchymal stem cells in bone marrow mesenchymal stem cells has not been reported. Therefore, it is clear that the interaction between epigenetics and stem cells is beneficial to the mechanism of oral repair materials and cell action. The purpose of this study is to explore the effects of epigenetic enzymes on the morphology of TiO_2 nanotubes and the osteogenic differentiation of JMMSCs in vitro, and to explore whether epigenetics can play a role in the morphological biological ability of the implant surface and how to be in the implant. The internal mechanism of the surface micromorphology and the osteogenesis in the bone marrow stem cells of the jaw bone marrow stem cells can provide the corresponding theoretical support for the electrochemical treatment of implant surface, and provide a new direction for improving the bone binding ability of the implant. At the same time, it provides a certain basis for the epigenetic enzyme in the repair of bone binding in implant. Method 1, JMMSCs isolation, purification, culture and identification of bone marrow mesenchymal stem cells (JMMSCs) derived from bone marrow by tissue block culture and finite dilution method; flow cytometer was used to detect cell surface specific markers; methylthiazolyl tetrazolium (MTT) was used to detect the proliferation of stem cells; osteogenesis and lipid differentiation were used to detect stem cells. The multidirectional differentiation capacity.2, the effect of TiO_2 nanotube morphology on the ability of JMMSCs osteogenesis, using electrochemical anodizing method to prepare uniform and ordered TiO_2 nanotubes (NTs), and observe the surface morphology of the material by scanning electron microscope (SEM), to determine the nanoscale micromorphology, and to observe JMMS on the TiO_2 nanotube under the electron microscope. Cs morphology, Cell count 8 (CCK-8) method was used to detect the self proliferation ability of JMMSCs co cultured with TiO_2 nanotubes. After osteogenesis, the effect of TiO_2 nanotubes on JMMSCs osteogenesis differentiation was compared by alizarin red staining. The effect of epigenetic enzyme on TiO_2 nanotube morphology on the direction of JMMSCs osteogenesis was first through NTs and the bodies of JMMSCs. The epigenetic enzyme of specific expression was screened by qRT-PCR method at different time points, using small interference RNA to interfere with the selected specific genes, and through two ways: one was to induce 7d, 14d, and alkaline phosphorous by NTs co cultured JMMSCs after the application of small interference RNA. The staining of acid enzyme (ALP); one is to detect the expression of the osteogenic gene of the cell after the routine osteogenic differentiation induced by JMMSCs after the use of small interference RNA and NTs co culture to detect the qRT-PCR of the expression of the osteogenic gene of the cells, and to explore the changes of the bone differentiation ability of JMMSCs after specific gene interference, and all data are statistically analyzed by the SPSS 17 software system for statistics. Analysis. The two groups were compared with two samples t test; the multiple groups were compared with single factor variance analysis. The measurement data were expressed with mean mean difference of standard deviation. The difference of bilateral P0.05 was statistically significant. The experimental results were 1, all the samples were successful to obtain the original JMMSCs; the cells were long shuttle shape under the microscope; the flow cytometry results showed that the results showed that The specific surface markers (CD105, CD90) of JMMSCs mesenchyme stem cells were all positive, while the specific markers (CD34, CD14, CD45) of hematopoietic stem cells were negative expression. The results of MTT experiment showed that JMMSCs had the ability of self proliferation, and the osteogenic differentiation induction detection could see the deposition of calcified nodules, lipid differentiation induction detection of lipid droplets formation. The result shows that JMMSCs has the potential of multiple differentiation of stem cells,.2. Under the condition of a certain concentration of electrolysis solution, the morphology of TiO_2 nanotubes was successfully prepared by scanning electron microscope (SEM) by anodic oxidation method. The CCK8 kit detection found that the TiO_2 nanotube had a certain promoting effect on the proliferation of JMMSCs, and the osteogenic induction was induced. The results of alizarin red after 28d showed that the NTs group had a more significant effect on the osteogenic differentiation of JMMSCs. The results showed that the morphology of the TiO_2 nanotube had the effect of promoting the osteogenic differentiation of JMMSCs,.3, according to qRT-PCR in 3D, 7d detection of the gene expression of acetyltransferase gene, and a comprehensive comparison analysis showed that KAT2A in the acetyltransferase family was in TiO. The specific expression of _2 nanotube and JMMSCs co culture process; after the use of small interference RNA, compared to the undisturbed group, the osteogenic gene ALP, COL-1, OCN on the JMMSCs on the NTs of the interference group decreased, and the alkaline phosphatase staining results indicated that the interference with JMMSCs of the NTs co culture did not interfere with the decrease of the bone differentiation ability. The results show that KAT2A plays a key role in promoting the ability of JMMSCs osteogenesis in nanotube morphology. Conclusion 1, the culture of JMMSCs has the characteristics of stem cells in vitro, and can be applied to.2 in vitro. The morphology of TiO_2 nanotubes promotes the osteogenic differentiation of JMMSCs in.3, and KAT2A plays an important role in the process of promoting the osteogenic differentiation of the cells in TiO_2 nanotubes.

