miRNA与lncRNA调控牵张成骨快速成骨机制及其表达差异性分析研究
发布时间：2018-08-11 21:19
【摘要】：目的牵张成骨(Distraction Osteogenesis,DO)是一种内源性骨组织工程技术(Endogenous Bone Engineering),是通过机械力量将骨离断,造成创伤,在两骨断端安装牵引装置,按一定的速度牵开骨断端,使两骨断端间新骨形成的技术。DO后新骨形成的质量与正常骨接近,且新骨形成是有自限性和可控性。DO的成骨速度惊人,但成骨质量良好,其快速成骨的机制尚未清楚。血管形成贯穿整个成骨过程,血管在DO成骨过程中快速发生的机制也尚未阐明。现今,对DO的新骨形成和血管新生的机制研究开始进入基因网络调控时代。不编码RNA的一类基因——非编码RNA(non-coding RNA,nc RNA)可调控生物体发生发展的生物进程。nc RNA不仅参与细胞的增殖、分化、衰老、凋亡,更在肿瘤、心血管疾病、神经退行性疾病中起着重要调控作用。目前,对于非编码RNA的研究主要集中在微小RNA(micro RNA,miRNA)和长链非编码RNA(Long non-coding RNA,lnc RNA)上。有关miRNA和lnc RNA在DO过程中调控新骨形成和血管形成的作用已成为热点之一。本研究通过利用第二代高通量测序技术对牵张后即刻和固定2周的下颌骨牵张区新骨组织、胚胎犬、骨折固定后14天和28天断端区组织、0周/2周/4周幼犬以及正常成年犬的下颌骨骨组织进行miRNA及lnc RNA的基因组表达谱测序。通过对牵张组和非牵张组下颌骨骨组织的miRNA和lnc RNA进行基因组层面的表达差异分析,寻找牵张成骨新骨形成和血管新生相关联的miRNA和lnc RNA,探索牵张成骨快速成骨的发生机制,且希望从中找到牵张成骨和胚胎发育相关的因子及信号通路。方法选取健康成年中华田园犬12只,年龄1.5-2.0岁,雌雄不限,体重12.5kg±2.5kg(平均13.5kg),其中随机选取3只纳入牵张固定0周组(DO-I),另随机选取3只为牵张固定2周组(DO-2),随机选取3只纳入骨折固定14天组(BF1),另随机选取3只为骨折固定28天组(BF2)。另取幼犬出生0周、2周和4周幼犬各3只,以及健康成年犬3只,雌雄不限,分别纳入新生犬组(NB)、2周幼犬组(PUP-2)、4周幼犬组(PUP-4)、和正常对照组(AC)。将广西医科大学动物实验中心所提供的怀孕的实验中华田园犬共9只,分成3组:E1组,胚胎1组3只(孕30天);E2组,胚胎2组3只(孕35天);E3组,胚胎3组3只(孕50天)。牵张固定0周组和牵张固定2周组的受试犬全部行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,获取牵张区周围骨组织标本,骨折组的全部受试犬行下颌骨离断术后固定14天和固定28天后处死,获得骨折区愈合组织标本,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以及骨折愈合效果。取新生组、2周幼犬组、4周幼犬组和正常对照组受试犬单侧下颌骨正常骨组织。分别取孕30天、35天和50天的胚胎犬下颌骨组织。提取各组骨组织标本的总RNA,并行所提取RNA浓度及纯度的检测,合格后,分别建立用于miRNA及lnc RNA测序所需c DNA文库,使用Illumina公司Hiseq4000测序平台进行测序。通过高通量测序获得miRNA表达谱,将原始数据行数据质控、比对统计和miRNA基因表达量统计。随后将11组样本分成55个两两对比组,对各对比组行miRNA差异表达量统计分析,以及差异miRNA的GO富集分析和KEGG富集分析。通过高通量测序获得lnc RNA表达谱,将原始数据行数据质控、比对统计、novel lnc RNA预测及表达量统计。将11组样本分为55个两两对比组,对各对比组进行差异表达lnc RNA统计分析及靶基因预测,并行差异表达lnc RNA靶基因的GO富集分析和KEGG富集分析。结果1、DO-I组,新生组织内血管丰富,细胞丰富;DO-2组中,血管进一步发育成熟,分布均匀。胚胎各组的新生组织血管丰富,细胞丰富,而骨折组纤维组织较多,骨形成不良。2、11组样品中共检测到354个miRNAs、3967个lnc RNAs和3813个novel-lnc RNAs。表达有差异的miRNAs一共有279个,靶基因12990个;有差异的lnc RNAs一共有161个,靶基因177个。与胚胎组相比,DO-I组有差异的miRNAs以下调为主,DO-I组有差异的lnc RNAs以上调为主。DO-2组有差异的miRNAs以下调为主,lnc RNAs以上调为主。与正常组(AC)相比,DO-I组有差异的miRNAs和lnc RNAs以上调为主,而DO-2组有差异的miRNAs以下调为主,lnc RNAs以上调为主。与骨折组相比,DO-I组有差异的miRNAs以上调为主,DO-2组有差异的miRNAs和lnc RNAs以上调为主。与幼犬组相比,DO-I组有差异的miRNAs以下调为主,lnc RNAs以上调为主。DO-2组有差异的miRNAs以下调为主,lnc RNAs以上调为主。3、在牵张组、胚胎组与正常对照组、骨折组比较后得出的表达有显著差异的miRNAs和lnc RNAs中,miRNA-411,miRNA-410,miRNA-495,miRNA-487b,miRNA-487a,miRNA-369,miRNA-136,TCONS_00000934,TCONS_00170595等这些基因极有可能通过调控靶基因来调控牵张期的血管新生和新骨形成。结论1、通过对miRNA和lnc RNA表达谱的高通量测序结果的分析,可以初步推测牵张成骨过程中激活了胚胎发育相关的基因,调控了牵张成骨过程中的血管新生和新骨形成。2、miRNA-411,miRNA-410,miRNA-495,miRNA-487b,miRNA-487a,miRNA-369,miRNA-136,TCONS_00000934,TCONS_00170595这些基因极有可能通过调控靶基因来调控牵张期的血管新生和新骨形成。但是其具体的作用和机制仍需进一步探讨。
[Abstract]:Objective Distraction Osteogenesis (DO) is an endogenous bone tissue engineering (Endogenous Bone Engineering). It is a technique that breaks the bone and causes trauma by mechanical force. A distraction device is installed at the broken ends of the two bones, and the broken ends of the two bones are pulled apart at a certain speed. The quality of new bone formation after DO. The osteogenesis rate of DO is remarkable, but the quality of osteogenesis is good. The mechanism of rapid osteogenesis of DO is unclear. Vascular formation runs through the whole osteogenesis process, and the mechanism of rapid osteogenesis of blood vessels in the process of DO has not been clarified. Non-coding RNA (nc RNA), a class of genes that do not encode RNA, can regulate the biological process of organism development. NCRNA not only participates in cell proliferation, differentiation, senescence, apoptosis, but also plays