利用维甲酸诱导及Wnt5a基因敲除两种颌面发育畸形模型研究腭裂及舌发育异常的分子调控机制及相关性

发布时间：2017-12-31 17:06

本文关键词：利用维甲酸诱导及Wnt5a基因敲除两种颌面发育畸形模型研究腭裂及舌发育异常的分子调控机制及相关性　出处：《大连医科大学》2012年博士论文　论文类型：学位论文

【摘要】：以唇腭裂为代表的颌面部发育畸形发病率位于先天性发育畸形的首位。作为多基因易感性疾病，腭裂是由多重环境因素与多个基因交互作用所导致，因此病因复杂，机制不明。一直以来，大多数的腭裂病因学研究者均把目光集中在腭突自身的发育异常，然而，腭裂发生的原因，，一是由于腭突内部的缺陷，二是继发于颌面部其他组织的缺陷，如临床上颌发育不足和下颌后缩，导致舌位置异常，从而物理性的阻碍腭突上抬而导致腭裂。但目前颌面部其他组织器官，尤其是作为与腭组织结构、生理功能以及生长发育密切相关的舌发育分子调控机制、及其发育异常与腭裂发生的关联均少见报道。维甲酸（Retinoic acid，RA）是维生素A的衍生物，作为化妆品添加剂以及治疗皮肤病、癌症等疾病的药物，成为较常见的致畸的环境因素。同时维甲酸通路与WNT及SHH等信号分子通路相互作用共同参与颌面部发育的调控。本研究利用维甲酸诱导及Wnt5a基因敲除两种颌面发育畸形动物模型，通过体内和体外实验，探究腭裂以及舌发育异常的分子调控机制以及二者的相关性。以期加深对舌发育的分子机制的了解，并为腭裂及舌发育异常的预防以及临床治疗提供可能的科学依据。目的：利用维甲酸诱导及Wnt5a基因敲除小鼠两种颌面部发育畸形动物模型，研究腭裂以及舌发育异常分子调控机制以及二者的相关性。方法：建立和应用维甲酸诱导及Wnt5a基因敲除两种小鼠颌面部发育畸形模型，体视显微镜及组织染色观察表型的组织形态学变化，BrdU掺入法检测细胞增殖，TUNEL染色方法检测细胞凋亡，Masson染色检测舌肌纤维化，基因芯片筛选腭裂和舌发育异常的差异基因，实时定量PCR验证基因芯片结果，免疫组织化学实验进一步探查WNT信号分子通路GSK3β蛋白以及成肌调节因子家族成员Myf5和MyoD在舌肌中的表达变化。利用Trowell培养系统和去除舌组织的体外器官培养进行腭突的直接接触融合实验，探讨舌与腭发育相关性。应用稳定转染Wnt5a的C2C12细胞系体外检测Wnt5a对骨骼肌发育的影响，CCK-8法检测C2C12细胞增殖水平及血清饥饿法诱导凋亡后细胞的活性，流式细胞术Annexin V-FITC/PI双染检测细胞凋亡水平，诱导C2C12细胞肌向分化后，Real-Time PCR检测成肌调节因子成员Myf5、MyoG和Myogenin表达变化，免疫细胞化学法检测骨骼肌特异标志物Myosin的表达，以探讨Wnt5a对肌向分化的影响。结果：1、利用维甲酸诱导腭裂模型研究颌、舌与腭的发育（1）腭突组织基因芯片研究分析提示差异基因主要集中在转录调节、生长调节、细胞运动以及细胞骨架等功能，且与WNT和SHH通路相关，尤其是GSK3β和β-TrCP等既参与WNT通路又与SHH信号通路，与腭的发育畸形相关。实时定量PCR技术证明GSK3β及β-TrCP等表达下调，FZD3受体表达无明显变化。免疫组织化学结果显示GSK3β蛋白在E14腭突快速增殖期的腭突上皮和间充质中均有表达，在E15.5腭裂组上颌骨发育明显延迟，GSK3β表达在大量未分化的间充质细胞中。在E14-14.5，腭突开始上抬至发生接触融合的关键时期，实验组腭突仍垂直于舌的两侧，与对照组相比在麦克尔软骨边缘有多个TUNEL阳性凋亡细胞。（2）在E13.5去除舌组织并利用Trowell系统进行腭器官体外培养3天后，实验组以及对照组双侧腭突都发生了融合，双侧腭突的间充质相互贯通。在E14-16.5侧腭突发育全过程中，腭裂组与对照组颏舌肌细胞核数量无明显差异，但腭裂组肌束横截面的总面积明显小于对照组，差异有统计学意义（P0.05），舌肌细胞增殖、凋亡水平无明显差异，未见舌异常纤维化。舌组织基因芯片分析提示，舌发育畸形与维甲酸相关的信号网络相关，DTNA，CAMK2D等表达上调2倍以上。 2、Wnt5a基因对舌及骨骼肌发育的影响（1）Wnt5a在正常舌发育中的表达呈动态变化，在E13.5舌组织中呈高表达，在E15.5舌组织中的表达明显下调。而且在E15.5，即舌肌分化成熟期，维甲酸诱导腭裂组Wnt5a的表达高于对照组，差异有统计学意义（P0.05）。（2）Wnt5a基因敲除小鼠舌体以及颏舌肌中BrdU阳性率与正常对照组相比无显著差异，对照组颏舌肌的舌间充质出现区域性密集，实验组则不明显。成肌调节因子成员Myf5和MyoD蛋白在舌内外肌群中的表达水平均下降，差异有统计学意义（P0.05）。（3）应用Wnt5a稳定转染C2C12小鼠成肌细胞系，Wnt5a基因上调后第24h、48h和72h，C2C12细胞增殖能力无明显变化（P0.05），经过血清饥饿24h后细胞均发生凋亡，但Wnt5a上调对细胞凋亡和活性无明显影响（P0.05），细胞迁移也无明显变化（P0.05）。在体外成功诱导C2C12细胞系肌肉方向分化后，Wnt5a基因上调可引起成肌调节因子的变化，Myf5在分化第2，3天表达下降（P0.05），MRF4在分化第2天表达明显下降（P0.05），Myog的表达在分化第2天（P0.05）及第4天（P0.01）均下调。细胞免疫化学显示两组细胞都有Myosin的表达，均可形成多核肌管。结论：过量维甲酸可导致以腭裂为代表的口腔颌面部多器官发育异常，但不影响腭突中嵴上皮细胞接触后的融合。维甲酸诱导腭裂小鼠伴发舌发育延迟、上颌骨发育迟缓以及下颌麦克尔软骨异常凋亡，提示了过量维甲酸可干扰颌面系统协同发育。维甲酸诱导的腭发育异常与WNT和SHH信号通路相关，维甲酸相关信号网络参与舌的发育异常。体内敲除及体外转染上调Wnt5a基因均可延迟骨骼肌的肌向分化，提示了Wnt5a基因具有多向调控作用。
[Abstract]:With cleft lip and palate as the representative of the maxillofacial deformity in the incidence of congenital malformation of the first development. As the disease susceptibility gene, cleft palate is caused by multiple factors and multiple gene interactions, thus causes complex, unknown mechanisms. Since most of the etiology of cleft palate who focus their abnormal the development of the palatal cleft palate reason, however, is due to a defect of palate internal defects, two is secondary to other organizations such as clinical maxillofacial, maxillary hypoplasia and mandibular retrusion, leading to abnormal tongue position, and physical barriers caused cleft palate shelves. But the other the organization of maxillofacial organs, especially as with palatal tissue structure, physiological function and growth is closely related to the molecular mechanism of tongue development, and abnormalities associated with cleft palate were rarely reported.