【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R783.6

【相似文獻】

相關(guān)期刊論文 前10條

1 卞泗善;肖毅;徐展望;;骨髓間充質(zhì)干細胞成骨分化的研究進展[J];內(nèi)蒙古中醫(yī)藥;2013年31期

2 王運濤;骨髓間充質(zhì)干細胞成骨分化調(diào)控的研究進展[J];國外醫(yī)學(生物醫(yī)學工程分冊);2003年01期

3 井燕;李良;李毅;陳孟詩;吳文超;陳槐卿;劉小菁;;力學應(yīng)變對大鼠骨髓間充質(zhì)干細胞增殖和成骨分化能力的影響[J];生物醫(yī)學工程學雜志;2006年03期

4 崔向榮;蘇偉;黃釗;覃萬安;;電磁場促進骨髓間充質(zhì)干細胞成骨分化的研究進展[J];中國醫(yī)學物理學雜志;2011年04期

5 彭飛;鄭亞東;徐西強;虞冀哲;吳華;;620nm低能量紅光對骨髓間充質(zhì)干細胞成骨分化的影響[J];激光生物學報;2011年03期

6 趙領(lǐng)洲;劉麗;吳織芬;;微米坑/納米管氧化鈦形貌對骨髓間充質(zhì)干細胞成骨分化的作用[J];醫(yī)學爭鳴;2012年04期

7 楊姣;夏雷;湯郁;陸化;費小明;;腫瘤壞死因子預(yù)處理促進骨髓間充質(zhì)干細胞成骨分化潛能[J];南京醫(yī)科大學學報(自然科學版);2014年03期

8 王晶;張洹;;-80℃一步法凍存對成人骨髓間充質(zhì)干細胞成骨分化能力的影響[J];廣東醫(yī)學;2007年03期

9 陳翠平;羅新;;17β-雌二醇對大鼠骨髓間充質(zhì)干細胞成骨分化的作用[J];中國骨質(zhì)疏松雜志;2007年05期

10 ;骨髓間充質(zhì)干細胞的成骨分化[J];中國組織工程研究與臨床康復(fù);2010年10期

相關(guān)會議論文 前10條

1 鄭亮;鄭雅元;崔燎;劉鈺瑜;;四羥基二苯乙烯-2-O-β-D-葡萄糖苷對大鼠骨髓間充質(zhì)干細胞成骨分化的作用及機制研究[A];中華醫(yī)學會第七次全國骨質(zhì)疏松和骨礦鹽疾病學術(shù)會議論文匯編[C];2013年

2 吳江;陳槐卿;K-LP.SUNG;;鈦顆粒負荷影響骨髓間充質(zhì)干細胞成骨分化能力的機理[A];全國首屆青年復(fù)合材料學術(shù)交流會論文集[C];2007年

3 吳江;陳槐卿;李良;尹光福;K-L P．SUNG;;鈦顆粒負荷影響骨髓間充質(zhì)干細胞成骨分化能力的機理[A];中國復(fù)合材料學術(shù)研討會論文集[C];2005年

4 陳海嘯;洪盾;萬曉晨;李繼承;;腸毒素C聯(lián)合維生素C對骨髓間充質(zhì)干細胞成骨分化的影響研究[A];2008年浙江省骨科學學術(shù)年會論文匯編[C];2008年

5 袁風紅;鄒耀紅;高愷言;俞可佳;;地塞米松對體外人骨髓基質(zhì)細胞增殖及成骨分化的影響[A];全國自身免疫性疾病專題研討會暨第十一次全國風濕病學學術(shù)年會論文匯編[C];2006年

6 宋忠臣;束蓉;董家辰;李松;;低氧對牙周膜成纖維細胞增殖和成骨分化的影響[A];第十次全國牙周病學學術(shù)會議論文摘要匯編[C];2014年

7 董家辰;束蓉;宋忠臣;;炎癥微環(huán)境對人牙周膜成纖維細胞增殖與成骨分化的影響[A];第十次全國牙周病學學術(shù)會議論文摘要匯編[C];2014年

