犬下颌骨牵张成骨与胚胎成骨RNA表达谱关联性分析

发布时间：2018-06-01 18:35

  本文选题：牵张成骨 + 胚胎成骨　； 参考：《广西医科大学》2017年博士论文

【摘要】：研究背景牵张成骨(Distraction osteogenesis,DO)是指在低能量创伤致使的人为骨折情况下,对骨折端之间通过特制的钛金属牵张器来施加一个缓慢的牵引力,使逐渐牵张开的间隙中产生新骨的过程,通常被用来为骨发育不全、骨缺损制造新骨修复,在临床上已经被证明是极其有效的治疗手段。牵张成骨的成骨效率高、效果较稳定,每日可在牵张间隙成骨达到1mm,速率达到婴幼儿生长发育速度4-6倍,并且暂时未发现有成骨量的限制。牵张成骨的机制非常复杂,其成骨的高效率,超越了过去人们所有对骨质生成的认识。牵张成骨的分子学机制,是否和骨折愈合(healing)相似,还是另一种形式的骨再生(regeneration),亦或是两者共存的发展形式,至今仍然未完全清楚。研究目的牵张成骨的效率极高,达到婴幼儿生长发育速度的4至6倍,因此我们提出假想,牵张成骨中牵引机械力或者微创伤等因素,可能激活了机体仅在胚胎发育期或新生发育期才活跃的某种因子或信号通路,从而主导了一种与骨修复甚至骨再生不同的骨生成过程,这可能是一种近似于返祖的生物学过程。为探明其中的机制,本研究构建了犬下颌骨牵张成骨模型及妊娠犬孕胚胎犬模型,利用第二代高通量测序技术对牵张后即刻取材和固定2周取材的下颌骨牵张区骨组织、胚胎犬下颌骨组织、骨折犬骨折区组织、新生犬、2周/4周幼犬和正常成年犬的下颌骨骨组织进行mRNA及miRNA的全基因组表达谱测序。通过对牵张组和其他组下颌骨骨组织的mRNA和miRNA进行全基因组表达差异与关联性分析,更全面地了解牵张成骨与胚胎发育、骨折愈合成血管成骨的异同点,并探索牵张成骨可能的启动因素、新骨形成的独特性、血管发生等相关联的mRNA和miRNA表达情况及其参与信号通路的情况,筛选出具有关键作用的基因,为后续的验证研究提供关键基因表达谱。研究方法1、广西医科大学动物实验中心提供实验中华田园犬共33只。分成11组:(1)牵张后即刻取材组(DO-1),3只;(2)牵张后固定2周组(DO-2),3只;(3)新生犬组(P-0),3只;(4)2周幼犬组(P-2),3只;(5)4周幼犬组(P-4),3只;(6)孕期30天胚胎组(E-1),3只;(7)孕期35天胚胎组(E-2),3只;(8)孕期50天胚胎组(E-2),3只;(9)正常对照组(AC),3只;(10)骨折1组(BF-1),3只;(11)骨折2组(BF-2),3只。牵张各组处置:牵张即刻取材组和牵张固定2周组的实验动物均接受单侧下颌骨单线牵张成骨术,并于牵张结束即刻和牵张结束固定2周后处死,取材牵张区骨组织,通过标本大体观察、处死前CBCT影像学观察和HE染色组织学观察以确定牵张效果。胚胎组处置:经过术前B超确认孕期,对孕期30天、35天、50天的妊娠犬执行剖腹引产手术,取材各组活体胚胎,对标本进行大体观察和HE染色,剖腹后妊娠犬回笼饲养。骨折组处置:对实验犬执行骨折手术,通过钛板保持骨折间隙,骨折两组对应牵张组以作对照,骨折1组指骨折固定14天后取材(对应DO-1组5天的间隙期、7天的牵张期)、骨折2组指骨折固定28天后取材(对应DO-2组5天的间隙期、7天的牵张期以及2周的固定期)。另取新生半小时内幼犬3只,出生2周、4周幼犬各3只,以及非手术健康成年犬3只,分别纳入新生犬组(P-0)、2周幼犬组(P-2)、4周幼犬组(P-4)和正常对照组(AC),予以处死取单侧下颌骨骨组织为标本。以上动物除妊娠犬外均雌雄不限,成年犬年龄1.5-2.0岁,体重12.5kg±2.5kg(平均13.5kg)。2、提取各组骨组织标本的总RNA,经质检明确其浓度及纯度合格后,同时建立mRNA及miRNA测序c DNA文库,文库回收纯化后,由Illumina公司Hiseq4000测序平台进行测序。3、通过高通量测序获得mRNA、miRNA表达谱,将原始数据行mRNA、miRNA基因表达量标准化统计后,将11组样本分成55个两两对比组(1-vs-1)及36组的三实验组间比对(1-vs-11),对各对比组行mRNA、miRNA差异基因筛选,以及差异mRNA的GO富集分析和KEGG富集分析,并在此差异基因库里二次筛选能够提示牵张组、胚胎组、骨折组、正常对照组之间生物学层面异同点的兴趣基因,并作差异性与关联性分析。