Retinoic acid (Retinoic acid RA) is a derivative of vitamin A, as cosmetic additives and drug treatment of skin diseases, cancer and other diseases, environmental factors become more common teratogenic. At the same time with retinoic acid pathway WNT and SHH signal pathways involved in the regulation of interaction of maxillofacial development. This study used retinoic acid induction of Wnt5a gene knockout and two kinds of maxillofacial malformation animal model, through in vivo and in vitro experiments, correlation between the molecular mechanism of abnormal development of cleft palate and tongue research as well as the two. In order to deepen our understanding of the molecular mechanism of the development of the understanding of the tongue, and may provide scientific basis for the prevention of abnormal development of cleft palate and tongue and clinical treatment.
Objective: To study the molecular mechanism of cleft palate and tongue dysplasia and the correlation between the two kinds of animal models by using retinoic acid induced and Wnt5a knockout mice to develop two kinds of animal models of maxillofacial deformities.
Methods: the Wnt5a and the establishment and application of retinoic acid inducible gene knockout mice two maxillofacial malformation model to observe histomorphological changes the phenotype of stereoscopic microscope and staining, BrdU incorporation assay to detect cell proliferation, cell apoptosis TUNEL staining detection method, Masson staining of tongue muscle fibrosis, gene microarray screening differences of cleft palate and tongue abnormal development of the real-time PCR to validate the results of gene chip, immunohistochemistry experiment to further explore the WNT signal pathways of GSK3 beta protein and expression of myogenic regulatory factor family members of Myf5 and MyoD in tongue muscle. Using Trowell culture system and the removal of tongue tissue organ culture in vitro were in direct contact with palatal fusion experiments. To investigate the correlation between tongue and palate. Effect of C2C12 cell line in vitro Wnt5a stably transfected with Wnt5a on skeletal muscle development, CCK-8 was detected by C2 The activity of cell apoptosis induced by C12 cell proliferation and the level of serum starvation, flow cytometry Annexin V-FITC/PI staining to detect cell apoptosis, C2C12 cells induced by muscle differentiation, Real-Time PCR detection of myogenic regulatory factors Myf5, expression of MyoG and Myogenin, the immune cell chemical detection of skeletal muscle specific marker expression Myosin, to discuss the effects of Wnt5a on myogenic differentiation.