8 孫楠;楊力;張振;張樺;陳宏;蔡德鴻;;晚期氧化蛋白產(chǎn)物對大鼠骨髓間充質(zhì)干細胞增殖及向成骨分化的影響[A];中華醫(yī)學會第十二次全國內(nèi)分泌學學術(shù)會議論文匯編[C];2013年

9 馬雪;孟靜茹;賈敏;王寧;胡靜;周穎;羅曉星;;胰高血糖素樣肽-1類似物對大鼠骨髓間充質(zhì)干細胞增殖與成骨分化的影響[A];第十一屆全國青年藥學工作者最新科研成果交流會論文集[C];2012年

10 林和敏;;成骨分化的人臍血間充質(zhì)干細胞免疫原性研究[A];2013年全國激光醫(yī)學學術(shù)聯(lián)合會議暨2013年浙江省醫(yī)學會整形美容學術(shù)年會論文匯編[C];2013年

相關(guān)博士學位論文 前10條

1 龔逸明;microRNAs調(diào)控Satb2介導(dǎo)的骨髓基質(zhì)干細胞成骨分化的作用研究[D];復(fù)旦大學;2014年

2 李松濤;EphB信號在軸向仿生壓應(yīng)力調(diào)控MSC成骨分化中的作用和機制研究[D];第三軍醫(yī)大學;2015年

3 方文;純鈦表面WNT信號通路調(diào)控BMSCs成骨分化的機制研究[D];浙江大學;2014年

4 劉世宇;移植外源性MSCs持久恢復(fù)宿主MSCs功能的機制研究[D];第四軍醫(yī)大學;2015年

5 張莉;Fgfr2~(S252W/+)小鼠骨量、骨結(jié)構(gòu)特性及BMSCs成骨分化調(diào)控機制的研究[D];第三軍醫(yī)大學;2015年

6 劉旭杰;材料性質(zhì)調(diào)控干細胞成骨分化及殼聚糖基骨修復(fù)材料[D];清華大學;2015年

7 文采;多孔礦化水凝膠材料誘導(dǎo)人胚胎干細胞成骨分化的研究[D];東南大學;2015年

8 吳毛;胸腰段椎體骨折形態(tài)與椎管狹窄相關(guān)性研究及Sclerostin對成骨分化的影響[D];蘇州大學;2016年

9 黃江;激活HIF-1α對骨折愈合的影響及誘導(dǎo)miR-429對MC3T3-E1細胞成骨分化的作用機制[D];首都醫(yī)科大學;2016年

10 侯秋科;龜板有效成分促間充質(zhì)干細胞成骨分化的miRNA-VDR網(wǎng)絡(luò)機制[D];廣州中醫(yī)藥大學;2016年

相關(guān)碩士學位論文 前10條

1 錢鈞;KAT2A對二氧化鈦納米管形貌促進頜骨骨髓間充質(zhì)干細胞成骨分化的作用研究[D];廣州醫(yī)科大學;2017年

2 吳小瑩;腦源性神經(jīng)營養(yǎng)因子對人脂肪干細胞增殖與成骨分化的作用[D];北京協(xié)和醫(yī)學院;2015年

3 秦子順;淫羊藿苷對人牙周膜干細胞的增殖和成骨分化的影響[D];蘭州大學;2015年

4 劉可;miR-106bE靶向調(diào)控BMP2參與間充質(zhì)干細胞成骨分化與體內(nèi)骨形成[D];蘇州大學;2015年

5 付雪杰;MSCs與去分化MSCs在成骨分化過程中免疫原性上調(diào)的研究[D];蘇州大學;2015年

6 崔陽陽;蛋白激酶C在小鼠骨髓間充質(zhì)干細胞成骨分化中的作用研究[D];新鄉(xiāng)醫(yī)學院;2015年

7 嚴一杰;體外誘導(dǎo)人腎間質(zhì)成纖維細胞成骨分化的實驗研究[D];廣西醫(yī)科大學;2015年

8 高羽萱;直流微電場對種植體周骨髓間充質(zhì)干細胞遷移和成骨影響的研究[D];中國人民解放軍醫(yī)學院;2015年

9 郭中豪;甲狀旁腺激素對大鼠骨髓間充質(zhì)干細胞成骨分化的影響[D];山西醫(yī)科大學;2015年

10 李慧垠;高脂環(huán)境下大鼠BMSCs成骨分化過程中Wnt通路相關(guān)因子的表達變化[D];山東大學;2015年

，

本文編號：1847594