an important regulatory role in tumor, cardiovascular disease, neurodegenerative disease. The research on non-coding RNA mainly focuses on micro RNA (micro RNA, micro RNA) and long non-coding RNA (lnc RNA). Genomic expression profiles of microRNAs and LNC RNA were sequenced in mandibular distraction zone at week 1, embryonic canine, 14 and 28 days after fracture fixation, 0/2/4 weeks puppy and normal adult canine. Methods Twelve healthy adult Chinese idyllic dogs, aged 1.5-2.0 years, male and female, weighing 12.5, were selected. Three dogs were randomly selected as DO-I group for 0 weeks, three for DO-2 group for 2 weeks, three for BF1 group for 14 days, and three for BF2 group for 28 days. Nine pregnant Chinese pastoral dogs were divided into three groups: E1 group, embryo 1 group (30 days of gestation), E2 group, embryo 2 group (35 days of gestation), E3 group, embryo 3 group (50 days of gestation). All dogs in the 0-week group and 2-week group underwent unilateral mandibular distraction osteogenesis and were sacrificed immediately after distraction and 2 weeks after distraction. Bone tissue samples around distraction area were obtained. All dogs in the fracture group were sacrificed 14 days after mandibular interruption and 28 days after distraction osteogenesis. The unilateral mandibular normal tissues of newborn, 2-week puppy, 4-week puppy and normal control groups were obtained. The mandibular tissues of 30-day pregnant, 35-day pregnant and 50-day pregnant embryonic dogs were extracted. The total RNA of each group of bone tissue samples was detected by the concentration and purity of extracted RNA. After qualified, the C DNA libraries needed for the sequencing of microRNAs and LNC RNA were established and sequenced using Illumina Hiseq4000 sequencing platform. After that, 11 groups of samples were divided into 55 pairwise control groups. The differential expression of microRNAs, GO enrichment analysis and KEGG enrichment analysis were performed in each group. The expression profiles of LNC RNA were obtained by high-throughput sequencing. The original data were controlled by quality control, comparison, novel LNC RNA prediction and expression statistics. The samples were divided into 55 pairwise control groups, and the differential expression of LNC RNA was statistically analyzed and the target gene was predicted. The GO enrichment analysis and KEGG enrichment analysis of the target gene of the differential expression of LNC RNA were performed. A total of 354 microRNAs, 3967 LNC RNAs and 3813 novel-lnc RNAs were detected in 11 groups. 279 microRNAs and 12 990 target genes were differentially expressed, and 161 LNC RNAs and 177 target genes were found in the differentially expressed groups. Compared with the normal group (AC), the differences in the DO-I group were mainly up-regulation of microRNAs and LNC RNAs, while the differences in the DO-I group were mainly down-regulation of microRNAs and up-regulation of LNC RNAs. Compared with the normal group (AC), the differences in the DO-I group were mainly down-regulation of microRNAs and up-regulation of LNC RNAs, while the differences in the DO-2 group were mainly down-regulation of microRNAs and up-regulation of LNC RNAs. Compared with the puppy group, the microRNAs in the DO-I group were mainly down-regulated, while the LNC RNAs in the DO-2 group were mainly up-regulated. Compared with the puppy group, the microRNAs in the DO-I group were mainly down-regulated and the LNC RNAs up-regulated. MiNA-411, microNA-410, microNA-495, microNA-487b, microNA-487a, microNA-369, microNA-136, TCONS_00000934, TCONS_00170595 and other genes with significantly different expression profiles were most likely to regulate angiogenesis and new bone formation during distraction by regulating target genes. Analysis of high-throughput sequencing results of expression profiles may preliminarily speculate that genes involved in embryonic development are activated during distraction osteogenesis, regulating angiogenesis and new bone formation during distraction osteogenesis. 