犬下頜牽張成骨中與成骨成血管相關(guān)的microRNA初步研究
發(fā)布時間：2018-07-14 19:13
【摘要】：目的:建立犬下頜骨牽張成骨模型,利用高通量測序技術(shù)對牽張后即刻和固定2周的下頜骨牽張區(qū)骨組織及犬胚胎、新生犬、骨折犬以及正常對照組犬的下頜骨組織進行microRNA的全基因組表達譜測序,獲得microRNA的差異性表達,預(yù)測差異性micro RNA靶基因,并通過KEGG富集分析找到牽張成骨中成骨、成血管作用相關(guān)的因子信號通路。方法:設(shè)立11組實驗組分別為牽張固定0周組(DOI)、牽張固定2周組(DO2)、骨折固定12天(對應(yīng)牽張組間歇期7天及牽張期7天)組(MF1)、骨折固定26天(對應(yīng)牽張組間歇期7天、牽張期7天及固定期14天)組(MF2)、犬30天胚胎組(E1)、犬35胚胎組(E2)、犬50天胚胎組(E3)、新生犬組(NB)、幼犬2周組(P2)、幼犬4周組(P4)、對照組(AC)。每組實驗動物為3只。DOI組和DO2組分別選取3只健康成年中華田園犬,于其右側(cè)下頜骨施行下頜骨牽張成骨術(shù),間歇期7天后開始以1mm/天速度將右下頜骨向近中方向牽張7天,并于牽張結(jié)束后即刻和固定2周后處死、收集右側(cè)下頜骨牽張區(qū)標(biāo)本。MF1和MF2組同樣選取健康成年中華田園犬各3只,截斷右側(cè)下頜骨、骨折間隙為7mm,分別于術(shù)后12天和26天處死、取材納入實驗,固定時間對應(yīng)牽張各組。E1、E2、E3組分別選取孕30、35、50天犬各3只,確認孕期后取各自胚胎納入實驗。NB組選擇3只出生后當(dāng)日新生犬,P2和P4組分別選取同種出生2周、4周的健康幼犬各3只納入實驗組。對照組選取健康成年中華田園犬,處死E1、E2、E3、NB、P2、P4組及AC組,收集右下頜骨標(biāo)本。提取這幾組骨組織microRNA,進行高通量microRNA測序檢測。結(jié)果:1)大體觀:實驗犬均成功完成手術(shù)過程及術(shù)后隨訪,傷口愈合良好,無嚴重并發(fā)癥。DOI、DO2組牽張器與固位釘牢固,未見松動脫落。右下頜骨牽張區(qū)內(nèi)新生骨組織顏色鮮紅,質(zhì)軟,多為纖維組織及類骨質(zhì),未見明顯硬組織形成。2)從11組樣本中均提取出足量的總RNA,并成功完成了各組高通量microRNA測序,總共檢測到354個microRNA有表達。3)在牽張成骨組、胚胎組、新生組與對照組的對比中,共發(fā)現(xiàn)27個差異表達microRNA,相對正常對照組均有表達上調(diào)的共18個micro RNA、均有下調(diào)的共9個microRNA。其中miR-494的靶基因PTEN,可能為造成牽張過程中成骨速度加快的原因之一。結(jié)論:miR-494在牽張成骨組、胚胎組及新生組表達而在正常對照組不表達,提示牽張成骨與胚胎發(fā)育及幼犬快速生長發(fā)育具有相似的調(diào)節(jié)方式�？赡芡ㄟ^負調(diào)控其靶基因PTEN,從而激活PI3K-Akt信號通路促進成血管、成骨。
[Abstract]:Objective: to establish a canine mandibular distraction osteoblast model, and to determine the bone tissue of mandibular distraction zone and the embryo of dog and newborn dog immediately after distraction and 2 weeks after distraction by high throughput sequencing. The genomic expression profiles of microRNAs were sequenced in mandibular tissues of fracture dogs and normal dogs. The differentially expressed microRNAs were obtained, the differentially expressed micro target genes were predicted, and osteogenesis in distraction osteogenesis was found by KEGG enrichment analysis. The factor signaling pathway associated with angiogenesis. Methods: eleven experimental groups were divided into two groups: distraction fixation group (DOI), distraction fixation group (DO2), fracture fixation group (MF1) for 12 days (7 days for distraction group and 7 days for distraction period), and 26 days for fracture fixation (7 days for distraction group). The distraction period was 7 days and 14 days fixed (MF2), 30 days embryo group (E1), 35 embryo group (E2), 50 day embryo group (E3), newborn dog group (NB), puppies 2 weeks group (P2), puppies 4 weeks group (P4), control group (AC). Three healthy adult Chinese pastoral dogs were selected from each group of experimental animals. Mandibular distraction osteogenesis was performed on the right mandible of the dogs. After 7 days of distraction osteogenesis, the right mandible was stretched to the proximal Chinese side at the rate of 1mm/ day for 7 days. At the end of distraction and 2 weeks after fixation, the right mandibular distraction zone specimens. MF1 and MF2 groups were also selected to amputate the right mandible. The fracture space was 7mm. The animals were killed 12 days and 26 days after operation. The fixed time of each group was corresponding to the distraction time of each group. Three dogs were selected from each group on the 30th day of pregnancy, 35 days and 50 days, respectively. Three puppies (P 2 and P 4) of postnatal day were selected and 3 healthy puppies of 2 weeks and 4 weeks of homologous birth were selected to be included in the experimental group. The healthy adult Chinese pastoral dogs were selected in the control group. The right mandibular specimens were collected from the right mandibular specimens in the E1OE2E3NBP2P4 group and AC group. These groups of bone microRNAs were extracted for high throughput microRNA sequencing. Results (1) General observation: all the dogs successfully completed the operation and followed up after operation. The wound healed well. There was no serious complication. The distractor and retainer were firm and no loosening and falling off. In the right mandibular distraction zone, the new bone tissue was bright red, soft, mostly fibrous tissue and bony, no obvious hard tissue formation .2) the total RNAs were extracted from 11 groups of samples, and the high throughput microRNA sequencing of each group was successfully completed. A total of 354 microRNAs were detected. In the distraction osteogenesis group, embryonic group, newborn group and control group, a total of 27 differentially expressed microRNAs were found. Compared with the normal control group, 18 micro RNAs were up-regulated and 9 microRNAs were down-regulated. Among them, the target gene PTEN of miR-494 may be one of the causes of accelerated osteogenesis during distraction. Conclusion: the expression of miR-494 is expressed in distraction osteogenesis group, embryonic group and newborn group, but not in normal control group, suggesting that distraction osteogenesis is similar to embryonic development and rapid growth and development of puppies. It is possible that PI3K-Akt signaling pathway can be activated by negative regulation of its target gene PTEN to promote angiogenesis and osteogenesis.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R782
