發(fā)布時(shí)間：2018-06-01 18:35

  本文選題：牽張成骨 + 胚胎成骨　； 參考：《廣西醫(yī)科大學(xué)》2017年博士論文

【摘要】：研究背景牽張成骨(Distraction osteogenesis,DO)是指在低能量創(chuàng)傷致使的人為骨折情況下,對(duì)骨折端之間通過(guò)特制的鈦金屬牽張器來(lái)施加一個(gè)緩慢的牽引力,使逐漸牽張開的間隙中產(chǎn)生新骨的過(guò)程,通常被用來(lái)為骨發(fā)育不全、骨缺損制造新骨修復(fù),在臨床上已經(jīng)被證明是極其有效的治療手段。牽張成骨的成骨效率高、效果較穩(wěn)定,每日可在牽張間隙成骨達(dá)到1mm,速率達(dá)到嬰幼兒生長(zhǎng)發(fā)育速度4-6倍,并且暫時(shí)未發(fā)現(xiàn)有成骨量的限制。牽張成骨的機(jī)制非常復(fù)雜,其成骨的高效率,超越了過(guò)去人們所有對(duì)骨質(zhì)生成的認(rèn)識(shí)。牽張成骨的分子學(xué)機(jī)制,是否和骨折愈合(healing)相似,還是另一種形式的骨再生(regeneration),亦或是兩者共存的發(fā)展形式,至今仍然未完全清楚。研究目的牽張成骨的效率極高,達(dá)到嬰幼兒生長(zhǎng)發(fā)育速度的4至6倍,因此我們提出假想,牽張成骨中牽引機(jī)械力或者微創(chuàng)傷等因素,可能激活了機(jī)體僅在胚胎發(fā)育期或新生發(fā)育期才活躍的某種因子或信號(hào)通路,從而主導(dǎo)了一種與骨修復(fù)甚至骨再生不同的骨生成過(guò)程,這可能是一種近似于返祖的生物學(xué)過(guò)程。為探明其中的機(jī)制,本研究構(gòu)建了犬下頜骨牽張成骨模型及妊娠犬孕胚胎犬模型,利用第二代高通量測(cè)序技術(shù)對(duì)牽張后即刻取材和固定2周取材的下頜骨牽張區(qū)骨組織、胚胎犬下頜骨組織、骨折犬骨折區(qū)組織、新生犬、2周/4周幼犬和正常成年犬的下頜骨骨組織進(jìn)行mRNA及miRNA的全基因組表達(dá)譜測(cè)序。通過(guò)對(duì)牽張組和其他組下頜骨骨組織的mRNA和miRNA進(jìn)行全基因組表達(dá)差異與關(guān)聯(lián)性分析,更全面地了解牽張成骨與胚胎發(fā)育、骨折愈合成血管成骨的異同點(diǎn),并探索牽張成骨可能的啟動(dòng)因素、新骨形成的獨(dú)特性、血管發(fā)生等相關(guān)聯(lián)的mRNA和miRNA表達(dá)情況及其參與信號(hào)通路的情況,篩選出具有關(guān)鍵作用的基因,為后續(xù)的驗(yàn)證研究提供關(guān)鍵基因表達(dá)譜。研究方法1、廣西醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心提供實(shí)驗(yàn)中華田園犬共33只。分成11組:(1)牽張后即刻取材組(DO-1),3只;(2)牽張后固定2周組(DO-2),3只;(3)新生犬組(P-0),3只;(4)2周幼犬組(P-2),3只;(5)4周幼犬組(P-4),3只;(6)孕期30天胚胎組(E-1),3只;(7)孕期35天胚胎組(E-2),3只;(8)孕期50天胚胎組(E-2),3只;(9)正常對(duì)照組(AC),3只;(10)骨折1組(BF-1),3只;(11)骨折2組(BF-2),3只。牽張各組處置:牽張即刻取材組和牽張固定2周組的實(shí)驗(yàn)動(dòng)物均接受單側(cè)下頜骨單線牽張成骨術(shù),并于牽張結(jié)束即刻和牽張結(jié)束固定2周后處死,取材牽張區(qū)骨組織,通過(guò)標(biāo)本大體觀察、處死前CBCT影像學(xué)觀察和HE染色組織學(xué)觀察以確定牽張效果。胚胎組處置:經(jīng)過(guò)術(shù)前B超確認(rèn)孕期,對(duì)孕期30天、35天、50天的妊娠犬執(zhí)行剖腹引產(chǎn)手術(shù),取材各組活體胚胎,對(duì)標(biāo)本進(jìn)行大體觀察和HE染色,剖腹后妊娠犬回籠飼養(yǎng)。骨折組處置:對(duì)實(shí)驗(yàn)犬執(zhí)行骨折手術(shù),通過(guò)鈦板保持骨折間隙,骨折兩組對(duì)應(yīng)牽張組以作對(duì)照,骨折1組指骨折固定14天后取材(對(duì)應(yīng)DO-1組5天的間隙期、7天的牽張期)、骨折2組指骨折固定28天后取材(對(duì)應(yīng)DO-2組5天的間隙期、7天的牽張期以及2周的固定期)。另取新生半小時(shí)內(nèi)幼犬3只,出生2周、4周幼犬各3只,以及非手術(shù)健康成年犬3只,分別納入新生犬組(P-0)、2周幼犬組(P-2)、4周幼犬組(P-4)和正常對(duì)照組(AC),予以處死取單側(cè)下頜骨骨組織為標(biāo)本。以上動(dòng)物除妊娠犬外均雌雄不限,成年犬年齡1.5-2.0歲,體重12.5kg±2.5kg(平均13.5kg)。2、提取各組骨組織標(biāo)本的總RNA,經(jīng)質(zhì)檢明確其濃度及純度合格后,同時(shí)建立mRNA及miRNA測(cè)序c DNA文庫(kù),文庫(kù)回收純化后,由Illumina公司Hiseq4000測(cè)序平臺(tái)進(jìn)行測(cè)序。3、通過(guò)高通量測(cè)序獲得mRNA、miRNA表達(dá)譜,將原始數(shù)據(jù)行mRNA、miRNA基因表達(dá)量標(biāo)準(zhǔn)化統(tǒng)計(jì)后,將11組樣本分成55個(gè)兩兩對(duì)比組(1-vs-1)及36組的三實(shí)驗(yàn)組間比對(duì)(1-vs-11),對(duì)各對(duì)比組行mRNA、miRNA差異基因篩選,以及差異mRNA的GO富集分析和KEGG富集分析,并在此差異基因庫(kù)里二次篩選能夠提示牽張組、胚胎組、骨折組、正常對(duì)照組之間生物學(xué)層面異同點(diǎn)的興趣基因,并作差異性與關(guān)聯(lián)性分析。研究結(jié)果1、牽張即刻組和牽張固定2周組實(shí)驗(yàn)犬均手術(shù)過(guò)程順利,術(shù)后無(wú)不良反應(yīng)。牽張器固定牢靠,無(wú)松脫、斷裂及變形。觀察所有實(shí)驗(yàn)犬尖牙咬合關(guān)系較牽張前呈下頜左偏突頜改變,下頜骨牽張間隙有類骨樣新生物質(zhì),說(shuō)明右側(cè)頜骨成功通過(guò)牽張延長(zhǎng)。胚胎各組通過(guò)剖腹引產(chǎn)術(shù)取得活體犬胚胎,手術(shù)過(guò)程順利,取得胎體完整,形態(tài)符合各孕期應(yīng)有的發(fā)育形態(tài),妊娠犬經(jīng)引產(chǎn)后無(wú)不良反應(yīng),持續(xù)生存。骨折各組手術(shù)過(guò)程順利,術(shù)后尖牙咬合關(guān)系能顯示下頜左偏突頜改變,術(shù)區(qū)未出現(xiàn)嚴(yán)重感染,鈦板及鈦釘無(wú)松脫、斷裂及變形,骨折兩端見纖維樣組織增生,無(wú)類骨樣物質(zhì)可被辨識(shí)。新生犬幼犬組和正常對(duì)照犬組取材過(guò)程順利,無(wú)特殊。2、從11組樣本中均提取出足量的總RNA,并分別成功構(gòu)建了用于mRNA及miRNA測(cè)序所需的c DNA文庫(kù)。