Cleft palate formation after palatal fusion occurs due to the rupture of epithelial basement membranes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and hydronephrosis in the mouse embryo. Cleft palate occurs due to failure in palatal grow, but the underlying mechanisms are unclear. We investigated the mechanisms of cleft palate development in TCDD-exposed mouse embryos. We administered olive oil (control group) or TCDD diluted in olive oil (40 μg/kg) via gastric tubes to pregnant mice on gestational day (GD) 12. Embryos of control and TCDD-exposed groups were removed from pregnant mice on GD 14 and GD 15, respectively. One mouse embryo from the control group had anteroposterior palatal fusion. Palatal fusion was observed in three TCDD-exposed mouse embryos. Palates of TCDD-exposed mice fused from the interior to the middle of the palates, while the palates were separated in the posterior region. The middle of the embryonic palatal shelves in TCDD-exposed animals was narrow and split at the fusional position. At this position, palatal and blood cells were dispersed from the palatal tissue and the epithelium was split, with a discontinuous basement membrane. The results suggest that decreased intercellular adhesion or insufficient tissue strength of the palatal shelves may be involved in the development of cleft palate following palatal fusion.     

腭裂相关基因mcpr1在小鼠腭突发育及腭裂发生中的表达研究

目的检测mcpr1基因的蛋白在正常腭突及小鼠腭裂模型腭突发育中的时空表达模式。方法实验组管饲含全反式维甲酸的植物油诱导建立小鼠腭裂模型,对照组小鼠管饲植物油。2组取胎龄12、13、14、16 d的胎鼠各3只,包埋切片,HE染色观察2组胎鼠腭部发育的形态学变化,免疫组织化学方法观察MCPR1蛋白在2组胎鼠腭突中的表达差异。结果成功建立小鼠腭裂模型。切片HE染色观察发现实验组双侧腭突体积较对照组小,两侧腭突未接触;而对照组腭突融合,腭骨形成。免疫组织化学检查结果表明对照组小鼠胎龄12 d时MCPR1蛋白在腭突上皮细胞呈强阳性表达;而实验组小鼠胎龄12 d时腭突间充质细胞内MCPR1蛋白的表达较强,实验组胎龄16 d小鼠仅在上皮细胞附近的间充质细胞中有阳性表达。同一时间点比较,MCPR1蛋白的表达在实验组均强于对照组（P〈0.05）。结论 MCPR1蛋白的表达变化随腭部发育存在时空表达差异。     

叶酸对小鼠腭突间充质细胞增殖的影响

目的:从细胞水平对维A酸致小鼠腭裂畸形作用和叶酸是否有拮抗维A酸的致畸作用及其机制进行研究。方法:给予孕鼠过量维A酸,诱导胚胎腭裂模型,不完全消化法提取孕期第14天、17天(GD14、17)的胚胎腭突间充质细胞(EPM cells)进行培养并鉴定细胞;采用四甲基偶氮唑盐(MTT)法检测加入不同浓度叶酸与未加入叶酸细胞增殖的情况。采用SPSS16.0软件包对数据进行统计学处理。结果:①在GD10、GD12管饲孕鼠50 mg/kg维A酸,可致胎鼠腭裂畸形;②不完全消化法提纯EPM细胞,纯度达到98%;③维A酸导致GD14的EPM细胞增殖受到抑制,20 μg/mL、40 μg/mL浓度叶酸可拮抗这种抑制作用。结论:①过量维A酸可致小鼠腭裂畸形,腭突间充质细胞增殖受抑制是其致畸机制之一;②叶酸可拮抗维A酸对小鼠GD14 EPM细胞增殖的抑制作用。     

腭发育不同时期维甲酸对腭突细胞增殖和凋亡的影响

目的研究维甲酸对小鼠腭突融合期细胞增殖和细胞凋亡的影响。方法在SPF级C57BL/6J近交系母鼠妊娠10 d和12 d给予维甲酸（RA）建立小鼠腭裂模型,利用BrdU免疫组化方法和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测胚胎15 d（即腭突融合期）小鼠腭突中细胞增殖及细胞凋亡的表达和分布。结果10 d给药组腭胚间充质细胞及腭中嵴上皮细胞中BrdU阳性细胞率和TUNEL阳性细胞率均低于对照组,12 d给药组和对照组BrdU阳性细胞率和TUNEL阳性细胞差异无统计学意义。结论维甲酸作用于腭发育的不同时期对腭突细胞增殖及凋亡水平有不同的影响,作用于腭突发生前期可引起腭间充质细胞增殖抑制、凋亡过度而发生腭裂,作用于腭突快速生长期可能影响腭中嵴上皮细胞的上皮间充质转化和迁移等其他转归形式。     

