肌相关microRNAs在维甲酸诱导舌发育异常中对舌肌分化调控的机制研究
发布时间：2019-06-18 21:22
【摘要】：背景：腭裂是口腔颌面部常见的先天畸形之一，是通过基因与环境因素交互作用造成的多基因易感性疾病。在本课题组前期研究中，我们发现维甲酸（retinoicacid, RA）诱导小鼠胚胎腭裂发生同时常伴有舌发育的畸形。发生腭裂的小鼠舌体在胚胎第14.5天并未下降，仍充满口腔，使两侧腭突仍位于舌体的两侧，无法转入水平生长期，进而无法在中线处融合。该时期腭裂胎鼠不仅舌体形态以及舌体中舌肌的纤维走形发生异常，而且其相关成肌调节因子（Myogenic RegulatoryFactors，MRFs）的表达量也异于正常的胎鼠。舌肌是舌组织的主体，而舌肌属于骨骼肌，因此，对骨骼肌发生发育起到调控作用的一系列基因也在舌肌的分化过程中起到一定的作用。肌相关microRNAs，即MyomiRs，就是一类在肌肉中富集的microRNAs，它们对成肌过程起到一定的调控作用。研究表明：在分化过程中，miR-206直接靶向结合Pax7，Pax7抑制相关MRFs的表达，miR-206抑制Pax7之后使得MRFs表达上升，从而达到促进分化的作用；miR-1和miR-206有着高度相似的核苷酸序列，在骨骼肌的分化中也有着相似的作用。但是，关于MyomiRs在舌发育过程中，尤其是维甲酸诱导腭裂小鼠舌异常的发育过程中对于MRFs和Pax3/Pax7的相关调控作用，尚未见报道。 目的：通过检测维甲酸诱导小鼠胚胎腭裂模型中胎鼠舌体发育过程中相关MyomiRs、MRFs以及Pax3/Pax7的表达变化，来探究MyomiRs在舌肌分化过程中的调控作用，推测维甲酸致胎鼠腭裂伴发舌异常的可能机制，为腭裂机制的研究以及舌发育异常的机制提供相关理论依据。 方法：选取10周龄左右SPF级ICR孕鼠，在胚胎10天时按孕鼠体重100mg/kg灌胃维甲酸建立胎鼠腭裂动物模型。分别在E13.5、E14.5、E15.5和E18.5用颈部脱臼法处死孕鼠，在用DEPC水配置的PBS中取出并收集胚胎小鼠的舌体组织。利用异硫氰酸胍法（Trizol法）提取组织总RNA；用非特异性染料（SYBR GreenⅠ）实时定量PCR技术检测舌体中相关成肌因子和盒转录因子的表达；用特异性染料（TaqMan探针）实时定量PCR技术检测舌体中相关MyomiRs的表达。 结果： 1、小鼠胚胎发育过程中，miR-1和miR-206在小鼠胚胎舌体发育过程中的相对表达量均持续上升，且表达均在出生前，即E18.5达到最高峰。 2、RA诱导小鼠腭裂组的小鼠舌体中miR-1及miR-206的表达变化趋势与正常组相似，，但相对表达量在舌体发育各个阶段均低于正常组，miR-1的结果在E14.5和E15.5具有统计学意义（p0.01）；miR-206的结果在E13.5具有统计学意义（p0.05）。 3、胚胎发育过程中，正常组和RA诱导小鼠腭裂组的小鼠舌体中MyoD和Myf5的相对表达量都在E14.5达到峰值，随后下降。在E13.5和E14.5，腭裂组MyoD的表达均低于正常组，且在E14.5的结果有统计学意义（p0.05）；而在E15.5，腭裂组MyoD的表达却高于正常组，且结果有统计学意义（p0.01）。在E14.5和E15.5，腭裂组Myf5的相对表达量均低于正常组，且在E15.5的结果有统计学意义（p0.05）。 4、胚胎发育过程中，正常组和RA诱导小鼠腭裂组的小鼠舌体中Pax3的表达均在E14.5达到峰值，而Pax7的表达均在E15.5达到峰值。腭裂组舌体中Pax3的相对表达量在E14.5显著高于正常组（p0.05）；而腭裂组Pax7的相对表达量则在E13.5显著高于正常组（p0.01）。 结论：在舌肌分化过程中，肌相关microRNAs，即miR-1/miR-206与盒转录因子Pax3/Pax7及成肌调节因子Myf5/MyoD的表达趋势具有相关性。在维甲酸诱导的舌发育异常中，仍然存在这种相关性，而维甲酸可能通过下调miR-1/miR-206而靶向上调Pax3/Pax7，进而达到下调MyoD/Myf5表达，从而抑制维甲酸诱导腭裂胎鼠的舌肌分化，导致舌肌发育异常。
[Abstract]:Background: Fissure is one of the most common congenital malformations in the oral and maxillofacial region, which is a multi-gene susceptibility disease caused by the interaction of the gene with the environmental factors. In the earlier study of the research group, we found that retinoic acid (RA) induced the development of the mouse embryo and often accompanied the development of the tongue. In the day 14.5, the tongue body of the mouse was still filled with the oral cavity, so that both sides of the tongue were still on both sides of the tongue, and could not be transferred to the horizontal growth phase and could not be fused at the midline. In this period, not only the shape of the tongue body and the abnormal of the fiber of the tongue in the tongue body, but also the expression of the related myogenic regulating factor (MFRs) was different from that of the normal rat. The tongue muscle is the main body of the tongue tissue, and the tongue muscle belongs to the skeletal muscle. Therefore, a series of genes which play a role in regulating the development of the skeletal muscle play a certain role in the differentiation of the tongue muscle. The muscle-related microRNAs, that is, MyomiRs, is a kind of microRNAs enriched in the muscle, which play a certain regulatory role in the muscle process. The results showed that, in the course of differentiation, miR-206 was directly targeted to the expression of Pax7 and Pax7, and the expression of MFRs was inhibited by the expression of miR-206, and the expression of MFRs was increased after the expression of miR-206, so that the effect of promoting differentiation was achieved; and miR-1 and miR-206 had a highly similar nucleotide sequence, which also played a similar role in the differentiation of skeletal muscle. However, the related regulation of MFRs and Pax3/ Pax 7 was not reported in the course of the tongue development of MyomiRs, especially in the course of the development of the tongue abnormality induced by retinoic acid. Objective: To study the effect of retinoic acid on the expression of MyomiRs, MFRs and Pax 3/ Pax 7 in the development of fetal rat's tongue in mouse embryo-breaking model induced by retinoic acid. The mechanism of the mechanism of the tongue dehiscence and the mechanism of the abnormal tongue development provide the relevant theory. According to the method, a 10-week-old SPF-grade