成功完成了犬下頜骨牽張區(qū)及正常下頜骨骨組織的高通量測(cè)序。3、本次測(cè)序共獲得22523個(gè)表達(dá)mRNA基因,綜合55個(gè)兩兩對(duì)比組和36組三組間比較(1-vs-11),通過(guò)對(duì)差異表達(dá)mRNA進(jìn)行分析后,我們發(fā)現(xiàn)了在牽張即刻組和胚胎犬組都共同表達(dá)上調(diào)、而正常組相對(duì)下調(diào)的mRNA共50個(gè),以CXCL12、MINOS1、OGN、POSTN、PTN、SFRP2、MFAP5、IGFBP5、HSPB6等最具特征性,其中基因MINOS1、MFAP5、HSPB6均在正常成年犬中幾乎無(wú)表達(dá)。以上基因功能囊括EMT調(diào)控、血管發(fā)生調(diào)控、間充質(zhì)干細(xì)胞及成骨細(xì)胞動(dòng)員等。牽張組與骨折組之間具有聯(lián)系的基因有1921個(gè),以ATF4、CD99L2、IGFBP5、RPL15等特征性最強(qiáng),這些基因在牽張組和骨折組中具有不同的特征性表達(dá)趨勢(shì),主要為EMT調(diào)控,且相對(duì)于正常組中均顯著表達(dá)(上調(diào)或下調(diào)),代表了牽張成骨與骨折愈合的不同特點(diǎn)。4、本次測(cè)序還獲得354個(gè)表達(dá)miRNA,根據(jù)以上對(duì)比組,牽張各組與胚胎各組共同高表達(dá)、并在正常組相對(duì)下調(diào)表達(dá)的基因共46個(gè),尤以mi R-136、mi R-299、mi R-369、mi R-376a、mi R-380、mi R-382、mi R-410、mi R-432、mi R-503、mi R-20a、mi R-23a、mi R-126、mi R-370、mi R-433最具特征性,其中基因cfa-mi R-370、cfa-mi R-433均在正常成年犬中無(wú)表達(dá)。以上基因功能表達(dá)囊括血管發(fā)生與血管生成調(diào)控;胚胎干細(xì)胞、內(nèi)皮祖細(xì)胞與間充質(zhì)干細(xì)胞動(dòng)員;血管內(nèi)皮細(xì)胞遷移;成骨細(xì)胞與破骨細(xì)胞分化調(diào)控;成肌調(diào)控等。此外,我們篩選出牽張組與骨折組之間具有聯(lián)系的基因,并對(duì)AC組亦同等量級(jí)表達(dá)的基因進(jìn)行排除。獲得了候選基因mi R-203、mi R-204、mi R-205、mi R-338、mi R-9和mi R-34a,這些基因在牽張組和骨折組中具有特征性的表達(dá)趨勢(shì),并相對(duì)于AC組有顯著表達(dá)(上調(diào)或下調(diào)),主要為EMT調(diào)控,提示了牽張成骨與骨折愈合之間基因異同性表達(dá)的時(shí)空特點(diǎn),揭示牽張成骨與骨折愈合各自的成血管化及成骨化特點(diǎn)。結(jié)論牽張成骨中,受初期微創(chuàng)傷造成的缺血性環(huán)境影響、炎癥反應(yīng)刺激、機(jī)械牽張力刺激或固定期靜態(tài)牽張力刺激所啟動(dòng),出現(xiàn)以下生物學(xué)過(guò)程:大量基因自胚胎發(fā)育期后二次顯著表達(dá)并調(diào)控以血管發(fā)生(vasculogenesis)為主的成血管化,同時(shí)牽張成骨的早期機(jī)體的血管生成(Angiogenesis)機(jī)制受抑制,固定期后期有多個(gè)基因參與了對(duì)過(guò)載發(fā)育的血管網(wǎng)的限控;免疫性單核細(xì)胞受誘導(dǎo)向血管內(nèi)皮細(xì)胞分化;上皮-間充質(zhì)細(xì)胞轉(zhuǎn)化(EMT)被顯著抑制,大量的間充質(zhì)細(xì)胞轉(zhuǎn)化為上皮及內(nèi)皮細(xì)胞以形成新的血管網(wǎng);熱休克蛋白受啟動(dòng),維持細(xì)胞骨架穩(wěn)定來(lái)強(qiáng)化成血管化及成骨化過(guò)程;多個(gè)基因調(diào)控的大量骨髓間充質(zhì)干細(xì)胞快速增殖向成骨細(xì)胞分化、既有的成骨細(xì)胞募集、牽張區(qū)肌肉組織分泌特異蛋白促進(jìn)骨代謝,維持著新骨快速生成。相較之下,骨折愈合過(guò)程中表達(dá)基因與牽張成骨差異較明顯,部分基因啟動(dòng)較牽張成骨緩慢,EMT進(jìn)程相較于牽張成骨顯著活躍,愈合過(guò)程缺乏快速成血管化的特征。
[Abstract]:Background distraction osteogenesis (Distraction osteogenesis, DO) refers to the process of applying a slow traction force between the end of the fracture by a special titanium metal distraction in the human fracture caused by low energy trauma and the process of producing new bone in the gradually stretched gap, which is usually used for bone development and new bone defects. Bone repair has been proved to be an extremely effective treatment in clinic. The osteogenesis of the distraction osteogenesis is high, the effect is more stable, the osteogenesis can reach 1mm daily in the distraction space, the rate reaches 4-6 times the rate of growth and development of the infant, and the bone formation is not limited. The mechanism of distraction osteogenesis is very complex, and its osteogenesis is highly efficient. It is beyond all understanding of bone formation in the past. The mechanism of distraction osteogenesis, whether it is similar to fracture healing (healing), or another form of bone regeneration (regeneration), or the coexistence of the two forms, is still not completely clear. The rate of birth is 4 to 6 times, so we suggest that factors such as traction mechanical force or minimally invasive injury in the distraction osteogenesis may