本文編號：2122677
[Abstract]:Objective: to establish a canine mandibular distraction osteoblast model, and to determine the bone tissue of mandibular distraction zone and the embryo of dog and newborn dog immediately after distraction and 2 weeks after distraction by high throughput sequencing. The genomic expression profiles of microRNAs were sequenced in mandibular tissues of fracture dogs and normal dogs. The differentially expressed microRNAs were obtained, the differentially expressed micro target genes were predicted, and osteogenesis in distraction osteogenesis was found by KEGG enrichment analysis. The factor signaling pathway associated with angiogenesis. Methods: eleven experimental groups were divided into two groups: distraction fixation group (DOI), distraction fixation group (DO2), fracture fixation group (MF1) for 12 days (7 days for distraction group and 7 days for distraction period), and 26 days for fracture fixation (7 days for distraction group). The distraction period was 7 days and 14 days fixed (MF2), 30 days embryo group (E1), 35 embryo group (E2), 50 day embryo group (E3), newborn dog group (NB), puppies 2 weeks group (P2), puppies 4 weeks group (P4), control group (AC). Three healthy adult Chinese pastoral dogs were selected from each group of experimental animals. Mandibular distraction osteogenesis was performed on the right mandible of the dogs. After 7 days of distraction osteogenesis, the right mandible was stretched to the proximal Chinese side at the rate of 1mm/ day for 7 days. At the end of distraction and 2 weeks after fixation, the right mandibular distraction zone specimens. MF1 and MF2 groups were also selected to amputate the right mandible. The fracture space was 7mm. The animals were killed 12 days and 26 days after operation. The fixed time of each group was corresponding to the distraction time of each group. Three dogs were selected from each group on the 30th day of pregnancy, 35 days and 50 days, respectively. Three puppies (P 2 and P 4) of postnatal day were selected and 3 healthy puppies of 2 weeks and 4 weeks of homologous birth were selected to be included in the experimental group. The healthy adult Chinese pastoral dogs were selected in the control group. The right mandibular specimens were collected from the right mandibular specimens in the E1OE2E3NBP2P4 group and AC group. These groups of bone microRNAs were extracted for high throughput microRNA sequencing. Results (1) General observation: all the dogs successfully completed the operation and followed up after operation. The wound healed well. There was no serious complication. The distractor and retainer were firm and no loosening and falling off. In the right mandibular distraction zone, the new bone tissue was bright red, soft, mostly fibrous tissue and bony, no obvious hard tissue formation .2) the total RNAs were extracted from 11 groups of samples, and the high throughput microRNA sequencing of each group was successfully completed. A total of 354 microRNAs were detected. In the distraction osteogenesis group, embryonic group, newborn group and control group, a total of 27 differentially expressed microRNAs were found. Compared with the normal control group, 18 micro RNAs were up-regulated and 9 microRNAs were down-regulated. Among them, the target gene PTEN of miR-494 may be one of the causes of accelerated osteogenesis during distraction. Conclusion: the expression of miR-494 is expressed in distraction osteogenesis group, embryonic group and newborn group, but not in normal control group, suggesting that distraction osteogenesis is similar to embryonic development and rapid growth and development of puppies. It is possible that PI3K-Akt signaling pathway can be activated by negative regulation of its target gene PTEN to promote angiogenesis and osteogenesis.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R782
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