ICR pregnant rat was selected, and the fetal rat was established by intragastric administration of retinoic acid at the weight of 100 mg/ kg of the pregnant rat on 10 days of the embryo. The mice were sacrificed by the cervical dislocation method in E13.5, E14.5, E1.5 and E18.5, respectively, and the tongue of the embryonic mice was taken out and collected in PBS with DEPC water. Body tissue: The total RNA of tissue was extracted by the Trizol method; the expression of related myogenic factor and cassette transcription factor in the tongue was detected by real-time quantitative PCR with a non-specific dye (SYBR Green I); the related MyomiRs in the tongue were detected by real-time quantitative PCR with a specific dye (TaqMan probe). expression Results: During the development of mouse embryos, the relative expression of miR-1 and miR-206 in the development of the tongue body of the mouse increased continuously, and the expression of miR-1 and miR-206 were all before the birth, that is, E18. .5. The expression trend of miR-1 and miR-206 in the tongue of mice induced by RA was similar to that of normal group, but the relative expression of miR-1 was lower than that of normal group, and the results of miR-1 were statistically significant in E14.5 and E15.5. Significance (p0.01); the results of miR-206 were statistically significant at E13.5 Significance (p0.05).3. The relative expression of MyoD and Myf5 in the tongue of mice induced by normal and RA-induced mice was E14 in the process of embryo development. The expression of MyoD in E13.5 and E14.5 was lower than that of normal group, and the expression of MyoD in E13.5 and E14.5 was higher than that of normal group. The relative expression of Myf5 in E14.5 and E1.5 was lower than that of normal group, and in E15.5, the relative expression of Myf5 was lower than that of normal group (P 0.01). In the process of embryo development, the expression of Pax 3 in the tongue of mice induced by normal and RA-induced mice reached a peak at E14.5, while Pax 7 The expression of Pax 7 in the tongue was significantly higher in E14.5 than in the normal group (p0.05), while the relative expression of Pax 7 in the cleavage group was higher than that in the normal group (p0.05). Conclusion: In the process of tongue dedifferentiation, the muscle-related microRNAs, that is, miR-1/ miR-206 and the box transcription factor, Pax 3/ Pax 7 and myostatin Myf5, The expression of/ MyoD has a correlation. In the development of the tongue developed by the retinoic acid, there is still such a correlation, and the retinoic acid may target up-regulate the Pax 3/ Pax 7 by down-regulating the miR-1/ miR-206, thereby reducing the expression of MyoD/ Myf5, thereby inhibiting the retinoic acid-induced fracture of the tongue.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.2
本文编号：2501801
[Abstract]:Background: Fissure is one of the most common congenital malformations in the oral and maxillofacial region, which is a multi-gene susceptibility disease caused by the interaction of the gene with the environmental factors. In the earlier study of the research group, we found that retinoic acid (RA) induced the development of the mouse embryo and often accompanied the development of the tongue. In the day 14.5, the tongue body of the mouse was still filled with the oral cavity, so that both sides of the tongue were still on both sides of the tongue, and could not be transferred to the horizontal growth phase and could not be fused at the midline. In this period, not only the shape of the tongue body and the abnormal of the fiber of the tongue in the tongue body, but also the expression of the related myogenic regulating factor (MFRs) was different from that of the normal rat. The tongue muscle is the main body of the tongue tissue, and the tongue muscle belongs to the skeletal muscle. Therefore, a series of genes which play a role in regulating the development of the skeletal muscle play a certain role in the differentiation of the tongue muscle. The muscle-related microRNAs, that is, MyomiRs, is a kind of microRNAs enriched in the muscle, which play a certain regulatory role in the muscle process. The