还原型谷胱甘肽稳态在腭裂发生中的作用

目的： 观察还原型谷胱甘肽稳态的改变对腭中嵴上皮细胞形态及腭裂发生率的影响,探讨氧化自由基在四氯二苯二噁英（TCDD）致畸过程中的作用。方法： 将GD10的SPF级C57BL/6J孕鼠随机分为4组,TCDD组：腹腔注射生理盐水和TCDD灌胃;TCDD+丁硫氨酸-亚砜胺(BSO)组：腹腔注射BSO 4 h后给予TCDD灌胃;BSO组：腹腔注射BSO和玉米油灌胃;对照组：腹腔注射生理盐水和玉米油灌胃。光学显微镜和电子扫描电镜下观察胎鼠腭胚突的发育及细胞表面形态。统计各组小鼠在GD17的腭裂发生率。免疫组织化学方法检测各组腭中嵴上皮各时期CYP1A1蛋白的表达。采用SPSS13.0软件包对数据进行统计分析。结果： TCDD成功构建高致畸率的腭裂模型。BSO增加了5%的腭裂发生率,但是相对于TCDD组无显著差异(P>0.05)。TCDD组腭胚突上抬时间较对照组推迟1 d,添加BSO的TCDD组腭胚突上抬过程较正常组减慢。电镜下胚胎发育的各发育时期,腭中嵴上皮表面TCDD组与对照组有显著差异。免疫组织化学检测发现,TCDD组腭中嵴上皮可见CYP1A1高表达。结论： 扰乱体内还原型谷胱甘肽稳态,可能会减少对小鼠体内氧化自由基的消耗,从而影响腭胚突的融合,继而加速了TCDD的毒性,影响腭裂发生。     

TCDD和B6 联合作用下小鼠腭板形态的扫描电镜观察

目的：二恶英（TCDD）和维生素B。联合作用下小鼠腭板形态的扫描电镜观察。方法：对孕期10d（GD10）的小鼠,分别胃饲TCDD24μg／kg,5mg、10mg、20mgB6／kg＋TCDD24μg／kg,对照组胃饲芝麻油50ml／kg,然后分别在GD12．5、GD13．5、GD14．5、GD15．5和GD17．5处死孕鼠,检查GDl7．5胎鼠有无腭裂的发生,GD12．5、GD13．5、GD14．5、GD15．5胎鼠用于扫描电镜观察。结果：TCDD组的腭裂发生率为55．56％,对照组未见腭裂发生,TCDD＋B6组浓度梯度下腭裂发生率为31．81％（5mg）,44．44％（10mg）,40．90％（20mg）。扫描电镜对照组腭中嵴上皮细胞形态规整,有大量微丝,伪足,随着孕期增加,融合的进行,微丝,伪足逐渐消失。TCDD组细胞肿胀变形,表面光滑,未见微丝,和伪足,而且形态并不随孕期的延长而改变,B6组腭中嵴上皮完全消失和TCDD组类似。结论：维生素B6不能恢复小鼠腭中嵴上皮细胞的表面超微结构,从而无法逆转TCDD导致腭裂效应。     

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。     

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。     

组蛋白去甲基酶UTX在2，3，7，8-四氯二苯并二噁英诱导胎鼠腭裂模型中的作用机制初步研究

目的    初步研究组蛋白去甲基酶UTX在2,3,7,8-四氯二苯并二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD）诱导胎鼠腭裂模型中的作用机制。方法    选取18只C57BL/6J孕鼠随机分为TCDD组和对照组,每组9只。在妊娠第10.5天,TCDD组孕鼠采用经口灌胃方式一次性给予64 μg/kg的TCDD（溶于0.2 mL的玉米油中）,对照组采用经口灌胃方式给予0.2 mL玉米油。每组分别于妊娠第13.5、14.5、15.5天各安乐处死3只孕鼠,分离胎鼠腭突组织。采用HE染色观察腭突组织形态,采用实时荧光定量PCR（qRT-PCR）和Western Blot检测两组胎鼠腭突组织中UTX的mRNA和蛋白表达水平。结果    HE染色结果显示,妊娠第15.5天对照组胎鼠双侧腭突组织已接触融合;而TCDD组胎鼠腭突组织未融合,且舌体下降出现明显延迟,提示腭裂初步形成。在不同妊娠时间,两组胎鼠腭突组织中UTX的mRNA和蛋白相对表达量均有变化（F值分别为28.683、7.927,均P < 0.05）;TCDD组胎鼠腭突组织中UTX的mRNA和蛋白相对表达量均低于对照组（F值分别为1167.309、348.274,均P < 0.05）;且两组UTX的mRNA相对表达量差异随着妊娠时间的延长而变大（F = 4.885,P = 0.017）,而两组UTX蛋白相对表达量的差异随妊娠时间的延长无明显变化（F = 3.158,P = 0.061）。结论    胎鼠腭突组织中UTX的表达水平降低可能是TCDD诱导腭裂发生的主要原因之一,UTX可能在腭部发育过程中具有重要作用。     

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.     

