肌相關(guān)microRNAs在維甲酸誘導(dǎo)舌發(fā)育異常中對(duì)舌肌分化調(diào)控的機(jī)制研究
發(fā)布時(shí)間：2019-06-18 21:22
【摘要】：背景：腭裂是口腔頜面部常見的先天畸形之一，是通過基因與環(huán)境因素交互作用造成的多基因易感性疾病。在本課題組前期研究中，我們發(fā)現(xiàn)維甲酸（retinoicacid, RA）誘導(dǎo)小鼠胚胎腭裂發(fā)生同時(shí)常伴有舌發(fā)育的畸形。發(fā)生腭裂的小鼠舌體在胚胎第14.5天并未下降，仍充滿口腔，使兩側(cè)腭突仍位于舌體的兩側(cè)，無法轉(zhuǎn)入水平生長(zhǎng)期，進(jìn)而無法在中線處融合。該時(shí)期腭裂胎鼠不僅舌體形態(tài)以及舌體中舌肌的纖維走形發(fā)生異常，而且其相關(guān)成肌調(diào)節(jié)因子（Myogenic RegulatoryFactors，MRFs）的表達(dá)量也異于正常的胎鼠。舌肌是舌組織的主體，而舌肌屬于骨骼肌，因此，對(duì)骨骼肌發(fā)生發(fā)育起到調(diào)控作用的一系列基因也在舌肌的分化過程中起到一定的作用。肌相關(guān)microRNAs，即MyomiRs，就是一類在肌肉中富集的microRNAs，它們對(duì)成肌過程起到一定的調(diào)控作用。研究表明：在分化過程中，miR-206直接靶向結(jié)合Pax7，Pax7抑制相關(guān)MRFs的表達(dá)，miR-206抑制Pax7之后使得MRFs表達(dá)上升，從而達(dá)到促進(jìn)分化的作用；miR-1和miR-206有著高度相似的核苷酸序列，在骨骼肌的分化中也有著相似的作用。但是，關(guān)于MyomiRs在舌發(fā)育過程中，尤其是維甲酸誘導(dǎo)腭裂小鼠舌異常的發(fā)育過程中對(duì)于MRFs和Pax3/Pax7的相關(guān)調(diào)控作用，尚未見報(bào)道。 目的：通過檢測(cè)維甲酸誘導(dǎo)小鼠胚胎腭裂模型中胎鼠舌體發(fā)育過程中相關(guān)MyomiRs、MRFs以及Pax3/Pax7的表達(dá)變化，來探究MyomiRs在舌肌分化過程中的調(diào)控作用，推測(cè)維甲酸致胎鼠腭裂伴發(fā)舌異常的可能機(jī)制，為腭裂機(jī)制的研究以及舌發(fā)育異常的機(jī)制提供相關(guān)理論依據(jù)。 方法：選取10周齡左右SPF級(jí)ICR孕鼠，在胚胎10天時(shí)按孕鼠體重100mg/kg灌胃維甲酸建立胎鼠腭裂動(dòng)物模型。分別在E13.5、E14.5、E15.5和E18.5用頸部脫臼法處死孕鼠，在用DEPC水配置的PBS中取出并收集胚胎小鼠的舌體組織。利用異硫氰酸胍法（Trizol法）提取組織總RNA；用非特異性染料（SYBR GreenⅠ）實(shí)時(shí)定量PCR技術(shù)檢測(cè)舌體中相關(guān)成肌因子和盒轉(zhuǎn)錄因子的表達(dá)；用特異性染料（TaqMan探針）實(shí)時(shí)定量PCR技術(shù)檢測(cè)舌體中相關(guān)MyomiRs的表達(dá)。 結(jié)果： 1、小鼠胚胎發(fā)育過程中，miR-1和miR-206在小鼠胚胎舌體發(fā)育過程中的相對(duì)表達(dá)量均持續(xù)上升，且表達(dá)均在出生前，即E18.5達(dá)到最高峰。 2、RA誘導(dǎo)小鼠腭裂組的小鼠舌體中miR-1及miR-206的表達(dá)變化趨勢(shì)與正常組相似，，但相對(duì)表達(dá)量在舌體發(fā)育各個(gè)階段均低于正常組，miR-1的結(jié)果在E14.5和E15.5具有統(tǒng)計(jì)學(xué)意義（p0.01）；miR-206的結(jié)果在E13.5具有統(tǒng)計(jì)學(xué)意義（p0.05）。 3、胚胎發(fā)育過程中，正常組和RA誘導(dǎo)小鼠腭裂組的小鼠舌體中MyoD和Myf5的相對(duì)表達(dá)量都在E14.5達(dá)到峰值，隨后下降。在E13.5和E14.5，腭裂組MyoD的表達(dá)均低于正常組，且在E14.5的結(jié)果有統(tǒng)計(jì)學(xué)意義（p0.05）；而在E15.5，腭裂組MyoD的表達(dá)卻高于正常組，且結(jié)果有統(tǒng)計(jì)學(xué)意義（p0.01）。在E14.5和E15.5，腭裂組Myf5的相對(duì)表達(dá)量均低于正常組，且在E15.5的結(jié)果有統(tǒng)計(jì)學(xué)意義（p0.05）。 4、胚胎發(fā)育過程中，正常組和RA誘導(dǎo)小鼠腭裂組的小鼠舌體中Pax3的表達(dá)均在E14.5達(dá)到峰值，而Pax7的表達(dá)均在E15.5達(dá)到峰值。腭裂組舌體中Pax3的相對(duì)表達(dá)量在E14.5顯著高于正常組（p0.05）；而腭裂組Pax7的相對(duì)表達(dá)量則在E13.5顯著高于正常組（p0.01）。 結(jié)論：在舌肌分化過程中，肌相關(guān)microRNAs，即miR-1/miR-206與盒轉(zhuǎn)錄因子Pax3/Pax7及成肌調(diào)節(jié)因子Myf5/MyoD的表達(dá)趨勢(shì)具有相關(guān)性。在維甲酸誘導(dǎo)的舌發(fā)育異常中，仍然存在這種相關(guān)性，而維甲酸可能通過下調(diào)miR-1/miR-206而靶向上調(diào)Pax3/Pax7，進(jìn)而達(dá)到下調(diào)MyoD/Myf5表達(dá)，從而抑制維甲酸誘導(dǎo)腭裂胎鼠的舌肌分化，導(dǎo)致舌肌發(fā)育異常。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R782.2
本文編號(hào)：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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R782.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 劉賢;吳靖芳;安峰;;舌發(fā)育異常與胎鼠地塞米松腭裂的關(guān)系[J];河北北方學(xué)院學(xué)報(bào)(醫(yī)學(xué)版);2009年03期
2 Wen Luo,;Qinghua Nie;Xiquan Zhang;;MicroRNAs Involved in Skeletal Muscle Differentiation[J];遺傳學(xué)報(bào);2013年03期
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