activate a certain factor or signal pathway that only activate the body only during the embryonic development or the new development period, thus leading a process of bone formation that is different from bone repair or even bone rebirth. This may be an approximation. To explore the biological process of ancestral, this study constructed a canine mandible distraction osteogenesis model and a gestation canine model. The second generation high-throughput sequencing technique was used to extract and fix the bone tissue of the mandible, the mandible tissue of the dog, the fracture area of the fractured dog, and the fracture area of the fractured dog. The whole genome expression of mRNA and miRNA was sequenced in the mandible bone tissue of young and normal adult dogs at 2 weeks /4 weeks. The whole genome expression difference and correlation analysis were carried out on the mRNA and miRNA of the distraction group and other group of mandible bone tissue. At the same time, and explore the possible initiation factors of distraction osteogenesis, the uniqueness of the formation of the bone, the expression of mRNA and miRNA associated with angiogenesis and its involvement in the signal pathway, screening the key genes to provide key gene expression profiles for subsequent validation studies. Method 1, Guangxi Medical University animal experiment Center A total of 33 Chinese pastoral dogs were provided, divided into 11 groups: (1) immediately after distraction (DO-1), 3; (2) 2 weeks after traction (DO-2), 3; (3) new dog group (P-0), 3 (4), 2 week puppy group (5), P-4, E-2, E-2 ) 3; (9) the normal control group (AC), 3; (10) fracture 1 group (BF-1), 3 only; (11) fracture 2 groups (BF-2), 3. The distraction and 2 weeks group of experimental animals were treated with unilateral mandibular unilateral distraction osteogenesis, and were executed immediately after the end of the distraction and the end of distraction was fixed for 2 weeks, and took the distraction bone tissue. Gross observation, CBCT imaging observation and histological observation of HE staining before death to determine the effect of distraction. The treatment of embryo group: by preoperative B-ultrasound confirmation of pregnancy, 30 days of pregnancy, 35 days, 50 days of pregnant dogs were performed by caesarean section, all groups of living embryos were obtained, the specimens were observed by gross and HE staining, and after caesarean section, the dog was fed back to the cage. Fracture group disposal: the fracture operation was performed in the experimental dogs. The fracture space was maintained through the titanium plate. The two groups of fracture were compared with the distraction group. The 1 groups of fractures were taken for 14 days (corresponding to the 5 days in the DO-1 group and 7 days of distraction), and the fracture of the 2 groups was fixed for 28 days after the fracture (corresponding to the gap of 5 days in the group of 5 days, and the time for the distraction of 7 days). In addition, 3 puppies in the newborn half an hour, 2 weeks of birth, 3 young dogs in 4 weeks, and 3 non-surgical healthy adult dogs were included in the newborn dog group (P-0), the 2 week young dog group (P-2), the 4 week puppy group (P-4) and the normal control