【摘要】 目的研究骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)对退变髓核细胞炎症反应和凋亡的影响,并探究其与PI3K/Akt信号通路的关系。方法通过氧糖剥夺(oxygen glucose deprivation,OGD)培养建立退变髓核细胞模型;重组腺相关病毒(adeno-associated virus,AAV)将BMP9转染入人髓核细胞内(human nucleus pulposus cells,HNPCs),实验共分5组:Control组(正常培养的HNPCs)、OGD组(氧糖剥夺模型)、AAV组(氧糖剥夺模型,转染AAV空载体)、AAV-BMP9组(氧糖剥夺模型,转染AAV-BMP9),AAV-BMP9+LY294002组(AAV-BMP9组基础上添加PI3K抑制剂LY294002)。免疫荧光方法检测蛋白聚糖(Aggrecan)和Ⅱ型胶原(typeⅡcollagen,Col2a1)的表达;Western blot检测p-Akt和p-m TOR的蛋白表达;酶联免疫吸附实验检测细胞培养上清液中TNF-α、IL-1β、IL-6和IL-8的表达;流式细胞术检测细胞的凋亡。结果在氧糖剥夺培养模型中Aggrecan和Col2a1表达明显降低;AAV-BMP9组中p-Akt和p-m TOR的表达明显高于OGD组和AAV组(P<0.05);此外,AAV-BMP9组HNPCs分泌的TNF-α、IL-1β、IL-6和IL-8明显减少,细胞凋亡被显著抑制(P<0.05);LY294002的加入能够显著逆转BMP9的上述效果。结论 BMP9能够抑制退变HNPCs的炎症反应和凋亡,并且这种作用是通过激活PI3K/Akt信号通路实现的。更多还原

【Abstract】 Objective To investigate the effects of bone morphogenetic protein 9( BMP9) on inflammatory response and apoptosis of degenerative nucleus pulposus cells and to mechanistically explore its association with PI3 K / Akt signaling pathway. Methods The degenerative nucleus pulposus cells were generated by oxygen glucose deprivation( OGD) method. BMP9 gene was transinduced into human nucleus pulposus cells( HNPCs) by recombinant adeno-associated virus( AAV) mediated gene transfer. According to the transfection and treatment,HNPCs were divided into 5 groups: Control group( cultured HNPCs without treatment),OGD group( OGD model),AAV group( OGD + AAV empty vector),AAV-BMP9 group( OGD+ AAV-BMP9),and AAV-BMP9 + LY294002 group( OGD + AAV-BMP9 + PI3 K inhibitor LY294002).Immunofluorescence staining was used to detect Aggrecan and type Ⅱ collagen( Col2a1). Western blot were performed to detect the protein expression of p-Akt and p-m TOR. Enzyme linked immunosorbent assay( ELISA) was carried out to measure the levels of TNF-α,IL-1β,IL-6 and IL-8 in the supernatant of medium. Flow cytometry was performed to detect cell apoptosis. Results The expression of Aggrecan and Col2a1 was significantly decreased in OGD model. The protein expression of p-Akt and p-m TOR was higher in AAV-BMP9 group than those in OGD and AAV groups( P < 0. 05). In addition,the levels of TNF-α,IL-1β,IL-6 and IL-8 were significantly decreased( P < 0. 05) and cell apoptosis was markedly reduced in HNPCs of AAV-BMP9 group. But,LY294002 treatment significantly reversed the above-mentioned effects induced by BMP9 overexpression in degenerative HNPCs. Conclusion BMP9 inhibits the inflammatory response and apoptosis of degenerative HNPCs through activating PI3 K / Akt signaling pathway.更多还原