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.     

MTHFR基因沉默对小鼠胚胎腭突间充质细胞增殖和凋亡的影响

目的:应用RNA干扰技术,从细胞水平研究5,10-亚甲基四氢叶酸还原酶(MTHFR)基因突变导致腭裂发生的机制.方法:构建MTHFR基因沉默载体,对13天胚胎的腭突间充质细胞进行RNA干扰实验.MTT法检测MTHFR基因沉默后细胞增殖的改变.流式细胞仪检测MTHFR基因沉默后对胚胎腭突间充质细胞凋亡的影响.实验结果采用SPSS11.0软件包进行分析,细胞增殖的比较用重复测量方差分析,凋亡率检测应用U检验.结果:MTHFR基因表达下调后,在无叶酸存在的情况下,腭突间充质细胞的增殖速度明显减慢,与对照组相比有显著性差异(P<0.001);实验组的凋亡率显著高于对照组(P<0.05).结论:在外源性叶酸补充不足的情况下,腭突间充质细胞增殖减弱并发生凋亡,这可能是MTHFR基因突变与先天性唇腭裂发生相关的病理学基础.     

Immunofluorescent detection of actin in non-muscle cells of the developing mouse palatal shelf

Cryostat sections of foetal mouse heads, taken at 14–15 days in utero, were incubated first in serum containing smooth muscle auto-antibody and then in fluorescein-conjugated rabbit or goat IgG directed against human IgG. A positive fluorescent reaction was observed in epithelial and mesenchymal cells of the secondary palate, oral mucosa, nasal septum and tongue. Incubating the auto-antibody-containing serum with highly purified actin blocked the auto-antibody and produced no reaction, suggesting that the contractile protein detected in non-muscle cells of the developing palatal shelf contain the muscle protein, actin.     

C57BL/6J小鼠胚胎腭突体外培养模型的建立

目的:建立近交系C57BL/6J小鼠胚胎腭突体外培养模型,为研究胚胎腭突发育提供条件。方法:无菌条件下,在体视显微镜下取怀孕13.5 d(GD13.5)的胚胎腭突,进行培养。分别于培养6、24、48 h后取出固定,行组织切片苏木精-伊红(HE)染色和扫描电镜(SEM)观察,每个时间点分别12对。结果:细胞核染色显示,6 h时,两侧腭突未融合;24 h时,两侧腭突开始融合,但是中间仍有部分腭中嵴上皮(MEE)细胞残留;48 h时,两侧腭突完全融合,中间的MEE完全消失。扫描电镜显示,6 h时,两侧腭突之间有一定裂隙;24 h时,两侧腭突已接触但能观察到腭中缝(MES);48 h时,两侧腭突已完全融合,MES消失。结论:本方法为研究腭裂发病机制提供了一个良好的体外模型。     

Cortisol inhibition of development of various lysosomal enzymes in cultured palatal shelves from mouse embryos

The lysosomal enzymes, non-specific esterases, β-glucuronidase, N-acetyl-β-glucosaminidase and aryl sulphatases A and B, were examined histochemically in medial-edge epithelia (MEE) of single palatal shelves; in vitro. Activities of most enzymes increased gradually in MEE with a peak at 24–26 h of culture. Aryl sulphatase B activities were lower than the others and aryl sulphatase A activities could not be detected in the palatal cells during the entire culture period. By 48 h, MEE cells degenerated and were lost. Cortisol suppressed increased activities of these hydrolytic enzymes and prevented programmed breakdown of the palatal epithelium.     

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。     

P21~(cipl/wafl)基因在C57BL/6N系小鼠腭突发育中对细胞增殖周期的调控作用

目的：探讨腭裂形成过程中腭突间充质细胞的增殖周期的变化及其调节基因Ｐ２１ｃｉｐｌ／ｗａｆｌ基因表达的意义。方法：用原位杂交、ＴＵＮＥＬ染色检测两组小鼠腭突间充质细胞增殖周期调控基因Ｐ２１ｃｉｐｌ／ｗａｆｌ基因的表达及细胞程序性死亡的发生。结果：在腭突发育的垂直生长期、上抬期,实验组中ＴＵＮＥＬ阳性指数明显高于对照组（Ｐ＜０．０１）。同时,Ｐ２１ｃｉｐｌ／ｗａｆｌ基因ｍＲＮＡ的转录水平也比同期对照组腭突间质细胞中Ｐ２１ｃｉｐｌ／ｗａｆｌ基因的表达增高（Ｐ＜０．０５）。结论：在腭突发育时期,实验组小鼠腭突间充质未分化细胞增殖周期抑制。Ｐ２１ｃｉｐｌ／ｗａｆｌ基因表达升高提示可能参与了细胞增殖周期抑制过程     

地塞米松对小鼠胚胎腭突间充质细胞增殖的影响

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 .2 5 %胰蛋白酶进行消化获得游离分散的EPM细胞 ,在DMEM培养基中进行培养 ,并采用反复贴壁法纯化EPM细胞。在培养基中加入 10 6M地塞米松 ,采用AgNOR染色、Feulgen染色及透射电子显微镜观察 ,检测地塞米松对EPM细胞增殖能力的影响。结果　地塞米松处理的EPM细胞银染颗粒数减少 (P <0 .0 1) ,核面积比及DNA合成能力下降 (P <0 .0 1) ,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     