group (AC). The adult dog age 1.5-2.0 years, weight 12.5kg + 2.5kg (mean 13.5kg).2, extracted the total RNA of all bone tissue specimens. After quality inspection, the concentration and purity were clear, mRNA and miRNA sequencing C DNA library was established. After the library was recovered and purified, the Illumina company Hiseq4000 sequencing platform was sequenced and obtained through high throughput sequencing. A expression spectrum, after standardized statistics of mRNA and miRNA gene expression of original data lines, 11 groups of samples were divided into 55 22 contrast groups (1-vs-1) and 36 groups of three experimental groups (1-vs-11), mRNA, miRNA differential gene screening, and GO rich set analysis and KEGG enrichment analysis of different mRNA, and two sieves in the differential gene reservoir. Select the interest genes that could indicate the differences and similarities of the biological level between the distraction group, the embryo group, the fracture group and the normal control group, and make a difference and correlation analysis. 1, the results of the study were smooth and no adverse reaction after the operation, and the distractor fixation, no loosening, fracture and deformation were observed. The relationship between the occlusion of the canine teeth was better than that before the distraction. The mandibular distraction space had a new kind of new bone like biomass, which indicated that the right jaw was successfully extended by distraction. The embryo of the living canine was obtained by cesarean section. The operation process was smooth and the body was complete. There was no adverse reaction and sustained survival after induction of labor in pregnant dogs. The operation process of each fracture was smooth. The relationship of the canine occlusion can show the change of the mandibular left partial maxillary jaw, no severe infection in the operation area, the titanium plate and titanium nail without loosening, fracture and deformation, the fibrous tissue of the fracture at both ends of the fracture, and the non osteoid substance can be identified. The young dog group and the newborn dog group can be identified. The normal control dog group had successfully obtained the total RNA from 11 groups of samples, and successfully constructed the C DNA library for mRNA and miRNA sequencing, and successfully completed the high throughput sequencing.3 of the canine mandible distraction area and the normal mandible bone tissue. This sequence obtained a total of 22523 mRNA genes. 55 22 contrast groups and 36 groups of three groups (1-vs-11), through the analysis of the differential expression of mRNA, we found that both the immediate group and the embryo group were up to be up up, and the normal group was relatively down-regulated in a total of 50 mRNA, with CXCL12, MINOS1, OGN, POSTN, PTN, SFRP2, MFAP5, IGFBP5, HSPB6 and so on. AP5, HSPB6 were almost no expression in normal adult dogs. The function of the above gene included EMT regulation, angiogenesis, mesenchymal stem cells and osteoblast mobilization. There were 1921 genes associated with fracture groups, including ATF4, CD99L2, IGFBP5, RPL15 and other genes. These genes were not in the distraction and fracture groups. The same characteristic