组织细胞内环—磷酸腺苷及前列腺素E2浓度变化对A系腭裂小鼠…

通过检测地塞米松（ＤＥＸ）作用后Ａ系小鼠腭、舌、颌骨组织的环－磷酸腺苷（ｃＡＭＰ）及前列腺素Ｅ２（ＰＧＥ２）的活性水平及其变化，以进一步探讨糖皮质激素诱发Ａ系小鼠腭裂的可能原因。结果，Ａ系胎鼠的腭胚突组织在闭合期之间（１４＾２０）实验组（ＤＥＸ组）低于对照组，腭闭合后期（１５＾８～２０）对照组浓度迅速下降而低于ＤＥＸ组。ＰＧＥ２腭闭合前ＤＥＸ组低于对照组，腭闭合后期出现极高的峰值。舌组织、下颌骨组     

A系小鼠腭裂动物模型的组织酶学变化及其与腭,舌,颌发育关…

采用酶组织化学方法检测ＤＥＸ作用后的Ａ系小鼠，其舌、腭、颌组织的有关酶类活性的变化。结果表明ＤＥＸ作用后的小鼠，腭胚突骨钙代谢活动加强。在发育早期（１３＾１４～１４＾８），细胞有氧代谢酶类活性较高，而无氧代谢及核酸代谢在腭突发育后期较低，表明细胞能量代谢障碍，此与腭突发育不良有关。颌骨区骨钙代谢的酶活性较正常小鼠过早过强，有氧代谢酶类活性较高，无氧代谢及核酸代谢活性水平偏低，颌骨有早熟性改变。     

地塞米松对小鼠胚胎腭胚胎腭突间充质细胞增殖的细胞

目的 探讨地塞米松对小鼠胚胎腭突间充质（EPM）细胞增殖的影响。方法 在显微镜下解剖妊娠第13d的鼠胚腭突，用0．25％胰蛋白酶刊物消化获得游离分散的EPM细胞，在DEMF培养基中进行培养，并采用反复贴壁纯化EPM细胞。在培养基中加入10^-6M地塞米松，采用AgNOR染色、Feulen染色及透射电子量显微镜观察，检测地塞米松对EPM细胞增殖功能力的影响。结果 地塞米松处理的EPM细胞银梁颗粒数减少（P＜0．01），核面积比及DNA合成能力下降（P＜0．01），胞浆内线粒体数目减少，粗面内质网肿胀。结论 地塞米松对EPM细胞的增殖及生物合成有明显的抑制作用，影响腭突的正常发育，是腭裂发生的机制之一。     

地塞米松和维生素B_（12）交互作用对小鼠腭胚突超微结构影响的观察

目的:观察地塞米松和维生素B12作用后腭胚突超微结构的变化.方法:将孕鼠分为实验组(致畸组,拮抗组)和对照组,按体重分别注射地塞米松、地塞米松+维生素B12和生理盐水,分别在E14.5 d和E17.5 d处死孕鼠并获取胚胎,用扫描电镜观察E14.5 d的腭胚突表面结构,记录E17.5 d腭胚突的腭裂情况.结果:地塞米松作用后,小鼠胚胎的腭裂发生率从3.1%上升到42.1%(P<0.01),硬腭处细胞的伪足结构消失.维生素B12拮抗后,小鼠胚胎的腭裂发生率降低(P<0.05),伪足结构部分恢复.结论:维生素B12能部分恢复小鼠腭胚突细胞的表面超微结构,从而拮抗地塞米松的致腭裂效应.     

