维甲酸诱导腭裂发生中转化生长因子-β信号通路相关分子表达的研究

目的研究维甲酸诱导腭裂发生中转化生长因子-β信号通路相关分子的表达。方法采用器官体外培养法进行腭板培养,实验分为对照组和全反式维甲酸（all trans retinoic acid,ATRA）组。分别培养24h、48h、72h后,苏木索一伊红染色观察两组腭板在不同时间点的融合情况,原位末端转的凋亡,免疫组化检测各组腭板中caspase-9、转化生长因子-β3（transforming growth factor-β3,TGF-β3）．转化生长因子-β受体Ⅱ（transforming growth factor type-Ⅱ receptor,TGFβ RⅡ）、维甲酸核受体理（retirmic acid receptor α,RARα）、维甲酸核受体β（retinoi cacid receptorp,RARβ）及维甲酸X受体α（retinoi cacid X receptor α,RXRα）的表达。结果成功建立维甲酸诱导腭裂模型,培养24h、48h、72h后,对照组腭板的融合数分别为4个、8个、8个,而ATRA组未见腭板融合,差异具有统计学意义（P〈0.05）。与对照组相比,ATRA组凋亡率和caspase-9表达上升（P〈0．05）。ATRA能明显抑制TGFβRⅡ的表达（P〈0．05）,但对于TGF-β3的作用却较小。ATRA可明显上调RARD的表达,并抑制RXRa在间充质中的表达。结论ATRA可能通过诱导腭突间充质细胞的凋亡及转化生长因子-β信号通路相关分子和维甲酸受体的表达改变,而诱导腭裂形成。     

小鼠体外胚胎腭器官培养模型的建立及全反式维甲酸致畸

目的 探讨体外胚胎腭器官培养模型的建立,并研究全反式维甲酸(atRA)诱导形成腭裂的机制.方法 首先建立体外胚胎腭器官培养模型,并在培养体系内添加3.0 μmol/L atRA,对照组加入相应剂量的乙醇溶液.吖啶橙染色观察胚胎腭器官体外培养效果.TUNNEL法检测体胚胎腭细胞凋亡情况,免疫组化检测Smad2/3表达情况.结果 通过静止法体外培养基本可以重复体内腭突接触融合过程,融合率达到82.30%(93/113).对照组的腭突逐步发生接触,并在接触区上皮出现凋亡带,在体外培养24 h的腭突中嵴上皮细胞Smad2/3呈阳性表达.48 h腭突实现融合,腭突中嵴上皮消失,Smad2/3在腭上皮组织内的表达明显下降.而单侧腭突体外培养时,在中嵴上皮区域未出现凋亡现象.实验组3.0 μmol/L atRA可以阻碍腭突中嵴上皮带在接触后出现的凋亡现象,同时Smad2/3在中嵴上皮内的表达水平较对照组明显下降,中嵴上皮带不能消失,双侧腭突间充质无法融合.结论 本研究成功建立胚胎腭器官体外培养模型,证实两侧腭突接触触发了腭突中嵴上皮部位的凋亡现象.同时证实atRA干扰了转化生长因子β/Smad信号系统在腭突中嵴上皮带的消失过程中的调节作用,导致两侧腭突不能融合.     

siRNA抑制TGFβ3诱导小鼠腭裂机制的研究

目的:探讨siRNA抑制TGFβ3诱发腭裂的机制。方法:使用不同剂量(200、400、800pmol)经脂质体LipofectamineTM2000包裹的TGFβ3 StealthTMRNAi于交配后(days post coitum,dpc)12天做孕鼠宫腔注射,等体积生理盐水注射作为空白对照。14dpc时取部分胎鼠腭突,RT-PCR及Western印迹检测腭突中TGFβ3、Smad2、BMP2的表达情况。16dpc时再取部分胎鼠腭突,观察腭突融合情况,免疫组织化学及免疫荧光观察胚鼠腭突的TGFβ3、Smad2、BMP2的表达情况。实验数据均以SPSS13.0软件包进行分析,多组间比较采用单因素方差分析,两组间比较采用t检验。结果:宫腔注射800pmol的StealthTMRNAi,可引起TGFβ3基因mRNA和蛋白表达水平明显降低,400pmol组能显著降低Smad2基因的mRNA和蛋白表达水平,但BMP2的表达情况未受明显影响。20%～60%的实验组小鼠胚胎腭突不能融合,形成腭隐裂,与空白对照组相比,其TGFβ3、Smad2的表达量较低,而BMP2的表达未见显著变化。结论:在12dpc给予小鼠宫腔注射TGFβ3 StealthTMRNAi,能有效沉默TGFβ3基因,20%～60%小鼠胚胎形成腭隐裂,同时Smad2基因表达下调,但对BMP2基因表达无明显影响。     

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。     

胚鼠腭突细胞转化生长因子β3基因的RNA干扰及对TGFβ-Smad信号通路的影响

目的 探讨在胚鼠腭突培养细胞中应用StealthTM RNAi转染有效沉默转化生长因子β3(TGFβ3)基因的可行性.方法 采用Bal b/c近交系小鼠在交配后第14天取胚鼠腭突,离散后进行细胞培养,应用相差显微、MTT法观察传代细胞的形态和生物学特性.采用化学合成的StealthTM RNAi抑制原代胚鼠腭突上皮细胞...     

Sox9在腭发育中作用的动物实验研究进展

<正>唇腭裂是由环境和遗传因素共同作用而导致的一种多基因遗传病。腭裂的发生是腭发育不正常的结果。腭发育过程中有原发腭与继发腭之分,它们都是由神经嵴来源的间充质细胞形成。原发腭在人类胚胎第7周(鼠胚第12.5天,简写E12.5)由两侧的中鼻突融合完全后而形成。继发腭的发育开始于人类胚胎第45天(短吻鳄胚胎为第17天、鸡胚为第6天,鼠胚为第11.5天[1]),双侧上颌突生长形成腭突,双侧腭突于舌两侧垂直向生长;人类胚胎第9周左右(鼠胚第14天,即E14.0),由于舌的下降、下颌发育,双侧腭突上抬,水平向生长向中线靠     

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。     

转化生长因子βⅢ型受体在腭融合中的作用

为了探明腭裂的分子病理机制,腭形成的分子调节机制成为连续的研究热点。转化生长因子Ⅲ型受体(transforming growth factor-β type Ⅲ receptor,TβR-Ⅲ)在腭黏附的关键时期特异表达在中嵴上皮(medial edge epithelial,MEE)。本研究的目的是探明TβR-Ⅲ mRNA和蛋白在胎鼠体内的定位和表达水平,以及确定体外腭融合期间对TβR-Ⅲ表达的需求。     

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。     

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     

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.     

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.     

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.     

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。