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研究生:黃俞智
研究生(外文):Yu-Chih Huang
論文名稱:利用牙本質做為骨癒合填補材料之早期基因傳導機轉分析
論文名稱(外文):Transcription analysis for early healing bone treated with dentin powders.
指導教授:黃豪銘
口試委員:蕭裕源林哲堂
口試日期:2013-06-24
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生醫材料暨組織工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:112
中文關鍵詞:牙本質骨粉、基因晶片同源性基因方法生物反應途徑資料庫
外文關鍵詞:Dentin graftMicroarrayOrthologues approachIPA
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研究指出,以牙本質做為骨填補材極具有潛力。然而,使用分子生物學的角度來探討牙本質做為骨填補材的生物效應目前尚未明瞭。故本研究透過基因晶片技術探討牙本質做為骨填補材其早期骨癒合之基因表現情形,並探討可能的分子機轉與調控路徑。牙齒經過清洗、燒結、研磨與滅菌之後,製成直徑250-500μm之顆粒狀骨粉並進行XRD、FTIR、EDX等材料性質檢驗。本次實驗將骨粉填入12隻紐西蘭白兔之前額葉顱骨進行三、五、七天之實驗。Total RNA與基因晶片用來萃取與獲得完整之基因表現。基因整理與同源性基因轉換乃使用GeneSpring軟體與BetterBunny線上資料庫。基因表現分析使用IPA生物反應途徑軟體並使用Real-time PCR進行驗證。材料性質分析方面,本研究所製備之牙本質骨粉,其粗糙的表面特性適合細胞攀附與生長,表面組成也接近天然氫氧基磷灰石。基因表現方面,免疫與發炎基因在早期大量表現,IL1A與IL1B分別表現7.793與3.462倍,TNF與IL8分別有3.160與9.302倍表現。同時,抑制免疫與發炎基因IL1RN大量表現4.496倍,並活化CCL2基因之表現達2.317倍。七天時,骨母細胞分化相關基因BMP2、RUNX2分別表現1.717與1.831倍,骨母細胞生成基因COL1A1也有2.236倍之表現。本研究的結果證明,劇烈的免疫與發炎反應使得骨缺損處在第五天時活化了IL1RN基因,IL1RN基因不僅發揮了抑制發炎反應基因的角色,同時也活化了CCL2基因的表現。接著,CCL2基因會活化ERK-MAPK訊息傳導機轉,並藉由此訊息傳導路徑誘發RUNX2、BMP2與後續調控骨成長的基因表現。
In clinical jawbone formation, infilling proper graft material for greater strength before implant surgery is considered a golden standard. Of all graft material in world, human particulate dentin was reported to be an alternative material for bone grafting. There are many studies using histological approaches to evaluate the consequences between material and wound area at endpoint. However, the interactional biological effects of dentin powder on bone healing at molecular level are still unclear. The aim of this study was to assess the gene expression of bone tissues grafted with human particulate dentin at the early healing stage. Crown-removed tooth were cleaned, sintered and grounded to powder of 250-500μm particle size. X-ray diffraction was performed to confirm the crystal structure of dentin material with standard hydroxyapatite sample. The surface morphology and chemical characterization were examined by scanning electron microscopy and energy-dispersive x-ray spectroscopy. In this study, defects were created on the heads of twelve New Zeeland white rabbits. The x-ray sterilized dentin powders were used as grafting materials. Tissues and blood samples were taken for gene expression at 3, 5 and 7 days post-implantation and were then analyzed via microarray. Total RNA was isolated and tested using a 2100 Bioanalyzer. Overall changes in gene expression were evaluated using Agilent Rabbit Oligo 4×44K Microarray and analyzed with GeneSpring GX software. Only genes with a signal-to-noise ratio of above 2 in the experiments were included in the statistical analysis. Additional filtering (minimum 2-fold change) was applied to extract the most differentially expressed genes based on the study groups (Day 3: Control vs. Sample, Day 5: Control vs. Sample, Day 7: Control vs. Sample). The orthologues approach and functional annotation of Gene Ontology analysis were performed by Better Bunny coupled with DAVID online database. XRD showed that treated dentin material, in crystal structure, have great similarity with standard hydroxyapatite. On chemical characterization, the Ca/P ratio evaluated by SEM/EDX was 1.80, close to natural hydroxyapatite. Microarray analysis came out that human particulate dentin implantation led to a two-fold change in 1672 genes on day 3, 1652 genes on day 5, and 575 genes on day 7, compared to corresponding control. The immune and inflammatory genes were highly ecpressed. Our results showed that IL1A and IL1B expressed 7.793 and 3.462 at Day 5 respectively. And TNF and IL8 genes also highly expressed 3.160 and 9.302 compare to control. However, the immune and inflammatory response arose the antagonist gene IL1RN. IL1RN expressed 4.496 fold change compare to control. Moreover, the IL1RN gene activated the gene CCL2. CCL2 expressed 2.317 fold change than control group. The osteoblastogenesis related gene BMP2, RUNX2, COL1A1 expressed 1.717, 1.831 and 2.236 respectively. Our results established that the IL1RN inhibit the responses of immune and inflammatory and besides, enhance the expressed of CCL2 gene. Furthermore, CCL2 activate the osteoblastogenesis through ERK-MAPK pathway then establish the bone argumentation.
第一章、 緒論………………………………………………………1
第二章、 文獻回顧…………………………………………………3
第一節、 骨粉在臨床牙科的角色…………………………………3
第二節、 骨粉的特性……………………………………………4
第三節、 骨粉的分類………………………………………………6
第四節、 牙本質做為骨填補材料的歷史…………………………8
第五節、 基因晶片的檢驗技術……………………………………9
第六節、 兔子個體基因之同源性基因交叉比對法……………10
第七節、 高證據力生物反應資料庫之角色與應用……………11
第三章、 材料與方法……………………………………………13
第一節、 實驗設計與流程 ……………………………………13
第二節、 骨粉的製備……………………………………………13
第三節、 牙粉性質的檢驗………………………………………14
第四節、 骨粉細胞毒性測試……………………………………16
第五節、 動物實驗………………………………………………17
第六節、 Total RNA萃取與基因晶片實驗………………………18
第七節、 基因篩選及分析…………………………………………19
第八節、 Ingenuity Pathways Analysis 分析………………20
第九節、 即時聚合連鎖反應分析(Real-time PCR)…………22
第四章、 實驗結果………………………………………………26
第一節、 牙本質骨粉顏色分析…………………………………26
第二節、 XRD分析結果…………………………26
第三節、 FT-IR分析結果…………………………………………………………27
第四節、 SEM/EDX分析結果………………………………………………27
第五節、 細胞毒性測試結果………………………28
第六節、 RNA完整度測試結果…………………………………………29
第七節、 基因晶片的分析結果……………………………………………………30
第八節、 Gene Ontology功能性分類項分析……………………………31
第九節、 Ingenuity Pathway Analysis 資料庫分析…………………………34
第十節、 基因晶片之相關基因表現量表……………………………………36
第十一節、即時聚合連鎖反應分析結果………………….38
第五章、 討論………………………………………………………41
第六章、 結論………………………………………………………54
第七章、 研究文獻…………………………………………………55
附錄……………………………………………………………………64
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