维甲酸诱导小鼠腭裂发病机制的实验研究

目的 观察维甲酸（retinoic acid,RA)诱导胚鼠腭突发育的形态学变化，探讨胚鼠腭部转化生长因子（transforming growth factor,TGF)β1、TGFβ3、表皮生长因子（epidermal growth factor,EGF)及BCL2的表达在RA诱导腭裂发病机制中的意义。方法 采用显微镜观察胚腭的发育过程，原位杂交和免疫组化检测TGFβ1、TGFβ3、EGF及BCL2基因在胚腭中的表达。结果 RA引起双侧腭突的形成，使双侧腭突不能接触或接触后不能融合形成中嵴上皮带，以及可调控TGFβ1、TGFβ3、EGF及BCL2在鼠胚腭部的表达。结论 RA抑制中嵴上皮细胞（MEPM）增殖，引起双侧腭突的形成，诱导MEE细胞殿堂分化，使双侧腭突不能接触融合，而最终导致腭裂形成及TGFβ1、TGFβ3、EGF在鼠胚腭部表达的变化与RA诱导腭裂的形成密切相关。     

MEE细胞在鼠腭发育及腭裂形成过程中的分化与转归

目的 探讨MEE细胞在腭发育过程中的分化和转归特点及其与腭裂发生的关系。方法 以磷酸地塞米松诱导胎鼠腭裂畸形,并分别在光镜、透射电镜和扫描电镜下,观察实验组、正常组、对照组标本中MEE细胞各时期的分化特征。结果 腭突在垂直生长期,MEE细胞为多层不规则排列。腭突定向运动和水平生长早期,MEE细胞呈典型的两层排列,基底膜完整。腭突水平生长晚期,各组间MEE细胞分化开始出现差异。正常组与对照组MEE细胞表层开始凋亡脱落;腭突接触融合期,两侧腭突基底层MEE细胞发生接触,之后被分割成上皮岛于间充质中,基底膜断裂、消失,腭突融合。实验组腭突MEE细胞未出现上述演化过程,形成腭裂。结论 在腭的发育过程,MEE细胞表层转归为“细胞凋亡”,基底层转归为“上皮一间充质转化”。MEE细胞的异常转归将导致腭裂发生。     

C57BL/6N系小鼠腭裂模型的建立及扫描电镜观察

观察腭裂形成过程及腭突正常发育中形态发生、组织学变化。方法１６只妊娠１０天的Ｃ５７ＢＬ／６Ｎ系小鼠随机分为实验组、对照组、于ＧＤ１３＾１４,（１３ｄ１４ｈ略写为３＾１４以下类同）,ＧＤ１３＾３２,ＧＤ１４＾８,ＧＤ１４＾２２,ＧＤ１５＾８,ＧＤ１５＾２２,ＧＤ１６＾８剖腹取鼠头部标本分别做扫描电镜及ＨＥ染色。     

The mechanism of palatal clefting in the Col11a1 mutant mouse.

The occurrence of cleft palate in mutant mice offers an opportunity to understand the possible role of specific genes in palatogenesis. Here, cleft palate in mice carrying the chondrodysplasia (cho) defect, which consists of an autosomal-recessive mutation in the collagen gene Col11a1, was investigated. The proposed cause of cleft palate in cho homozygous mice is failure of the palatal shelves to adhere and make contact due to mandibular growth abnormalities. Another cause of cleft palate that has recently been demonstrated in other animal models is failure of the midline epithelial seam forming between the shelves to undergo epithelial-mesenchymal transformation (EMT). The present strategy to test the likelihood of this second possibility was to culture the unfused cho/cho palatal shelves at different stages of development to see if they were capable of adhering and undergoing EMT in vitro. By using carboxydichlorofluorescein succinimidyl ester to trace the fate of the medial-edge epithelium (MEE), it was shown that cho/cho palates have full potential for MEE adherence and EMT up to embryonic day 17.5/18.5, when epithelia keratinize before birth, preventing the adherence of both the normal and homozygous palatal shelves. Thus, the major effect of the mutant collagen gene on the palate is likely to be via mandibular growth disruption. The possibility that unfused palatal shelves in other clinical syndromes can adhere and undergo EMT if brought into contact at appropriate times before birth has important therapeutic implications.     