results showed that, in the course of differentiation, miR-206 was directly targeted to the expression of Pax7 and Pax7, and the expression of MFRs was inhibited by the expression of miR-206, and the expression of MFRs was increased after the expression of miR-206, so that the effect of promoting differentiation was achieved; and miR-1 and miR-206 had a highly similar nucleotide sequence, which also played a similar role in the differentiation of skeletal muscle. However, the related regulation of MFRs and Pax3/ Pax 7 was not reported in the course of the tongue development of MyomiRs, especially in the course of the development of the tongue abnormality induced by retinoic acid. Objective: To study the effect of retinoic acid on the expression of MyomiRs, MFRs and Pax 3/ Pax 7 in the development of fetal rat's tongue in mouse embryo-breaking model induced by retinoic acid. The mechanism of the mechanism of the tongue dehiscence and the mechanism of the abnormal tongue development provide the relevant theory. According to the method, a 10-week-old SPF-grade ICR pregnant rat was selected, and the fetal rat was established by intragastric administration of retinoic acid at the weight of 100 mg/ kg of the pregnant rat on 10 days of the embryo. The mice were sacrificed by the cervical dislocation method in E13.5, E14.5, E1.5 and E18.5, respectively, and the tongue of the embryonic mice was taken out and collected in PBS with DEPC water. Body tissue: The total RNA of tissue was extracted by the Trizol method; the expression of related myogenic factor and cassette transcription factor in the tongue was detected by real-time quantitative PCR with a non-specific dye (SYBR Green I); the related MyomiRs in the tongue were detected by real-time quantitative PCR with a specific dye (TaqMan probe). expression Results: During the development of mouse embryos, the relative expression of miR-1 and miR-206 in the development of the tongue body of the mouse increased continuously, and the expression of miR-1 and miR-206 were all before the birth, that is, E18. .5. The expression trend of miR-1 and miR-206 in the tongue of mice induced by RA was similar to that of normal group, but the relative expression of miR-1 was lower than that of normal group, and the results of miR-1 were statistically significant in E14.5 and E15.5. Significance (p0.01); the results of miR-206 were statistically significant at E13.5 Significance (p0.05).3. The relative expression of MyoD and Myf5 in the tongue of mice induced by normal and RA-induced mice was E14 in the process of embryo development. The expression of MyoD in E13.5 and E14.5 was lower than that of normal group, and the expression of MyoD in E13.5 and E14.5 was higher than that of normal group. The relative expression of Myf5 in E14.5 and E1.5 was lower than that of normal group, and in E15.5, the relative expression of Myf5 was lower than that of normal group (P 0.01). In the process of embryo development, the expression of Pax 3 in the tongue of mice induced by normal and RA-induced mice reached a peak at E14.5, while Pax 7 The expression of Pax 7 in the tongue was significantly higher in E14.5 than in the normal group (p0.05), while the relative expression of Pax 7 in the cleavage group was higher than that in the normal group (p0.05). Conclusion: In the process of tongue dedifferentiation, the muscle-related microRNAs, that is, miR-1/ miR-206 and the box transcription factor, Pax 3/ Pax 7 and myostatin Myf5, The expression of/ MyoD has a correlation. In the development of the tongue developed by the retinoic acid, there is still such a correlation, and the retinoic acid may target up-regulate the Pax 3/ Pax 7 by down-regulating the miR-1/ miR-206, thereby reducing the expression of MyoD/ Myf5, thereby inhibiting the retinoic acid-induced fracture of the tongue.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.2
【参考文献】
相关期刊论文 前2条
1 刘贤;吴靖芳;安峰;;舌发育异常与胎鼠地塞米松腭裂的关系[J];河北北方学院学报(医学版);2009年03期
2 Wen Luo,;Qinghua Nie;Xiquan Zhang;;MicroRNAs Involved in Skeletal Muscle Differentiation[J];遗传学报;2013年03期
本文编号：2501801
本文链接：https://www.wllwen.com/yixuelunwen/kouq/2501801.html
最近更新
教材专著
