犬下颌牵张成骨中与新骨快速形成相关IncRNA的初步研究

发布时间：2018-07-01 12:52

本文选题：牵张成骨 + 胚胎成骨　；参考：《广西医科大学》2017年硕士论文

【摘要】：目的:建立犬下颌骨牵张成骨、下颌骨骨折模型及幼犬下颌骨及胚胎下颌骨模型,通过高通量测序找到牵张成骨期间可能激活的与新骨快速形成相关的因子及信号通路。方法:实验共分为7组,其中实验组分别为牵张固定0周组(DO1)、牵张固定2周组(DO2)、犬30天胚胎组(E)、幼犬2周组(P)、骨折固定2周(对应牵张组间歇期1周及牵张期1周)组(MF1)、骨折固定4周(对应牵张组间歇期1周、牵张期1周及固定期2周)组(MF2)及对照组(AC)。每小组实验动物为3只。DO1和DO2组中分别随即选取3只健康成年中华田园犬,体重约15公斤,于其右侧下颌骨施行下颌骨牵张成骨术。间歇期1周后开始以1mm/天速度将右下颌骨向近中方向牵张,牵张期1周共预计牵张7mm后牵张结束,处死DO1组实验犬并收集右下颌骨牵张区标本。DO2组完成牵张后固定2周,固定期结束后拍摄锥形束CT观察成骨情况,然后处死动物并收集右侧下颌骨牵张区新生标本,记录标本大体观。MF1和MF2组分别随即选取3只健康成年中华田园犬犬,体重约15公斤,于其右侧下颌骨施行下颌骨骨折手术,术式参考下颌骨牵张成骨,骨折间隙为7mm。分别于2周及4周后拍摄CBCT及处死取材,收集右下颌骨骨折间隙标本。E组为孕30天的母犬,拍摄B超确认天数后纳入实验组。P组为随机选取3只同种出生2周健康幼犬纳入实验组。AC组随机选取3只健康成年中华田园犬,体重约15公斤。处死E组、P组及AC组并收集各组右下颌骨标本。将7组标本进行高通量LncRNA测序检测。结果:1、DO1、DO2、MF1、MF2组动物均顺利完成手术及术后随访观察。术后没有实验犬出现相关严重并发症,牵张器及钛板固定牢固,未见松脱,亦未发现牵张器及钛板的断裂及变形。观察所有实验犬行牵张前和牵张后右侧尖牙牙位及骨折手术前后右侧尖牙牙位,可发现牵张后及骨折后下颌尖牙相对于上颌尖牙有明显的近中前移,说明右侧颌骨成功通过牵张及人为骨折延长。2、从7组样本中均提取出足量的总RNA,并分别成功构建了用于lncRNA测序所需的cDNA文库。成功完成了各组高通量lncRNA测序。3、本项目7组样品数据,总共有3504个lincRNA和3731个novel_lncRNA有表达。4、牵张组、胚胎组、幼犬组同正常对照组及骨折组相比差异表达的lncRNA中,TCONS_00170590有可能调控其靶基因Lix1参与牵张成骨的快速成骨过程。结论:1、Lnc RNA TCONS_00170590在DO1组、E组、P组中表达同AC组和MF1组相比有显著差异,提示牵张成骨与胚胎及幼犬发育有共同点且不同于骨折愈合。2、LncRNA TCONS_00170590负调控其靶基因LIX1,并且可能参与调控牵张成骨过程中的血管新生和新骨快速形成。3、牵张成骨中除与胚胎及幼犬共同的生长调节因素外,牵张力、微创伤等因素可能是其更快速成骨的原因。
[Abstract]:Objective: to establish canine mandibular distraction osteogenesis, mandibular fracture model and pup mandibular and embryonic mandibular model, and to find out the factors and signal pathways related to the rapid formation of new bone during distraction osteogenesis by high-throughput sequencing. Methods: the experiment was divided into 7 groups. The experimental groups were: distraction fixation 0 week group (do 1), distraction fixation group 2 weeks group (do 2), dog 30 days embryo group (E), puppies 2 weeks group (P), fracture fixation 2 weeks group (corresponding to distraction group 1 week and distraction period 1 week) group (MF1), fracture fixation 4 weeks (P). The intermission period of the distraction group was 1 week. The distraction period of 1 week and fixed period of 2 weeks) group (MF2) and control group (AC). Three healthy adult Chinese pastoral dogs, weighing about 15 kg, were selected from each group. The mandibular distraction osteogenesis was performed on the right mandible. One week after the intermission period, the right mandible was stretched to the proximal Chinese side at the rate of 1mm/ day, and the distraction was expected to end after the distraction of 7mm in one week. The dogs of group DO1 were killed and the specimens of the distraction area of right mandible were collected. The specimens of the distraction area of the right mandible were fixed for 2 weeks after distraction in group DO2. At the end of the fixation period, the osteogenesis was observed by tapered CT, then the animals were killed and the newborn specimens of the right mandibular distraction region were collected. Three healthy adult Chinese pastoral dogs were selected immediately in the gross observation. MF1 and MF2 groups, respectively. The mandibular fracture was performed on the right mandible of the mandible, weighing about 15 kg. The mandibular distraction osteogenesis was referred to the procedure, and the fracture space was 7 mm. CBCT was taken at 2 and 4 weeks later, and the specimens of right mandibular fracture space were collected. Group E was 30 days pregnant. Three healthy puppies of the same type were randomly selected from the experiment group and AC group, and 3 healthy adult Chinese pastoral dogs were randomly selected, weighing about 15 kg. Group E, group P and group AC were sacrificed and the right mandibular specimens were collected. High throughput LncRNA sequencing was performed in 7 groups of specimens. Results all the animals in the two groups were successfully operated and followed up. There were no serious complications in dogs after operation. The tensioner and titanium plate were firmly fixed without loosening, and the fracture and deformation of the distractor and titanium plate were not found. By observing the right canine tooth position before and after distraction and the right canine tooth position before and after fracture operation, we found that the mandibular canine teeth after distraction and fracture had a significant proximal anterior movement to the maxillary canine teeth. The results showed that the right jaw bone was successfully lengthened by distraction and artificial fracture. The total RNAs were extracted from 7 groups of samples and the cDNA libraries for lncRNA sequencing were constructed successfully. High throughput lncRNA sequencing. 3 was successfully completed in each group. In this project, there were 3504 lincRNAs and 3731 Novelln cRNAs expressed in 7 groups of sample data, including distraction group, embryo group, distraction group and embryo group. TCONS00170590 in the differentially expressed lncRNA in the puppies, compared with the normal control group and fracture group, may be involved in the rapid osteogenesis of distraction osteogenesis by regulating its target gene Lix1. Conclusion the expression of TCONS00170590 in TCONS00170590 is significantly different from that in AC and MF1 groups, and the expression of TCONS00170590 in P group is significantly higher than that in AC group and MF1 group. These results suggest that distraction osteogenesis has something in common with the development of embryos and puppies and is different from fracture healing. 2LncRNA TCONS00170590 negatively regulates its target gene LIX1, and may be involved in the regulation of angiogenesis and rapid formation of new bone during distraction osteogenesis. In addition to common growth regulation factors with embryos and puppies, Some factors, such as stretch and microtrauma, may be the causes of faster 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】：R782.4

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