论文部分内容阅读
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3.0μmol/LatRA,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24h的腭突中嵴上皮细胞Smad2/3呈阳性表达。48h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3.0μmol/LatRA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。
Objective To investigate the establishment of in vitro embryo palatal organ culture model and to study the mechanism of atRA induced cleft palate formation. Methods The palatal organ culture model was established in vitro and 3.0 μmol / L ATRA was added into the culture system. The control group was given the corresponding dose of ethanol solution. Acridine orange staining observed embryonic palatal organ in vitro culture effect. TUNEL staining was used to detect the apoptosis of palatal cells and the expression of Smad2 / 3 was detected by immunohistochemistry. Results The in vivo palatal contact fusion process could be repeated by in vitro static culture. The fusion rate reached 82.30% (93/113). The control group had gradual contact with the palatal process, and apoptotic bands appeared in the contact epithelium. Positive Smad2 / 3 expression was observed in the midclavicular epithelial cells cultured in vitro for 24 h. 48h palatal protrusion fusion, disappear in the midgut epithelium, Smad2 / 3 expression in the palatal epithelium decreased significantly. However, when the unilateral palate process was cultured in vitro, no apoptotic phenomenon occurred in the midrib epithelial region. The experimental group 3.0μmol / LatRA can hinder the palatal midclavian epithelium in the contact after the emergence of apoptosis, while the expression of Smad2 / 3 in the midrib ridge epithelium significantly decreased compared with the control group, the crest of the crista can not disappear, both sides Palatal foraging mesenchyme can not be integrated. Conclusion This study successfully established embryonic palatal organ in vitro culture model confirmed that both sides of the palate contact triggered the cleft palate midgut epithelium apoptosis. Meanwhile, atRA was also found to interfere with the regulatory function of TGF-β / Smad signaling system during the disappearance of the upper crest of the palate process, resulting in the inability of both palatal fossa to fuse.