研究结果1、牵张即刻组和牵张固定2周组实验犬均手术过程顺利,术后无不良反应。牵张器固定牢靠,无松脱、断裂及变形。观察所有实验犬尖牙咬合关系较牵张前呈下颌左偏突颌改变,下颌骨牵张间隙有类骨样新生物质,说明右侧颌骨成功通过牵张延长。胚胎各组通过剖腹引产术取得活体犬胚胎,手术过程顺利,取得胎体完整,形态符合各孕期应有的发育形态,妊娠犬经引产后无不良反应,持续生存。骨折各组手术过程顺利,术后尖牙咬合关系能显示下颌左偏突颌改变,术区未出现严重感染,钛板及钛钉无松脱、断裂及变形,骨折两端见纤维样组织增生,无类骨样物质可被辨识。新生犬幼犬组和正常对照犬组取材过程顺利,无特殊。2、从11组样本中均提取出足量的总RNA,并分别成功构建了用于mRNA及miRNA测序所需的c DNA文库。成功完成了犬下颌骨牵张区及正常下颌骨骨组织的高通量测序。3、本次测序共获得22523个表达mRNA基因,综合55个两两对比组和36组三组间比较(1-vs-11),通过对差异表达mRNA进行分析后,我们发现了在牵张即刻组和胚胎犬组都共同表达上调、而正常组相对下调的mRNA共50个,以CXCL12、MINOS1、OGN、POSTN、PTN、SFRP2、MFAP5、IGFBP5、HSPB6等最具特征性,其中基因MINOS1、MFAP5、HSPB6均在正常成年犬中几乎无表达。以上基因功能囊括EMT调控、血管发生调控、间充质干细胞及成骨细胞动员等。牵张组与骨折组之间具有联系的基因有1921个,以ATF4、CD99L2、IGFBP5、RPL15等特征性最强,这些基因在牵张组和骨折组中具有不同的特征性表达趋势,主要为EMT调控,且相对于正常组中均显著表达(上调或下调),代表了牵张成骨与骨折愈合的不同特点。4、本次测序还获得354个表达miRNA,根据以上对比组,牵张各组与胚胎各组共同高表达、并在正常组相对下调表达的基因共46个,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均在正常成年犬中无表达。以上基因功能表达囊括血管发生与血管生成调控;胚胎干细胞、内皮祖细胞与间充质干细胞动员;血管内皮细胞迁移;成骨细胞与破骨细胞分化调控;成肌调控等。此外,我们筛选出牵张组与骨折组之间具有联系的基因,并对AC组亦同等量级表达的基因进行排除。获得了候选基因mi R-203、mi R-204、mi R-205、mi R-338、mi R-9和mi R-34a,这些基因在牵张组和骨折组中具有特征性的表达趋势,并相对于AC组有显著表达(上调或下调),主要为EMT调控,提示了牵张成骨与骨折愈合之间基因异同性表达的时空特点,揭示牵张成骨与骨折愈合各自的成血管化及成骨化特点。结论牵张成骨中,受初期微创伤造成的缺血性环境影响、炎症反应刺激、机械牵张力刺激或固定期静态牵张力刺激所启动,出现以下生物学过程:大量基因自胚胎发育期后二次显著表达并调控以血管发生(vasculogenesis)为主的成血管化,同时牵张成骨的早期机体的血管生成(Angiogenesis)机制受抑制,固定期后期有多个基因参与了对过载发育的血管网的限控;免疫性单核细胞受诱导向血管内皮细胞分化;上皮-间充质细胞转化(EMT)被显著抑制,大量的间充质细胞转化为上皮及内皮细胞以形成新的血管网;热休克蛋白受启动,维持细胞骨架稳定来强化成血管化及成骨化过程;多个基因调控的大量骨髓间充质干细胞快速增殖向成骨细胞分化、既有的成骨细胞募集、牵张区肌肉组织分泌特异蛋白促进骨代谢,维持着新骨快速生成。相较之下,骨折愈合过程中表达基因与牵张成骨差异较明显,部分基因启动较牵张成骨缓慢,EMT进程相较于牵张成骨显著活跃,愈合过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.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R782
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