腭突间充质细胞中TGFβ1信号通路介导维甲酸致腭裂的实验研究.docVIP

腭突间充质细胞中TGFβ1信号通路介导维甲酸致腭裂的实验研究 　　【摘要】 目的 探讨转化生长因子β1（TGFβ1）信号通路在维甲酸（RA）致腭裂过程中的作用。方法 选取8～10周龄野生型小鼠， 构建RA诱导腭裂的小鼠模型， 分别取E13.5（E13.5组）、E15.5（E15.5组）和E17.5（E17.5组）的腭突， 荧光定量PCR（q-PCR）检测TGFβ1基因的表达， 信号通路检测法（Western blot）检测下游p-Smad2（S467）的表达；RA处理原代培养的腭突间充质细胞（MEPM）， q-PCR检测TGFβ1基因的表达， Western blot检测下游p-Smad2（S467）的表达。RA和TGFβ1共同处理MEPM， CCK-8法检测细胞的增殖活性。结果 RA诱导的腭裂小鼠中， E13.5组和E15.5组腭突中TGFβ1的表达显著下降（P0.05）。p-Smad2（S467）的表达也呈现同样的趋势。RA显著抑制了MEPM中TGFβ1和p-Smad2（S467）的表?_， 且呈浓度依赖性。RA和TGFβ1共处理组的细胞增殖活性较RA单纯处理组显著上升， 体外添加TGFβ1重组蛋白能有效逆转RA对MEPM增殖的抑制作用。结论 MEPM中TGFβ1信号通路在RA致腭裂过程中发挥着重要作用。 　　【关键词】 维甲酸；腭裂；转化生长因子β1；腭突间充质细胞 　　DOI：10.14163/j.cnki.11-5547/r.2016.32.090 　　【Abstract】 Objective To investigate effect by signal channel of transforming growth factor β1 （TGFβ1） in embryonic palate mesenchymal cells in retinoic acid （RA）-induced cleft palate. Methods RA-induced cleft palate rat model was established on 8～10-week-old wild mice. Palatine process of E13.5（E13.5 group）， E15.5（E15.5 group） and E17.5（E17.5 group） were taken， along with quantitative PCR（q-PCR） for TGFβ1 gene expression detection and Western blot for downstream p-Smad2 （S467） expression detection. RA was used to process embryonic palate mesenchymal cells （MEPM） in primary culture， along with q-PCR for TGFβ1 gene expression detection and Western blot for downstream p-Smad2 （S467） expression detection. Combination of RA and TGFβ1 was used to process MEPM cells， along with CCK-8 for cellular proliferative activity detection. Results Among rats with RA-induced cleft palate， E13.5 group and E15.5 group had obviously reduced TGFβ1 expression （P0.05）. Their p-Smad2 （S467） expression showed the same pattern. Expression of TGFβ1 and p-Smad2 （S467） expressions in MEPM cells were obviously inhibited by RA， along with concentration dependent manner. Cellular proliferative activity in combined process by RA and TGFβ1 was higher than single RA. External addition of TGFβ1 recombinant protein effectively reversed inhibiting effect by RA on MEPM c