miRNA與lncRNA調(diào)控牽張成骨快速成骨機(jī)制及其表達(dá)差異性分析研究
發(fā)布時(shí)間：2018-08-11 21:19
【摘要】：目的牽張成骨(Distraction Osteogenesis,DO)是一種內(nèi)源性骨組織工程技術(shù)(Endogenous Bone Engineering),是通過(guò)機(jī)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(shù)。DO后新骨形成的質(zhì)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(zhì)量良好,其快速成骨的機(jī)制尚未清楚。血管形成貫穿整個(gè)成骨過(guò)程,血管在DO成骨過(guò)程中快速發(fā)生的機(jī)制也尚未闡明。現(xiàn)今,對(duì)DO的新骨形成和血管新生的機(jī)制研究開始進(jìn)入基因網(wǎng)絡(luò)調(diào)控時(shí)代。不編碼RNA的一類基因——非編碼RNA(non-coding RNA,nc RNA)可調(diào)控生物體發(fā)生發(fā)展的生物進(jìn)程。nc RNA不僅參與細(xì)胞的增殖、分化、衰老、凋亡,更在腫瘤、心血管疾病、神經(jīng)退行性疾病中起著重要調(diào)控作用。目前,對(duì)于非編碼RNA的研究主要集中在微小RNA(micro RNA,miRNA)和長(zhǎng)鏈非編碼RNA(Long non-coding RNA,lnc RNA)上。有關(guān)miRNA和lnc RNA在DO過(guò)程中調(diào)控新骨形成和血管形成的作用已成為熱點(diǎn)之一。本研究通過(guò)利用第二代高通量測(cè)序技術(shù)對(duì)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(jìn)行miRNA及l(fā)nc RNA的基因組表達(dá)譜測(cè)序。通過(guò)對(duì)牽張組和非牽張組下頜骨骨組織的miRNA和lnc RNA進(jìn)行基因組層面的表達(dá)差異分析,尋找牽張成骨新骨形成和血管新生相關(guān)聯(lián)的miRNA和lnc RNA,探索牽張成骨快速成骨的發(fā)生機(jī)制,且希望從中找到牽張成骨和胚胎發(fā)育相關(guān)的因子及信號(hào)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(jī)選取3只納入牽張固定0周組(DO-I),另隨機(jī)選取3只為牽張固定2周組(DO-2),隨機(jī)選取3只納入骨折固定14天組(BF1),另隨機(jī)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(duì)照組(AC)。將廣西醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心所提供的懷孕的實(shí)驗(yàn)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(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,獲取牽張區(qū)周圍骨組織標(biāo)本,骨折組的全部受試犬行下頜骨離斷術(shù)后固定14天和固定28天后處死,獲得骨折區(qū)愈合組織標(biāo)本,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(duì)照組受試犬單側(cè)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(biāo)本的總RNA,并行所提取RNA濃度及純度的檢測(cè),合格后,分別建立用于miRNA及l(fā)nc RNA測(cè)序所需c DNA文庫(kù),使用Illumina公司Hiseq4000測(cè)序平臺(tái)進(jìn)行測(cè)序。通過(guò)高通量測(cè)序獲得miRNA表達(dá)譜,將原始數(shù)據(jù)行數(shù)據(jù)質(zhì)控、比對(duì)統(tǒng)計(jì)和miRNA基因表達(dá)量統(tǒng)計(jì)。隨后將11組樣本分成55個(gè)兩兩對(duì)比組,對(duì)各對(duì)比組行miRNA差異表達(dá)量統(tǒng)計(jì)分析,以及差異miRNA的GO富集分析和KEGG富集分析。通過(guò)高通量測(cè)序獲得lnc RNA表達(dá)譜,將原始數(shù)據(jù)行數(shù)據(jù)質(zhì)控、比對(duì)統(tǒng)計(jì)、novel lnc RNA預(yù)測(cè)及表達(dá)量統(tǒng)計(jì)。將11組樣本分為55個(gè)兩兩對(duì)比組,對(duì)各對(duì)比組進(jìn)行差異表達(dá)lnc RNA統(tǒng)計(jì)分析及靶基因預(yù)測(cè),并行差異表達(dá)lnc RNA靶基因的GO富集分析和KEGG富集分析。結(jié)果1、DO-I組,新生組織內(nèi)血管豐富,細(xì)胞豐富;DO-2組中,血管進(jìn)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(xì)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(cè)到354個(gè)miRNAs、3967個(gè)lnc RNAs和3813個(gè)novel-lnc RNAs。表達(dá)有差異的miRNAs一共有279個(gè),靶基因12990個(gè);有差異的lnc RNAs一共有161個(gè),靶基因177個(gè)。與胚胎組相比,DO-I組有差異的miRNAs以下調(diào)為主,DO-I組有差異的lnc RNAs以上調(diào)為主。DO-2組有差異的miRNAs以下調(diào)為主,lnc RNAs以上調(diào)為主。與正常組(AC)相比,DO-I組有差異的miRNAs和lnc RNAs以上調(diào)為主,而DO-2組有差異的miRNAs以下調(diào)為主,lnc RNAs以上調(diào)為主。與骨折組相比,DO-I組有差異的miRNAs以上調(diào)為主,DO-2組有差異的miRNAs和lnc RNAs以上調(diào)為主。與幼犬組相比,DO-I組有差異的miRNAs以下調(diào)為主,lnc RNAs以上調(diào)為主。DO-2組有差異的miRNAs以下調(diào)為主,lnc RNAs以上調(diào)為主。3、在牽張組、胚胎組與正常對(duì)照組、骨折組比較后得出的表達(dá)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(guò)調(diào)控靶基因來(lái)調(diào)控牽張期的血管新生和新骨形成。結(jié)論1、通過(guò)對(duì)miRNA和lnc RNA表達(dá)譜的高通量測(cè)序結(jié)果的分析,可以初步推測(cè)牽張成骨過(guò)程中激活了胚胎發(fā)育相關(guān)的基因,調(diào)控了牽張成骨過(guò)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(guò)調(diào)控靶基因來(lái)調(diào)控牽張期的血管新生和新骨形成。但是其具體的作用和機(jī)制仍需進(jìn)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(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
本文編號(hào)：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(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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