Shh信号通路变化对小鼠胚胎腭突间充质细胞自噬的影响

The effect of Shh signaling pathway changes on autophagy in mouse embryonic palatal mesenchymal cells

摘要:

目的 探讨地塞米松（dexamethasone，DEX）及平滑激动剂（smoothened agonist，SAG）干预下小鼠胚胎腭突间充质细胞Shh信号通路改变后，小鼠胚胎腭突间充质细胞自噬状态的变化。方法 体外培养胚胎14.5 d的C57BL/6J小鼠胚胎腭突间充质细胞，根据干预方式分为4组：对照组、地塞米松组（DEX组）、地塞米松+SAG组（DEX+SAG组）、单独SAG干预组（SAG组）。通过透射电镜观察各组细胞自噬体或自噬溶酶体的数量，Western blot检测各组Shh、Ptch1、Smo、Gli3A/R、Cyclin D1及自噬标志物LC3Ⅱ/Ⅰ和P62、Beclin-1的表达情况。结果 电镜结果显示，对照组和DEX 组中自噬体/自噬溶酶体的数量无明显差异，而SAG干预后可观察到大量自噬体/自噬溶酶体。Western blot结果显示，与对照组相比，P62、Ptch1、Smo、Gli3A/R、CyclinD1在DEX组中明显降低（P<0.05），LC3Ⅱ/Ⅰ、Beclin1表达无明显变化（P>0.05）；DEX+SAG组及SAG组中Ptch1、Smo、Gli3A/R、CyclinD1的表达与DEX组相比明显升高（P<0.05），Shh表达无明显差异（P>0.05），Beclin1表达明显升高（P<0.05），P62表达明显降低（P<0.05）；与SAG组相比，DEX+SAG组中Smo、Gli3A/R、Ptch1、CyclinD1表达明显降低（P<0.05），Shh表达无明显差异（P>0.05），LC3Ⅱ/Ⅰ、Beclin1明显升高（P<0.05），P62表达明显降低（P<0.05）。结论 DEX可抑制小鼠胚胎腭突间充质细胞的Shh信号通路，激活Shh信号通路，诱发小鼠胚胎腭突间充质细胞产生自噬且Shh通路抑制后再激活，细胞自噬更加明显。

Abstract:

Objective To explore the changes of autophagy of mouse embryonic palatal mesenchymal cells after the changes of Shh signaling pathway in mouse embryonic palatal mesenchymal cells treated with dexamethasone（DEX） and smoothened agonist（SAG）.Methods C57BL/6J mouse palatal mesenchymal cells were cultured in vitro for 14.5 days and divided into 4 groups according to the interventions：blank control group，DEX group，DEX + SAG group and SAG alone group. The number of autophagosomes or autophagy lysosomes in each group was observed by transmission electron microscope，and the expression of Shh，Ptch1，Smo，Gli3A/R，Cyclin D1 and autophagy markers LC3 Ⅱ/Ⅰ，P62 and Beclin-1 were detected by Western blot.Results The results of electron microscopy showed that there was no significant difference in the number of autophagosomes/autophagy lysosomes between control group and DEX group， but a large number of autophagosomes/autophagy lysosomes could be observed after SAG intervention. The results of Western blot showed that compared with the control group，the expression of P62， Ptch1，Smo，Gli3A/R and Cyclin D1 in DEX group decreased significantly（P<0.05），while the expression of LC3Ⅱ/Ⅰ and Beclin1 had no significant change in DEX + SAG group and SAG group（P>0.05）. The expression of Ptch1，Smo，Gli3A/R and Cyclin D1 in DEX+SAG group and SAG group was significantly higher than that in DEX group（P<0.05），but there was no significant difference in Shh expression（P>0.05）. The Beclin1 expression increased significantly（P<0.05） and P62 expression significantly decreased（P<0.05）. Compared with SAG group，the expression of Smo，Gli3A/R，Ptch1 and Cyclin D1 in DEX+SAG group was significantly lowered down（P<0.05），while the expression of Shh was not significantly different（P>0.05）. And LC3Ⅱ/Ⅰ，Beclin1 and P62 expression were significantly increased（P<0.05）.Conclusion DEX can inhibit the Shh signaling pathway of mouse embryonic palatal mesenchymal cells and activate Shh signaling pathway. It can induce autophagy of mouse embryonic palatal mesenchymal cells and reactivate the Shh pathway，and autophagy turns more obvious.