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

Experimental research of signal channel of TGFβ1 in embryonic palate mesenchymal cells in mediation of retinoic acid-induced cleft palate

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(P<0.01),到E17.5时,与对照组的表达水平相当(P>0.05)。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)的表达,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。

Objective To investigate effect by signal channel of transforming growth factorβ1 （TGF β1） in embryonic palate mesenchymal cells in retinoic acid （RA）-indueed cleft palate. Methods RA-induced cleft palate rat model was established on 8-10-week-old wild mice. Palatine process of EI3.5（E13.5 group）, E15.5（E15.5 group） and E17.5（E17.5 group） were taken, along with quantitative PCR（q-PCR） for TGF β1 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 TGF β1 gene expression detection and Western blot for downstream p-Smad2 （S467） expression detection. Combination of RA and TGF β1was used to process MEPM ceils, along with CCK-8 for cellular proliferative activity detection.Results Anmng rats with HA-induced cleft palale, E13.5 group and E15.5 group had obvi^llsly ~＇educed TGFβ1 expressima （P〈0.01）, which was at similar level as the conlral group irl E17.5 （P〉0.05）. Their p-Snmd2 （S467） expression showed Ihe s,~ill＇le patlern. Expre.~sion of TGFβ1 and p-Smad2 （S467） expressiotls in MEPM cells were obviously inhibited },y RA, along wilh ~：oncentrati^m dependenl manner. Cellular proliferative aclivily in combined process by RA and TGFβ1was higher than si^gle I/A. Exler^ml addiiio~＊ ~t＂ T（~i!＇ ~ 1 revoml^inant pro｜ein effeelively reversed inhibiting ell＇eel by RA on MEPM cells pmliferali^m. Conclusion Signal channel af TCF 13 1 in MEPM shows significanl effect in relinoic aci（t-i^l（]uc^ed cleft palate.