expression trend was mainly regulated by EMT, and was significantly expressed (up or down) compared to the normal group (up or down), representing the different characteristics of distraction osteogenesis and fracture healing,.4, and 354 expressions of miRNA were obtained in this sequence. According to the above comparison group, the groups of the distraction and embryo fetal groups were highly expressed, and the expression was relatively downregulated in the normal group. There are 46 genes, especially mi R-136, MI R-299, MI R-369, MI R-376a, MI R-380, MI R-382. Embryonic stem cells, endothelial progenitor cells and mesenchymal stem cells mobilize, vascular endothelial cell migration, osteoblast and osteoclast differentiation regulation, and myoblast regulation. In addition, we screened the genes associated with the fracture group between the distraction group and the AC group, and obtained the candidate gene mi R-203, MI R -204, MI R-205, MI R-338, MI R-9 and MI R-34a, these genes have the characteristic expression trend in the stretch group and the fracture group, and have a significant expression relative to the AC group (up or down), mainly for EMT regulation, suggesting the spatio-temporal characteristics of the gene difference expression between the distraction osteogenesis and the fracture healing, and reveal the distraction osteogenesis and the fracture healing. Conclusion in the distraction osteogenesis, the effects of ischemic environment, inflammatory response, mechanical tension stimulation, or static tension stimulation at fixed stage were initiated in the distraction osteogenesis, and the following biological processes were observed: a large number of genes were expressed two times after the embryonic development and regulated by blood vessels (Vasculo Genesis) mainly vascularized, at the same time, the angiogenesis (Angiogenesis) mechanism of the early body of the distraction osteogenesis was inhibited. There were several genes involved in the restriction of the overloaded vascular network at the later stage of the fixation; the immune mononuclear cells were induced to differentiate into vascular endothelial cells, and the epithelial mesenchymal cell transformation (EMT) was significantly inhibited. Mesenchymal cells are transformed into epithelial and endothelial cells to form a new vascular network, and heat shock proteins are activated to maintain cytoskeleton stability to strengthen vascularization and osteogenesis, and a large number of genes regulate a large number of bone marrow mesenchymal stem cells to proliferate rapidly to osteoblasts, some osteoblasts are raised, and the muscle tissue in the stretch zone is traced. The secreting specific proteins promote bone metabolism and maintain the rapid formation of new bone. In comparison, the difference of expression gene and distraction osteogenesis is obvious during the process of fracture healing. Some genes start more slowly than distraction osteogenesis, EMT process is significantly active compared with distraction osteogenesis, and the healing process lacks the characteristics of rapid vascularization.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R782
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本文編號(hào)：1965216