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.     

骨形态发生蛋白受体2在维甲酸诱导腭裂小鼠胚胎腭突中的表达

目的 探讨全反式维甲酸（atRA）对小鼠胚胎腭突中骨形态发生蛋白受体 2（BMPR2）表达的影响。方法 通过 atRA灌胃的方法建立 atRA诱导的小鼠腭裂模型,取妊娠 15 d（GD15）和 17 d的（ GD17）的胚胎腭部进行苏木精-伊红染色,并用免疫组织化学及逆转录聚合酶链式反应技术检测 BMPR2在胚胎腭部的表达。结果 atRA诱导小鼠形成体积较小的畸形腭突和明显的中缝腭裂畸形。 BMPR2在GD15和GD17正常胚胎腭部有高水平的阳性表达,但在腭裂胚胎腭部的表达水平明显减弱。正常胚胎腭部 Bmpr2 mRNA在GD15和GD17的表达水平均明显高于腭裂胚胎（ P<0.05）。结论 atRA可导致小鼠胚胎腭突发育不良形成腭裂,并显著下调 BMPR2表达水平,从而影响腭部发育的正常分子调控过程。     

TCDD disrupts posterior palatogenesis and causes cleft palate

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity.ICR mice (10–12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue.Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate.TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion.     

Sox4基因在小鼠腭突发育过程中的表达

目的 探讨Sox4基因在BALB/c正常小鼠与腭裂模型腭突胚胎上皮中的表达差异,初步研究该基因在腭突发育过程中的作用.方法 维甲酸诱导实验组BALB/c孕鼠,建立胎鼠腭裂模型,对照组孕鼠给予等量玉米油喂养.在GD13(gestation day 13)、GD14、GD15将各组孕鼠处死,获取胎鼠标本,应用免疫细胞化学方法检测Sox4蛋白在实验组及对照组胎鼠腭突中的表达.结果 实验组及对照组GD13-GD15腭突被覆的上皮中均为阳性表达,在GD13,二组腭突无明显形态差别.在GD14对照组中胚胎腭中线处可见腭突上皮开始接触融合,胚胎腭中线上皮带(medial epithelial seam,MES)表达阳性.GD14实验组中腭突未发生接触及融合.GD15对照组可见腭中线上皮带基本消失,MES部分消失,中线处为阳性,间充质中也可见阳性表达,实验组依然未发生融合,腭突末端被覆上皮呈阳性表达.用平均光密度值对 Sox4蛋白进行半定量检测得出,GD13实验组与对照组无明显差异, GD14实验组表达高于对照组,GD15对照组表达较高.组内比较对照组Sox4表达峰值出现在GD14,实验组相邻两组间对比无差异.结论 Sox4在腭裂与正常腭突中GD14-GD15的表达存在明显差异,在腭突融合期发挥调节作用.     

Expression and function of patatin-like phospholipase domain-containing 2 in cleft palate induced by retinoic acid

Cleft palate is a common maxillofacial congenital malformation, and its mechanism still has not been fully illustrated. Recently, lipid metabolic defects have been observed in cleft palate. Patatin-like phospholipase domain-containing 2 (Pnpla2) is an important lipolytic gene. However, its effect on the formation of cleft palate remains unknown. In this research, we explored the expression of Pnpla2 in the palatal shelves of control mice. We also studied mice with cleft palates induced by retinoic acid and its effect on the embryonic palatal mesenchyme (EPM) cells phenotype. We found that Pnpla2 was expressed in the palatal shelves of both the cleft palate and control mice. Pnpla2 expression was lower in cleft palate mice than in the control mice. Experiments with EPM cells showed that knockdown of Pnpla2 inhibited cell proliferation and migration. In conclusion, Pnpla2 is linked to palatal development. We have indicated that low expression of Pnpla2 affects palatogenesis by inhibiting the proliferation and migration of EPM cells.     

腭器官培养在腭裂发病机制研究中的应用

唇腭裂是人类最常见的出生缺陷之一,其发病机理复杂.多学科的研究表明,唇腭裂的病因涉及众多因素,受到多个因素的调控.其中导致腭裂发生的原因之一是腭板未能融合,而对于腭板融合过程的研究,在体内受诸多因素的限制,因此可以采用体外腭器官培养的方法进行研究.长期实践证明,与体内实验相比,体外腭器官培养模型可以模拟体内腭裂形成的过程.本文就腭器官培养方法在腭裂发病机制研究中的作用作一综述.     

