犬下颌牵张成骨中与成骨成血管相关的microRNA初步研究

发布时间：2018-07-14 19:13

【摘要】：目的:建立犬下颌骨牵张成骨模型,利用高通量测序技术对牵张后即刻和固定2周的下颌骨牵张区骨组织及犬胚胎、新生犬、骨折犬以及正常对照组犬的下颌骨组织进行microRNA的全基因组表达谱测序,获得microRNA的差异性表达,预测差异性micro RNA靶基因,并通过KEGG富集分析找到牵张成骨中成骨、成血管作用相关的因子信号通路。方法:设立11组实验组分别为牵张固定0周组(DOI)、牵张固定2周组(DO2)、骨折固定12天(对应牵张组间歇期7天及牵张期7天)组(MF1)、骨折固定26天(对应牵张组间歇期7天、牵张期7天及固定期14天)组(MF2)、犬30天胚胎组(E1)、犬35胚胎组(E2)、犬50天胚胎组(E3)、新生犬组(NB)、幼犬2周组(P2)、幼犬4周组(P4)、对照组(AC)。每组实验动物为3只。DOI组和DO2组分别选取3只健康成年中华田园犬,于其右侧下颌骨施行下颌骨牵张成骨术,间歇期7天后开始以1mm/天速度将右下颌骨向近中方向牵张7天,并于牵张结束后即刻和固定2周后处死、收集右侧下颌骨牵张区标本。MF1和MF2组同样选取健康成年中华田园犬各3只,截断右侧下颌骨、骨折间隙为7mm,分别于术后12天和26天处死、取材纳入实验,固定时间对应牵张各组。E1、E2、E3组分别选取孕30、35、50天犬各3只,确认孕期后取各自胚胎纳入实验。NB组选择3只出生后当日新生犬,P2和P4组分别选取同种出生2周、4周的健康幼犬各3只纳入实验组。对照组选取健康成年中华田园犬,处死E1、E2、E3、NB、P2、P4组及AC组,收集右下颌骨标本。提取这几组骨组织microRNA,进行高通量microRNA测序检测。结果:1)大体观:实验犬均成功完成手术过程及术后随访,伤口愈合良好,无严重并发症。DOI、DO2组牵张器与固位钉牢固,未见松动脱落。右下颌骨牵张区内新生骨组织颜色鲜红,质软,多为纤维组织及类骨质,未见明显硬组织形成。2)从11组样本中均提取出足量的总RNA,并成功完成了各组高通量microRNA测序,总共检测到354个microRNA有表达。3)在牵张成骨组、胚胎组、新生组与对照组的对比中,共发现27个差异表达microRNA,相对正常对照组均有表达上调的共18个micro RNA、均有下调的共9个microRNA。其中miR-494的靶基因PTEN,可能为造成牵张过程中成骨速度加快的原因之一。结论:miR-494在牵张成骨组、胚胎组及新生组表达而在正常对照组不表达,提示牵张成骨与胚胎发育及幼犬快速生长发育具有相似的调节方式。可能通过负调控其靶基因PTEN,从而激活PI3K-Akt信号通路促进成血管、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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R782

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