Deficient cell proliferation in palatal shelf mesenchyme of CL/Fr mouse embryos

How secondary palate formation is affected in the cleft lip genotype remains poorly understood. The purpose of this study was to analyze regional patterns of cell proliferation in CL/Fr mouse embryos with or without cleft lip. Pairs of palatal shelves were dissected at E13.5 from CL/Fr normal embryos (CL/Fr-N), CL/Fr embryos with bilateral cleft lip (CL/Fr-BCL), and a control strain of C57BL embryos (C57BL). The explants were examined histologically after 48 hrs of organ culture. Cell kinetics for proliferation in the palatal shelves was examined at E13.5 by the bromodeoxyuridine method in vivo. The CL/Fr-BCL palates fused as well as the CL/Fr-N palates in vitro. There were inter-group differences in the absolute number of BrdU-positive cells and the ratio of positive/(positive+negative) cells in the palate's mesenchyme (C57BL > CL/Fr-N > CL/Fr-BCL) and epithelium (C57BL > CL/Fr-N = CL/Fr-BCL). These findings indicate that a cleft palate follows reduced cell proliferation of secondary palatal mesenchyme in CL/Fr mice.     

地塞米松对体外培养A系小鼠腭突腭中嵴上皮融合的影响

目的　研究在腭间充质存在的情况下,地塞米松对腭中嵴上皮细胞分化、转归的影响,以探讨地塞米松引 起腭裂畸形的发病机制。方法　A系小鼠妊娠14 d 8 h在体视显微镜下,解剖分离出A系小鼠胚胎腭突,应用半浸 静式腭突体外培养方法,通过光学显微镜和透射电镜观察在腭突接触融合时,地塞米松对腭中嵴上皮细胞的影响。 结果　地塞米松促进了腭中嵴上皮分化成复层鳞状上皮,加速了腭间充质分化过程,影响了腭突的正常发育,阻碍 腭突融合。结论　地塞米松可能通过加速腭间充质分化和抑制腭中嵴上皮正常转归的双重作用,阻碍腭的正常发 育过程,导致腭裂畸形。     

Association between palatal morphogenesis and Pax9 expression pattern in CL/Fr embryos with clefting during palatal development

The CL/Fr mouse strain develops cleft lip and palate (CLP) spontaneously. In this study, Pax9 mRNA expression was investigated in the palatal shelves during palatal morphogenesis to assess the correlation between secondary palatal morphogenesis and Pax9 expression of CL/Fr embryos with spontaneous cleft lip and palate. The expression of Pax9 mRNA was characterised using whole mount in situ hybridisation with a digoxygenin-labelled probe. In the control strain of C57BL/6 and CL/Fr normal embryos, Pax9 was expressed in the palate, especially along the medial edge (ME), on embryonic day 13.5 (E13.5) and E14.5 when the palatal shelves grew vertically down the side of the tongue and subsequently elevated to a horizontal position, and was down regulated on E15.5 when the palatal shelves met and began fusing. In the cleft embryo, Pax9 was expressed in the ME region but was not down regulated on E15.5. Furthermore, whole mount in situ hybridisation was performed after organ culture, using CL/Fr-N and CL/Fr-BCL palatal shelves dissected and approximated by pairs on E13.5. This showed that Pax9 was still expressed in the ME region in separated palatal shelves of CL/Fr-N and CL/Fr-BCL embryos, while Pax9 expression was down regulated in paired palatal shelves. These expression patterns of Pax9 in normal and cleft embryos during palatal fusion indicate that Pax9 expression is altered in spontaneous cleft lip and palate, and concludes that there is a direct correlation between Pax9 expression and palatal fusion.     

地塞米松诱导小鼠腭裂及E-钙黏素基因表达的研究

目的 建立地塞米松（DEX）诱发 C57BL/6J小鼠的腭裂模型,并在腭发育期间检测E-钙黏素基因的表达,探讨DEX诱发腭裂与E-钙黏素基因的相关性。方法 将孕鼠随机分为实验组和对照组,在小鼠E10.0—E12.0,连续3 d按体重分别给予孕鼠注射DEX（实验组）和生理盐水（对照组）,于E17.5在体视显微镜下检测各组腭裂的发生率;分别在E13.5、E14.5、E15.5、E17.5取胎鼠腭部组织行苏木精-伊红（HE）染色和免疫组织化学染色,观察腭部形态及E-钙黏素的表达情况;在E14.0、E14.5、E15.5时采用实时荧光定量聚合酶链反应检测2组腭突中E-钙黏素以及β-钙黏素 mRNA的表达水平。结果 DEX组腭裂发生率为43.59%（17/39）,对照组为3.03%（1/33）。DEX作用后,腭突体积明显缩小,上皮不能接触,E-钙黏素阳性表达于腭突间充质中。在E14.0、E14.5及E15.5,与对照组相比,实验组腭突E-钙黏素及β-钙黏素的表达均升高（P<0.05）。结论 DEX处理后,E-钙黏素在腭突间充质中异位表达,其基因表达上调,与其相结合的β-钙黏素表达量也增多,影响了间充质的增殖从而形成短小腭突导致腭裂。     