2, microRNA411, microRNA410, microRNA495, microRNA487b, microRNA487a, microRNA369, microRNA136, TCONS_00000934, TCONS_00170595 It is possible to regulate angiogenesis and new bone formation during distraction by regulating target genes. However, the specific role and mechanism of this regulation need to be further explored.
【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
本文编号：2178263
[Abstract]:Objective Distraction Osteogenesis (DO) is an endogenous bone tissue engineering (Endogenous Bone Engineering). It is a technique that breaks the bone and causes trauma by mechanical force. A distraction device is installed at the broken ends of the two bones, and the broken ends of the two bones are pulled apart at a certain speed. The quality of new bone formation after DO. The osteogenesis rate of DO is remarkable, but the quality of osteogenesis is good. The mechanism of rapid osteogenesis of DO is unclear. Vascular formation runs through the whole osteogenesis process, and the mechanism of rapid osteogenesis of blood vessels in the process of DO has not been clarified. Non-coding RNA (nc RNA), a class of genes that do not encode RNA, can regulate the biological process of organism development. NCRNA not only participates in cell proliferation, differentiation, senescence, apoptosis, but also plays an important regulatory role in tumor, cardiovascular disease, neurodegenerative disease. The research on non-coding RNA mainly focuses on micro RNA (micro RNA, micro RNA) and long non-coding RNA (lnc RNA). Genomic expression profiles of microRNAs and LNC RNA were sequenced in mandibular distraction zone at week 1, embryonic canine, 14 and 28 days after fracture fixation, 0/2/4 weeks puppy and normal adult canine. Methods Twelve healthy adult Chinese idyllic dogs, aged 1.5-2.0 years, male and female, weighing 12.5, were selected. Three dogs were randomly selected as DO-I group for 0 weeks, three for DO-2 group for 2 weeks, three for BF1 group for 14 days, and three for BF2 group for 28 days. Nine pregnant Chinese pastoral dogs were divided into three groups: E1 group, embryo 1 group (30 days of gestation), E2 group, embryo 2 group (35 days of gestation), E3 group, embryo 3 group (50 days of gestation). All dogs in the 0-week group and 2-week group underwent unilateral mandibular distraction osteogenesis and were sacrificed immediately after distraction and 2 weeks after distraction. Bone tissue samples around distraction area were obtained. All dogs in the fracture group were sacrificed 14 days after mandibular interruption and 28 days after distraction osteogenesis. The unilateral mandibular normal tissues of newborn, 2-week puppy, 4-week puppy and normal control groups were obtained. The mandibular tissues of 30-day pregnant, 35-day pregnant and 50-day pregnant embryonic dogs were extracted. The total RNA of each group of bone tissue samples was detected by the concentration and purity of extracted RNA. After qualified, the C DNA libraries needed for the sequencing of microRNAs and LNC RNA were