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。     

转化生长因子β1对A系小鼠胚腭突细胞增殖代谢的影响

目的 研究不同剂量转化生长因子β1(TGFβ1)对A系小鼠胚腭突细胞增殖和DNA、蛋白质及PGE2合成的影响。方法 实验分为9组，每组设平行4孔(瓶)，每组分别加入TGFβ1使其终浓度为0．005ng／ml，0．01ng／ml，0．05ng／ml，0．1ng／ml，0.5ng／ml，1ng／ml，5ng／ml，10ng／ml，50ng／ml，每组均设空白对照，在实验后第1、3、5天分别检测细胞的OD值、DNA、蛋白质和PGE2的含量。结果 在高浓度血清条件下，TGFβ1可显著刺激腭突细胞的增殖，但在低浓度血清条件下，TGFβ1对A系小鼠胚腭突细胞具有双重效应，即在其0．005～0．01ng／ml时促进腭突细胞增殖，而随着TGFβ1浓度的增大，逐渐抑制腭突细胞的增殖。TGFβ1使腭突细胞PGE2及蛋白质合成增加。结论 TGFβ1对A系小鼠胚腭突细胞的增殖具有双重作用，可能是腭突正常发育的重要调节因子。     

Epithelial-mesenchymal interaction in palatal shelf fusion.: An in vitro study

A bstract — Cleft palate is the result of a number of deviations from normal facial development, including failure of the paired palatal shelves to fuse in the midline. By the use of in vitro techniques it is possible to isolate and study fusion alone. This study investigates some of the epithelial-mesenchymal interactions involved and finds that contact between two epithelial covered surfaces is essential for fusion.     

Differences in the size of the palatal processes in mouse embryos with cleft palate induced in two critical periods.

Using planimetric measurements of projections of the space between the palatal processes of ICR-Velaz mouse embryos, we indirectly demonstrated that the pre-horizontalization size of the palatal processes after the i.m. administration of 7.5 mg cortsone acetate on the 12th day of gestation was smaller than in the controls. After horizontalization, the inadequate palatal processes were unable to meet in the midline as they do in the majority of normal embryos. The administration of 0.5 mg 6-amino-nicotinamide on the 14th day of gestation did not significantly affect the size of the palatal processes.     

A radioautographic study of deoxyribonucleic acid synthesis in embryonic rat palatal shelf epithelium with reference to the concept of programmed cell death

In order to study changes in the rate of mitosis and DNA synthesis of palatal shelf epithelium, radioautographs were made of frontal sections through the heads of 13, 14, 15 and 16-day in utero Sprague-Dawley rat embryos. The percentage of labelled epithelial cells was determined for four areas of oral and palatal shelf epithelium at each age.A declining trend in mitotic activity with time was observed in all areas. Palatal tip epithelium was found to be different from nasal, oral and tongue epithelium. The faster decline in mitotic activity of palatal tip epithelium, together with tissue culture and histochemical evidence from other studies, supports the concept of programmed cell death for this tissue.     

神经生长因子对体外培养的人牙乳头间充质细胞增殖和细胞周期的影响

目的：观察神经生长因子对人牙乳头间充质细胞增殖和细胞周期的影响。探讨其在牙胚生长发育过程中的可能作用。方法：采用^3H-TdR掺入和流式细胞仪分析法。结果：NGF（1－100U/ml)可明显促进培养的人牙乳头间充质细胞的^3H-TdR掺入率,NGF（100U／ml)刺激细胞增殖作用最强。NGF（1000U／ml)抑制细胞的增殖。在NGF（100U／ml)作用下,细胞S％明显升高,细胞增殖指数（S G2M)%也明显增高。结论：神经生长因子可促进人牙乳头间充质细胞DNA合成,刺激G1期细胞向S期转变。     

小鼠胚胎腭突间充质细胞体外培养及生物学特性研究

目的 研究小鼠胚胎腭突间充质（EPM）细胞的生物学特性。方法 在显微镜下解剖妊娠第13天的母鼠胚胎腭突，用0.25%胰蛋白酶进行消化获得游离分散的EPM细胞，在含10％胎牛血清的DMEM培养基中进行培养。采用免疫组化方法进行细胞鉴定，通过相差显微镜，生长曲线及透射显微镜观察细胞形态，增殖能力及超微结构。结果 EPM细胞为成纤维细胞样细胞，排列无序，细胞核大，核分裂象多，核膜内陷，核呈分叶状，胞浆含大量线粒体及粗面内质网。免疫组化显示角蛋白标记为阴性，S－100蛋白及波形蛋白标记为阳性。EPM细胞呈指数生长，有较强的增殖能力。结论 EPM细胞体外培养生长及增殖良好，可作为腭裂基础研究较理想的研究对象。     

亚甲基四氢叶酸还原酶基因在小鼠胚胎腭突间充质细胞中沉默效应的研究

目的构建亚甲基四氢叶酸还原酶（MTHFR）特异性小干扰RNA（siRNA）真核表达载体,体外观察其对MTHFR基因的沉默作用。方法采用基因克隆技术,将合成的特异性MTHFR RNA干扰寡核苷酸序列插入真核表达载体PsiRNA-Hh1neo_G2,构建MTHFR siRNA真核表达载体。应用核转染技术将PsiRNA-MTHFR重组质粒分别导入原代培养的小鼠胚胎腭突间充质（MEPM）细胞。48 h和5 d后用实时定量聚合酶链反应（Real-Time PCR）和Western blot技术检测MEPM细胞内MTHFR mRNA及蛋白水平的表达情况。结果成功构建PsiRNA-MTHFR真核表达载体。经检测48 h和5 d后转染PsiRNA-MTHFR的MEPM细胞MTHFR mRNA及蛋白表达均显著下调。结论构建的RNA干扰真核表达载体能明显干扰MTHFR mRNA及蛋白的表达,为进一步研究MTHFR的功能以及其调控胚胎腭突融合的机制奠定基础。