成人腭裂患者腭盖表面积和倾斜度的比较研究

目的:观察腭裂患者上颌骨是否具有正常的生长发育潜能.方法:应用三维CT扫描成像系统,对比分析正常(牙合)成人、单侧完全性唇腭裂均已手术修补组以及腭裂未手术修补组成人患者的腭盖表面积和倾斜度.结果:腭裂未手术组的腭盖倾斜度显著高于正常对照组,腭盖向颅侧旋转,位置更加垂直;无论腭裂手术修补与否,腭裂患者的腭盖面积均显著小于正常对照组.结论:单侧完全性腭裂患者存在内在性的组织发育不足.     

地塞米松对腭胚突上皮细胞PAR复合体和细胞极性的影响

目的 探讨地塞米松（DEX）是否可以影响腭中嵴上皮细胞（MES）PAR极性复合体基因的表达,并进一步扰乱其细胞极性而影响腭融合。方法 将孕鼠随机分为对照组和DEX组,DEX组按6 mg·kg^-1腹腔注射地塞米松磷酸钠注射液,对照组注射0.9%氯化钠0.1 mL。在E13.5、E14.0、E14.5、E15.5、E17.5断颈处死孕鼠获取腭胚突,观察腭裂的发生情况,并通过苏木精-伊红染色、扫描电子显微镜观察腭上皮的形态改变,通过免疫荧光染色、蛋白质印迹及实时荧光定量聚合酶链式反应检测PAR3、PAR6、aPKC基因和蛋白的表达。结果 DEX组腭裂发生率为46.15%,对照组腭裂发生率为3.92%,DEX组的腭裂发生率高于对照组（χ²=24.335,P=0.00）。与对照组相比,DEX组腭胚突发育延迟且短小,腭中嵴上皮为非极性排列,只由单层的上皮细胞组成,腭胚突表面平坦,球状结构减少;PAR3和PAR6蛋白仅在腭上皮中表达,aPKC则表达于腭上皮和腭间充质中;PAR3、PAR6及aPKC基因的表达均减少。DEX在蛋白和基因水平下调PAR3、PAR6、aPKC的表达。结论 DEX可以导致腭胚突的生长发育延迟,并造成PAR极性复合体在蛋白和基因水平的表达下降,从而使MES极性丧失导致腭裂。     

四氯二苯对二恶英和地塞米松诱导小鼠腭裂及转化生长因子-β3和受体活化样激酶5的表达

目的建立四氯二苯对二恶英（TCDD）和地塞米松（DEX）联合诱导C57BL/6J小鼠腭裂模型,并在腭发育关键时期检测转化生长因子-β3（TGF-β3）和受体活化样激酶5（Alk5）基因的表达,探讨TCDD和DEX联合诱导胎鼠腭裂与TGF-β3和Alk5的相关性。方法在小鼠GD10 ～GD12,实验组小鼠连续3 d胃饲TCDD和腹腔注射DEX,空白对照组不做处理,于GD17.5体视显微镜下检测各组腭裂发生率,并于GD13.5、GD14.5、GD15.5 分别剪取胎鼠腭突提取RNA,采用实时荧光定量聚合酶链反应检测TGF-β3和Alk5基因表达。结果采用TCDD和DEX联合致畸,可诱导C57BL/6J胎鼠形成100%腭裂,建立了一种稳定适合分子生物学研究的腭裂动物模型。GD13.5时TGF-β3和Alk5基因表达水平在实验组与空白对照组之间差异均无统计学意义（P>0.05）,在GD14.5、GD15.5实验组TGF-β3表达均降低（P<0.05）,而Alk5表达均升高（P<0.05）。结论TCDD和DEX联合作用可诱导C57BL/6J胎鼠形成稳定腭裂,在腭融合关键时期诱导TGF-β3表达下降,Alk5表达升高,与腭裂的发生具有一定的相关性。     