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。     

三维重建观察四氯二苯并二恶英对胎鼠腭器官发育的影响

目的:三维重建观察四氯二苯并二恶英(tetrachlorodibenzo-p-dioxin,TCDD)对胎鼠腭器官发育的影响.方法:对妊娠12.5 d昆明鼠进行40 μg/kg TCDD灌胃处理作为实验组,等剂量的玉米油处理作为对照组.妊娠13.5、14.5、15.5 d时,取出胎鼠头部固定,制备腭器官石蜡连续切片,苏木精-伊红染色,采集腭器官的图像用Photoshop处理、3D-DOCTOR软件对腭器官进行三维重建.结果:3D-DOCTOR三维重建显示,对照组腭器官从舌两侧移至舌上方,逐渐靠拢并融合;实验组腭器官从舌两侧延迟于对照组移至舌上方渐靠拢,但并未融合,形成腭裂.结论:3D-DOCTOR重建可观察腭器官发育过程和TCDD作用于孕鼠导致胎鼠腭裂形成过程.     

Temporal and spatial expression of Pax9 and Sonic hedgehog during development of normal mouse palates and cleft palates in TGF-beta3 null embryos

Transforming growth factor-beta (TGF-beta3) gene disruption causes cleft secondary palate. Pax9 and Sonic hedgehog (Shh) genes are involved in the patterning of vertebrate embryonic tissues, including the facial skeleton. We investigated the expression of Pax9 and Shh genes during normal mouse palate development and in the developing cleft palates of TGF-beta3 null embryos. Whole mount in situ hybridization was conducted with use of Pax9 and Shh riboprobes for TGF-beta3 null, heterozygous and wild type mice at E12.5-E16.5. Histological analysis was processed by section in situ hybridization. In the wild type, Pax9 and Shh were expressed in the palate between E12.5-E15.5. Shh expression in the secondary palate was restricted to the rugae and the soft palate. Pax9 expression was predominantly in the palatal medial edge between E14.5 and E15.5. These patterns suggest that Shh and Pax9 may have different functions during palate development. In TGF-beta3 null mice, both genes expression patterns in the palate were different to those in wild type mice. In TGF-beta3 null mice, Pax9 expression was much reduced in the palatal medial edge at the critical time of palatal fusion (E14.5-E15.5). Shh expression in the palates of TGF-beta3 null mice was reduced throughout E12.5-E15.5, whilst Shh expression in heterozygous did not appear down regulated compared with the wild type. These results indicate that Pax9 and Shh expression are altered when the TGF-beta3 gene is deleted and suggest that Pax9 and Shh may be involved in the TGF-beta3 regulation of normal palatal fusion.     

In vitro investigation of the secondary palate development in two strains of mice

The pathogenesis of cleft lip and palate (CL/P) is studied in animal experiments. This study revealed significant differences in foetal secondary palate development in two strains of mice (NMRI, A/WySnJ) using a palatal organ model. Palatal shelves of 114 NMRI embryos, resistant to cleft occurrence, and 93 A/WySnJ embryos, a strain with a high spontaneous CL/P rate, were micro-dissected at 14.25 GD (gestational day), before palatal fusion takes place. After cultivation in serum-free medium, palatal development was investigated microscopically and scored in a six-step system. At death (14.25 GD) the palatal shelves of the NMRI embryos (mean 3.5) were significant more developed than those of A/WySnJ (mean 2.7; p = 0.05). After incubation, 53% (60/114) NMRI and 14% (13/93) A/WySnJ cultures had over two-thirds fusion to stage V-VI, therefore in 17% NMRI (19/114) and 1% A/WySnJ cultures (1/93) fusion was macroscopically complete. 62% of the A/WySnJ cultures showed no significant development in vitro (mean 2.84; p = 0.094). There is a significant palatal development difference between normally developed NMRI (mean 4.45, p = 0.05) and CL/P appearance in A/WySnJ mice (mean 2.84). Palatal development of both strains was significantly delayed in organ culture (p = 0.05). The A/WySnJ strain was more susceptible to manipulation and vulnerable.     

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.     

RA诱导小鼠腭裂动物模型的实验研究

目的 :分析维甲酸 (RA)在胚胎发育期间对胎鼠和母孕鼠的毒性作用 ,建立RA诱导腭裂动物模型。方法 :将C5 7BL近交系小鼠分为对照组和用药组 ,在GD8d、GD10d和GD 12d分别口饲灌胃植物油和RA ,其中在GD 10d用药组按照三种不同的剂量口饲灌胃给药。结果 :RA在不同的发育阶段均可诱导腭裂畸形 ,GD10d不同剂量的RA诱导胎鼠腭裂形成的比率不同。结论 :在GD 10d 10 0mg/kg维甲酸诱导C5 7BL近交系小鼠腭裂是一种较为理想的腭裂动物模型     

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。