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。     

细胞凋亡及上皮-间充质转化在腭胚突融合过程中的作用探讨

目的: 探讨细胞凋亡及上皮-间充质转化（epithelial-mesenchymal transition, EMT）在腭胚突融合中的作用。方法: 选用8周龄C57BL/6J近交系小鼠作为研究对象。于E13.5、E14.0、E14.5、E15.5及E17.5 d获取胎鼠腭胚突组织,在腭胚突融合的关键阶段,应用免疫组织化学和TUNEL方法检测EMT过程中蛋白变化和细胞凋亡率。每个时间点选择3张图片,应用image J软件进行图片灰度分析,采用SPSS12.0软件包进行统计学分析。结果: 免疫组织化学和TUNEL法检测发现在腭融合的关键阶段（E14.5）,腭中嵴上皮（medial edge epithelium, MEE）中出现纤连蛋白和波形蛋白表达和细胞凋亡,前中后腭部MEE细胞也出现细胞凋亡,中后部细胞凋亡率显著高于前部。结论: 在体内腭胚突融合的关键阶段,EMT过程和细胞凋亡均参与了腭中嵴上皮带的退化消失,且前中后腭部的融合机制基本一致。     

维生素B12阻抑地塞米松诱发小鼠腭裂的实验研究

木实验拟使用维生素Ｂ＿（１２）阻抑地塞米松诱导Ａ系小鼠腭裂的形成，探讨其作用机理，为进一步了解先天性腭裂的发病机制及其预防提供实验依据。结果表明：在小鼠妊娠第１２天１４小时，以地塞米松２０ｍｇ／ｋｇ一次剂量可诱发Ａ系小鼠腭裂，其诱发率为８２．２％，维生素Ｂ＿（１２）可有效阻抑地塞米松对Ａ系小鼠腭裂的诱导作用，使其腭裂发生率降低至４２．７％，下降率为４８％，Ｖｉｔ－Ｂ１２作用后的小鼠无明显胚胎毒性反应，并可增加胎鼠体重。实验提示Ｖｉｔ－Ｂ＿（１２）可促进胎鼠生长发育，拮抗地塞米松对胎鼠腭胚突细胞的增殖及腭间质分化的抑制，从而预防腭裂的发生。     

p63在侧腭突MEE／MES消失过程中的作用机制

目的：探讨p63及其亚型ANp63、TAp63在正常胎鼠和腭裂模型胎鼠侧腭突上皮，特别是在胚胎腭中线上皮（medialedgeepithelial，MEE）／胚胎腭中线上皮带（medialepithelialseam，MES）中的表达及变化情况，阐明p63及其亚型△Np63、TAp63在MEE／MES消失过程中的作用。方法：建立全反式维A酸（all—transretinoicacid，atRA）诱导的胎鼠腭裂模型，实验组在鼠孕期第10天（gestationday，GD10）按100mg，kg给予atRA灌胃，对照组则给予同等剂量的玉米油．分别在GD13．5、GD14．5、GD15．5、GD16．5解剖取得胎鼠的腭突，通过HE染色、扫描电镜进行形态学观察，采用免疫组化方法检测胎鼠侧腭突上皮p63、ANp63及TAp63的表达。结果：在GD14．5，对照组p63和ANp63在侧腭突MEE细胞内基本呈阴性表达．而实验组则呈阳性表达。在GD15．5．对照组MEE／MES消失，腭突基本融合，侧腭突所有被覆上皮基底层细胞再次出现p63和△NF．63阳性表达；实验组MEE／MES则未完全消退，p63和ANp63在消退异常的MEE细胞内仍呈阳性表达。在GD16．5对照组，腭突融合完成，侧腭突被覆上皮基底层细胞p63和△Np63仍呈阳性表达；而实验组未融合，呈腭裂，p63和△Np63在MEE细胞内呈阴性表达。结论：在胎鼠侧腭突上皮发育过程中，存在p63及其亚型ANp63特定时空的差异表达，提示ANp63参与侧腭突上皮的分化调控，尤其是参与了MEE／MES消失过程中的信号调控．     