Alteration in the expression of bone morphogenetic protein-2,3,4,5 mRNA during pathogenesis of cleft palate in BALB/c mice

To identify the function of these bone morphogenetic proteins (BMP) during pathogenesis of cleft palate, an experimental model was established in BALB/c mice. Cleft palate was induced by exposure to retinoic acid on embryonic day (E)12. The expression of BMP-2,3,4,5 mRNA in normal and abnormal embryonic palatal shelves was then examined from E13 to E16 by in situ hybridization. The results showed that BMP-4 mRNA was expressed strongly and uniformly in normal epithelial cells and dispersed mesenchymal cells on E13. BMP-2,5 mRNA expression appeared only in dispersed mesenchymal cells. With the development of shelves, the staining density of BMP-2,4,5 decreased gradually in mesenchymal cells outside of the condensation and increased inside the condensation. After shelves had fused on E16, no positive signals for BMP-2,4,5 were detected in dispersed mesenchymal cells, but their expression persisted in the condensation. Exposure to retinoic acid delayed the formation of the condensation and decreased BMP-2,4,5 mRNA dramatically in mesenchyme from E13 to E15. BMP-3 mRNA expression were almost negative in either control or retinoic acid-treated groups during all stages. It was concluded that spatial and temporal expression of BMP-2,4,5 was required during normal palatogenesis, and that a deficiency of their mRNA expression may contribute to the pathogenesis of cleft palate.     

Genetic background influences the capacity for medial edge epithelium disintegration and phenotype of cleft palate in TGFβ3 knockout mice

ObjectivesCleft palate is a frequent congenital craniofacial malformation of unknown etiology. Transforming growth factor (TGF) β3 is required for palatal shelf fusion. Although TGFβ3 knockout (KO) mice are widely used mouse models for cleft palate, cleft palate phenotypes differ among these mice. This study aimed to determine the effects of genetic background on the cleft palate phenotype in mice.MethodsWe produced TGFβ3 KO congenic mouse strains with five different genetic backgrounds. The phenotypes of the congenic strains were determined by visual examination. The capacity for disintegration of the medial edge epithelium (MEE) and basement membrane (BM) of palatal shelves of all five mouse strains was analyzed by using immunofluorescence staining after single palatal shelf suspension culture. The relationship between phenotype and disappearance of the MEE and BM was analyzed.ResultsAlthough the five congenic strains carried the same defective Tgfb3 gene, the fetal palate phenotypes differed among strains. The loss of the MEE cells and BM also differed with the genetic background, and the degree of such loss correlated with the cleft palate phenotype.ConclusionsThe cleft palate phenotype in mice is influenced by the genetic background, which governs the capacity for MEE and BM disintegration.     

Inhibition of human embryonic palatal mesenchymal cell cycle by secalonic acid D: a probable mechanism of its cleft palate induction

OBJECTIVE: To assess the mechanism(s) of cleft palate induction by secalonic acid D (SAD) in human embryonic palatal mesenchymal (HEPM) cells and compare them with those evaluated in the murine embryonic palate. DESIGN: Effect of SAD on HEPM cell proliferation was studied by obtaining dose response curves for cell numbers, uptake of 3H-thymidine and the expression of proliferating cell nuclear antigen (PCNA). Effects of SAD on cell cycle were assessed by flowcytometry. Cell-labeling with 3H-glucosamine and immunoblot analysis were conducted to study SAD effects on the synthesis of glycosaminogycans (GAG) and the expression of fibronectin and tenascin, respectively. RESULTS: SAD induced a concentration-dependent decrease in HEPM cell number and 3H-thymidine uptake beginning at 0.1 microg of SAD/ml. Expression of PCNA and progression of cell cycle from G1 to S phase were inhibited following SAD exposure. Cell viability was significantly reduced only at 7.5 microg/ml of SAD or higher indicating that the reduction in cell numbers by SAD at lower concentrations is likely due to reduced proliferation and at higher concentrations due to both reduced proliferation and cell death. Synthesis of extra cellular matrix components (GAGs, fibronectin or tenascin) by HEPM cells, however, was not inhibited by SAD. CONCLUSION: The results of these studies confirmed those of our previous studies with mice and the MEPM cells that SAD may induce cleft palate by reducing numbers of palatal mesenchymal cells by inhibition of their proliferation thereby leading to a reduction in the size of the developing palate shelves.