established and sequenced using Illumina Hiseq4000 sequencing platform. After that, 11 groups of samples were divided into 55 pairwise control groups. The differential expression of microRNAs, GO enrichment analysis and KEGG enrichment analysis were performed in each group. The expression profiles of LNC RNA were obtained by high-throughput sequencing. The original data were controlled by quality control, comparison, novel LNC RNA prediction and expression statistics. The samples were divided into 55 pairwise control groups, and the differential expression of LNC RNA was statistically analyzed and the target gene was predicted. The GO enrichment analysis and KEGG enrichment analysis of the target gene of the differential expression of LNC RNA were performed. A total of 354 microRNAs, 3967 LNC RNAs and 3813 novel-lnc RNAs were detected in 11 groups. 279 microRNAs and 12 990 target genes were differentially expressed, and 161 LNC RNAs and 177 target genes were found in the differentially expressed groups. Compared with the normal group (AC), the differences in the DO-I group were mainly up-regulation of microRNAs and LNC RNAs, while the differences in the DO-I group were mainly down-regulation of microRNAs and up-regulation of LNC RNAs. Compared with the normal group (AC), the differences in the DO-I group were mainly down-regulation of microRNAs and up-regulation of LNC RNAs, while the differences in the DO-2 group were mainly down-regulation of microRNAs and up-regulation of LNC RNAs. Compared with the puppy group, the microRNAs in the DO-I group were mainly down-regulated, while the LNC RNAs in the DO-2 group were mainly up-regulated. Compared with the puppy group, the microRNAs in the DO-I group were mainly down-regulated and the LNC RNAs up-regulated. MiNA-411, microNA-410, microNA-495, microNA-487b, microNA-487a, microNA-369, microNA-136, TCONS_00000934, TCONS_00170595 and other genes with significantly different expression profiles were most likely to regulate angiogenesis and new bone formation during distraction by regulating target genes. Analysis of high-throughput sequencing results of expression profiles may preliminarily speculate that genes involved in embryonic development are activated during distraction osteogenesis, regulating angiogenesis and new bone formation during distraction osteogenesis. 2, microRNA411, microRNA410, microRNA495, microRNA487b, microRNA487a, microRNA369, microRNA136, TCONS_00000934, TCONS_00170595 It is possible to regulate angiogenesis and new bone formation during distraction by regulating target genes. However, the specific role and mechanism of this regulation need to be further explored.
【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
【参考文献】
相关期刊论文 前10条
1 瞿帅;宋杨;梅金玉;曹卫;陶冶;王雅堂;杨见明;;IL-17及其受体调控JAK/STAT3信号通路促进喉癌血管生成的作用[J];安徽医科大学学报;2015年10期
2 高艳;程晨;李静;肖伟凡;潘秋辉;;LncRNA AK051397在BMP-2诱导的C2C12成骨分化中的作用[J];同济大学学报(医学版);2014年04期
3 程晨;高艳;李静;潘秋辉;;骨形态发生蛋白2诱导C3H10T1/2细胞成骨分化中长链非编码RNA的作用[J];中国组织工程研究;2014年20期
4 周诺;黄旋平;江献芳;宋继传;李华;谢庆条;;骨形态发生蛋白-2基因修饰骨髓间充质干细胞复合对氧化聚乙烯和聚丙烯共聚物移植促进兔下颌骨牵张成骨的实验研究[J];华西口腔医学杂志;2013年03期
5 黄旋平;周诺;杨媛媛;江献芳;李华;谢庆条;;hBMP-2基因修饰自体BMSCs移植促进兔下颌骨牵张成骨新骨形成的X线分析[J];实用口腔医学杂志;2012年04期
6 周庄;张柳;;NF-κB信号通路在骨性关节炎发生发展中作用机制的研究[J];中国骨质疏松杂志;2012年01期
7 李涛;;B超法诊断犬妊娠日龄的研究[J];黑龙江动物繁殖;2012年01期
8 黄耿文;丁翔;;ESM-1作为肝癌血管内皮标志物的意义[J];肿瘤防治研究;2011年09期
9 周诺;;牵张成骨研究中的几个热点问题[J];口腔颌面外科杂志;2011年04期
10 周诺,赵亮,梁飞新,韦山良;血管内皮细胞生长因子、胰岛素样生长因子-1在下颌骨牵张成骨与骨缺损中的表达和意义[J];口腔医学;2005年05期
相关博士学位论文 前1条
1 王亮;人骨髓间充质干细胞成骨及成软骨分化中长链非编码RNA的初步研究[D];北京协和医学院;2015年
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