细胞凋亡及p5 3基因的表达在C5 7BL／6N系小鼠腭裂形成中的意义

目的：探讨细胞程序性死亡和相关基因ｐ５３的表达变化在腭裂发生中的意义。方法：利用建立的腭裂模型，采用原位末端转移酶标记法（ＴＵＮＥＬ）和基因探针原位杂交法，原位观察了二种阳性信号的表达。结果：实验组小鼠腭突发育的垂直期、上抬期中细胞程序性死亡明显多于对照组（Ｐ〈０．０１）。但两组间ｐ５３ｍＲＮＡ转录水平的变化均无明显差异（Ｐ〉０．０５）。对照组小鼠生长早期ｐ５３基因的表达高于同组融合期的腭突。结论     

60例腭部小涎腺癌临床分析

６０例腭部小涎腺癌临床分析ＣＡＲＣＩＮＯＭＡＯＦＭＩＮＯＲＳＡＬＩＶＡＲＹＧＬＡＮＤＳＩＮＴＨＥＰＡＬＡＴＥ－ＡＣＬＩＮＩＣＡＬＡＮＡＬＹＳＩＳＯＦ６０ＣＡＳＥＳ王志勇叶炳飞徐明耀作者单位：南京市口腔医院颌面外科（２１０００８）小涎腺癌是腭部最常见的...     

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。     

地塞米松对A系小鼠胚腭突间充质细胞表皮生长因子基因表达的影响

目的:研究地塞米松(DEX)对A系小鼠胚腭突间充质细胞表皮生长因子(EGF)基因mRNA表达的影响。方法：将DEX(10ng/ml)加入培养的A系小鼠胚腭突间充质细胞，并在培养第1、3、5天时收集细胞，提取RNA，应用RT-PCR技术半定量检测腭突问充质细胞EGF基因mRNA的表达水平。结果：DEX可显著促进腭突间充质细胞EGF mRNA的表达，并在加入DEX培养第3天时，这种促进EGF mRNA表达的作用最强。结论：DEX显著促进腭突间充质细胞EGF基因的表达，可能干扰腭突间充质细胞EGF基因的表达而影响了腭发育。     

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。     

Folic acid rescue of ATRA‐induced cleft palate by restoring the TGF‐β signal and inhibiting apoptosis

J Oral Pathol Med (2010) 40 : 433–439 Background: Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF‐β signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all‐trans retinoic acid (ATRA)‐induced cleft palate in organ culture were tested. Methods: C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFβ3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. Results: Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase‐9 in MEPM cells were detected in the ATRA group than in the control or the ATRA + FA group. Compared with the control or the ATRA + FA group, ATRA had little effect on TGF‐β3 in MEPM cells but significantly inhibited TGF‐β receptor II. Conclusions: Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFβ signaling in MEPM cells were involved in folic acid rescued ATRA‐induced cleft palate.     

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.     

Cell proliferation and apoptosis in the fusion of human primary and secondary palates

The markers of cell proliferation (Ki-67) and apoptosis [caspase-3, TdT-mediated biotin-dUTP nick-end labelling (TUNEL)] and the expression of syndecan-1 and heat shock protein 70 (Hsp70) were analyzed immunohistochemically in 11 developing human palates, from developmental weeks 6 to 10. During fusion of the primary palate, the proportion of proliferating cells decreased from 42 to 32% and the proportion of apoptotic cells decreased from 11 to 7% in the medial-edge epithelium. At later stages, the proportions of both types of cells decreased in the ectomesenchyme, except for proliferating cells in its non-condensing part. At developmental weeks 9-10, the epithelial seam in the secondary palate comprised 28% proliferative cells and 5% apoptotic cells. While condensing ectomesenchyme contained more apoptotic cells than proliferating cells, the opposite was observed for the non-condensing ectomesenchyme. Co-expression of syndecan-1 and Hsp70 was detected in cells budding from the epithelial seam. Our study indicates similar principles for human primary palate and secondary palate fusion, and parallel persistence of proliferation and apoptotic activity. While proliferation enables growth and fusion of different palatal primordia, apoptosis results in the removal of of large numbers epithelial cells at the fusion point. The disintegration of seam remnants seems to be executed through the processes of change in protein content and cell migration, probably leading to cell death as their final outcome.