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目的探讨过表达骨形成蛋白和激活素的跨膜抑制剂(BMP and activin membrane-bound inhibitor,BAMBI)对谷氨酸诱导的大鼠脊髓背角神经元和星形胶质细胞凋亡的影响。方法原代培养新生Wistar大鼠脊髓背角神经元和星形胶质细胞,将细胞分为6组:正常对照组,谷氨酸组,空载组,BAMBI过表达组,si RNA对照组和BAMBI沉默组。Western blot检测各组细胞中BAMBI的表达,流式检测细胞凋亡,Western blot检测自噬标志分子微管相关蛋白轻链3(microtubule associated protein light chain 3,LC3),Beclin1和p62的表达,荧光显微镜检测GFP-LC3的含量,最后Western blot检测肿瘤坏死因子-α(TNF-α),白介素-1β(IL-1β),裂解后的半胱天冬酶(cleaved-Caspase3)的表达。结果与对照组相比,谷氨酸诱导的细胞凋亡增加,LC3-Ⅰ的表达降低,LC3-Ⅱ,Beclin1和p62的表达增加,GFP-LC3的含量增加,TNF-α、IL-1β和cleaved-Caspase3的表达增加(P<0.05);与谷氨酸组相比,转染pc DNABAMBI的细胞凋亡减少,LC3和Beclin1的表达显著增加,LC3-Ⅰ和p62的表达降低,细胞内可见大量的GFP-LC3堆积,TNF-α、IL-1β和cleaved-Caspase3的表达显著下降(P<0.05)。BAMBI沉默组的结果与过表达组相反。结论过表达BAMBI可增强自噬,抑制谷氨酸诱导的大鼠脊髓背角神经元和星形胶质细胞凋亡。
Objective To investigate the effects of BAMBI overexpression on the apoptosis of neurons and astrocytes induced by glutamate in spinal dorsal horn of rats. Methods Primary cultured neonatal Wistar rats spinal dorsal horn neurons and astrocytes were divided into 6 groups: normal control group, glutamate group, no-load group, BAMBI overexpression group, si RNA control group and BAMBI silence group. Western blot was used to detect the expression of BAMBI in each group. Flow cytometry was used to detect the apoptosis of cells. Western blot was used to detect the expression of autophagy marker protein LC3, Beclin1 and p62, The content of GFP-LC3 was detected by microscope. The expression of TNF-α, IL-1β and cleaved-Caspase3 were detected by Western blot. Results Compared with the control group, glutamate-induced apoptosis increased, the expression of LC3-Ⅰ decreased, the expression of LC3-Ⅱ, Beclin1 and p62 increased, the content of GFP-LC3 increased, and the levels of TNF-α, IL- The expression of cleaved-Caspase3 increased (P <0.05). Compared with glutamate group, the apoptosis of pcDNABAMBI transfected cells decreased, the expression of LC3 and Beclin1 increased significantly, while the expression of LC3-Ⅰ and p62 decreased A large number of GFP-LC3 accumulation, TNF-α, IL-1β and cleaved-Caspase3 expression was significantly decreased (P <0.05). Results from the BAMBI silencing group were the opposite of the over-expression group. Conclusion Overexpression of BAMBI can enhance autophagy and inhibit glutamate-induced apoptosis of spinal dorsal horn neurons and astrocytes.