Results: 1, using the retinoic acid induced cleft palate model to study the development of the jaw, the tongue and the palate.
(1) study of gene chip analysis showed that the differences of the palatal tissue mainly in gene transcription regulation, growth regulation, cell motility and cytoskeleton function, and the WNT and SHH pathway, especially GSK3 beta and beta -TrCP is involved in WNT pathway and SHH pathway, and palatine malformation related in real time. Quantitative PCR proved that GSK3 beta and beta -TrCP expression, FZD3 expression had no obvious change. The immunohistochemistry results showed that GSK3 protein were expressed in mesenchymal E14 in palatal fast proliferating of the palatal epithelium and in the E15.5 group, cleft palate maxillary growth was significantly delayed, the expression of GSK3 in a large number of undifferentiated mesenchymal cells. In E14-14.5, palatal begins to rise to the occurrence of critical period of contact fusion, on both sides of the experimental group is perpendicular to the palatal tongue, compared with the control group there were more TUNEL positive apoptotic cells in Meckel's cartilage edge.
(2) E13.5 in the removal of tongue tissue and the use of Trowell system were cultured palatal organ in vitro after 3 days, the experimental group and the control group have undergone bilateral palatal fusion, bilateral palatal mesenchymal coalescence. On the E14-16.5 side in the whole process of the development of palate, cleft palate group and control group the number of genioglossus muscle cells the obvious difference, but the total area of the cross section of the cleft palate group was significantly lower than that of control group, the difference was statistically significant (P0.05), proliferation of tongue muscle cells, no significant difference between the levels of apoptosis, no abnormal tongue tongue tissue fibrosis. Gene chip analysis showed that tongue malformation associated with retinoic acid signaling network, DTNA, CAMK2D etc. expression of more than 2 times.
2, the effect of Wnt5a gene on the development of tongue and skeletal muscle
(1) the expression of Wnt5a in normal tongue in development is dynamic, high expression of E13.5 in tongue tissues, expression of E15.5 in tongue tissue was significantly reduced. And in E15.5, namely the tongue muscle differentiation in mature stage, the expression of Wnt5a in retinoic acid induced cleft palate group than in the control group, the difference was statistically significant (P0.05).
(2) Wnt5a gene knockout mice tongue and the positive rate of BrdU in genioglossus muscle compared with normal control group had no significant difference between the control group and the genioglossus tongue mesenchymal regional intensive, the experimental group was not obvious. The expression of myogenic regulatory factors Myf5 and MyoD protein in tongue and muscles in the fall, the difference was statistically significant (P0.05).
(3) the application of Wnt5a stable transfection of C2C12 mouse myoblast cell line, Wnt5a gene up-regulated after 24h, 48h and 72h, C2C12 cell proliferation had no significant change (P0.05), after serum starvation after 24h cells underwent apoptosis, but the upregulation of Wnt5a has no obvious effect on cell apoptosis and activity (P0.05), and cell migration no significant change (P0.05). The success of induced differentiation of C2C12 cells in vitro after muscle, up-regulated Wnt5a gene can induce changes of myogenic regulatory factor, decrease the expression of Myf5 in differentiation of day 2,3 (P0.05), the expression of MRF4 decreased significantly in second days of differentiation (P0.05), the expression of Myog in differentiation (second days P0.05) and 4 days (P0.01) were down regulated. Immunocytochemistry showed that the expression of the two groups of cells is Myosin, can form multinucleated myotubes.
Conclusion: excess retinoic acid can lead to cleft palate as the representative of the oral and maxillofacial multiple organ abnormalities, but does not affect the fusion of epithelial cells in the palatal ridge after contact with the tongue. Delayed development of cleft palate in mice induced by retinoic acid, maxillary growth retardation and mandibular cartilage abnormalities Meckel apoptosis, suggesting that excess retinoic acid can be coordinated development interference and system.
Retinoic acid induced velopharyngeal dysplasia is related to WNT and SHH signaling pathway. Retinoic acid related signal network is involved in the development of tongue. In vivo knockout and in vitro transfection of Wnt5a gene can delay skeletal muscle myogenic differentiation, suggesting that Wnt5a gene has multiple regulatory effects.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R-332;R440;R782.22

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