miRNA與lncRNA調控牽張成骨快速成骨機制及其表達差異性分析研究
發(fā)布時間：2018-08-11 21:19
【摘要】：目的牽張成骨(Distraction Osteogenesis,DO)是一種內源性骨組織工程技術(Endogenous Bone Engineering),是通過機械力量將骨離斷,造成創(chuàng)傷,在兩骨斷端安裝牽引裝置,按一定的速度牽開骨斷端,使兩骨斷端間新骨形成的技術。DO后新骨形成的質量與正常骨接近,且新骨形成是有自限性和可控性。DO的成骨速度驚人,但成骨質量良好,其快速成骨的機制尚未清楚。血管形成貫穿整個成骨過程,血管在DO成骨過程中快速發(fā)生的機制也尚未闡明�，F(xiàn)今,對DO的新骨形成和血管新生的機制研究開始進入基因網(wǎng)絡調控時代。不編碼RNA的一類基因——非編碼RNA(non-coding RNA,nc RNA)可調控生物體發(fā)生發(fā)展的生物進程。nc RNA不僅參與細胞的增殖、分化、衰老、凋亡,更在腫瘤、心血管疾病、神經退行性疾病中起著重要調控作用。目前,對于非編碼RNA的研究主要集中在微小RNA(micro RNA,miRNA)和長鏈非編碼RNA(Long non-coding RNA,lnc RNA)上。有關miRNA和lnc RNA在DO過程中調控新骨形成和血管形成的作用已成為熱點之一。本研究通過利用第二代高通量測序技術對牽張后即刻和固定2周的下頜骨牽張區(qū)新骨組織、胚胎犬、骨折固定后14天和28天斷端區(qū)組織、0周/2周/4周幼犬以及正常成年犬的下頜骨骨組織進行miRNA及l(fā)nc RNA的基因組表達譜測序。通過對牽張組和非牽張組下頜骨骨組織的miRNA和lnc RNA進行基因組層面的表達差異分析,尋找牽張成骨新骨形成和血管新生相關聯(lián)的miRNA和lnc RNA,探索牽張成骨快速成骨的發(fā)生機制,且希望從中找到牽張成骨和胚胎發(fā)育相關的因子及信號通路。方法選取健康成年中華田園犬12只,年齡1.5-2.0歲,雌雄不限,體重12.5kg±2.5kg(平均13.5kg),其中隨機選取3只納入牽張固定0周組(DO-I),另隨機選取3只為牽張固定2周組(DO-2),隨機選取3只納入骨折固定14天組(BF1),另隨機選取3只為骨折固定28天組(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,分別納入新生犬組(NB)、2周幼犬組(PUP-2)、4周幼犬組(PUP-4)、和正常對照組(AC)。將廣西醫(yī)科大學動物實驗中心所提供的懷孕的實驗中華田園犬共9只,分成3組:E1組,胚胎1組3只(孕30天);E2組,胚胎2組3只(孕35天);E3組,胚胎3組3只(孕50天)。牽張固定0周組和牽張固定2周組的受試犬全部行單側下頜骨單線牽張成骨術,并于牽張結束即刻和牽張結束固定2周后處死,獲取牽張區(qū)周圍骨組織標本,骨折組的全部受試犬行下頜骨離斷術后固定14天和固定28天后處死,獲得骨折區(qū)愈合組織標本,通過標本大體觀察、處死前CBCT影像學觀察和HE染色組織學觀察以確定牽張以及骨折愈合效果。取新生組、2周幼犬組、4周幼犬組和正常對照組受試犬單側下頜骨正常骨組織。分別取孕30天、35天和50天的胚胎犬下頜骨組織。提取各組骨組織標本的總RNA,并行所提取RNA濃度及純度的檢測,合格后,分別建立用于miRNA及l(fā)nc RNA測序所需c DNA文庫,使用Illumina公司Hiseq4000測序平臺進行測序。通過高通量測序獲得miRNA表達譜,將原始數(shù)據(jù)行數(shù)據(jù)質控、比對統(tǒng)計和miRNA基因表達量統(tǒng)計。隨后將11組樣本分成55個兩兩對比組,對各對比組行miRNA差異表達量統(tǒng)計分析,以及差異miRNA的GO富集分析和KEGG富集分析。通過高通量測序獲得lnc RNA表達譜,將原始數(shù)據(jù)行數(shù)據(jù)質控、比對統(tǒng)計、novel lnc RNA預測及表達量統(tǒng)計。將11組樣本分為55個兩兩對比組,對各對比組進行差異表達lnc RNA統(tǒng)計分析及靶基因預測,并行差異表達lnc RNA靶基因的GO富集分析和KEGG富集分析。結果1、DO-I組,新生組織內血管豐富,細胞豐富;DO-2組中,血管進一步發(fā)育成熟,分布均勻。胚胎各組的新生組織血管豐富,細胞豐富,而骨折組纖維組織較多,骨形成不良。2、11組樣品中共檢測到354個miRNAs、3967個lnc RNAs和3813個novel-lnc RNAs。表達有差異的miRNAs一共有279個,靶基因12990個;有差異的lnc RNAs一共有161個,靶基因177個。與胚胎組相比,DO-I組有差異的miRNAs以下調為主,DO-I組有差異的lnc RNAs以上調為主。DO-2組有差異的miRNAs以下調為主,lnc RNAs以上調為主。與正常組(AC)相比,DO-I組有差異的miRNAs和lnc RNAs以上調為主,而DO-2組有差異的miRNAs以下調為主,lnc RNAs以上調為主。與骨折組相比,DO-I組有差異的miRNAs以上調為主,DO-2組有差異的miRNAs和lnc RNAs以上調為主。與幼犬組相比,DO-I組有差異的miRNAs以下調為主,lnc RNAs以上調為主。DO-2組有差異的miRNAs以下調為主,lnc RNAs以上調為主。3、在牽張組、胚胎組與正常對照組、骨折組比較后得出的表達有顯著差異的miRNAs和lnc RNAs中,miRNA-411,miRNA-410,miRNA-495,miRNA-487b,miRNA-487a,miRNA-369,miRNA-136,TCONS_00000934,TCONS_00170595等這些基因極有可能通過調控靶基因來調控牽張期的血管新生和新骨形成。結論1、通過對miRNA和lnc RNA表達譜的高通量測序結果的分析,可以初步推測牽張成骨過程中激活了胚胎發(fā)育相關的基因,調控了牽張成骨過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這些基因極有可能通過調控靶基因來調控牽張期的血管新生和新骨形成。但是其具體的作用和機制仍需進一步探討。
[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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R782
本文編號：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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R782
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