Shh-BMSCs外泌体调控miR-133a对脂多糖诱导脊髓神经元细胞凋亡的影响  

Impact of Shh-BMSCs exosome regulating miR-133a on lipopolysaccharide-induced apoptosis of spinal cord neurons

作　　者：贾祎佳 陆廷盛 陈啟鸰 杨建文 欧阳北平 杨再松 罗春山 JIA Yijia;LU Tingsheng;CHEN Qiling;YANG Jianwen;OUYANG Beiping;YANG Zaisong;LUO Chunshan(Department of Spinal Orthopedics,Guizhou Orthopedics Hospital,Guiyang 510014,China)

机构地区：[1]贵州省骨科医院脊柱科,贵州贵阳550014

出　　处：《西部医学》2023年第6期822-829,共8页Medical Journal of West China

基　　金：贵州省科技计划项目[黔科合基础-ZK(2022)一般231]。

摘　　要：目的探讨音猬因子(Shh)-骨髓间充质干细胞(BMSCs)外泌体调控miR-133a对脂多糖(LPS)诱导脊髓神经元细胞凋亡的影响。方法体外培养大鼠脊髓神经元细胞,通过CCK-8实验检测0、100、300、500、700、900μg/mL的LPS对其细胞活力的影响,筛选最佳作用浓度。然后以0、20、40、80、120、160μg/mL的Shh-BMSCs外泌体处理LPS诱导的脊髓神经元细胞,通过同样方法筛选最佳作用浓度。将大鼠脊髓神经元随机分为对照组、模型组、Shh-BMSCs外泌体组、miR-133a inhibitor阴性对照组、miR-133a inhibitor组、Shh-BMSCs外泌体+miR-133a inhibitor组,除对照组外,其余各组以LPS诱导脊髓损伤(SCI)细胞模型,然后分组处理后,流式细胞术检测各组细胞凋亡情况;免疫荧光染色检测各组细胞轴突长度;酶联免疫吸附测定(ELISA)法检测各组细胞肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-10释放水平;实时荧光定量PCR检测各组细胞miR-133a和Shh mRNA表达;免疫印迹实验检测各组细胞Shh及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性信号相关蛋白表达。结果与对照组比较,模型组脊髓神经元细胞轴突长度、IL-10释放水平、miR-133a表达、Shh mRNA及蛋白表达明显降低(均P<0.05),凋亡率、TNF-α及IL-1β释放水平、NLRP3、ASC、caspase-1蛋白表达明显升高(均P<0.05)。与模型组比较,Shh-BMSCs外泌体组脊髓神经元细胞轴突长度、IL-10释放水平、miR-133a表达、Shh mRNA及蛋白表达升高(P<0.05),凋亡率、TNF-α及IL-1β释放水平、NLRP3、ASC、caspase-1蛋白表达降低(均P<0.05);miR-133a inhibitor组脊髓神经元细胞轴突长度、IL-10释放水平、miR-133a表达、Shh mRNA及蛋白表达降低(P<0.05),凋亡率、TNF-α及IL-1β释放水平、NLRP3、ASC、caspase-1蛋白表达升高(均P<0.05);miR-133a inhibitor阴性对照组脊髓神经元细胞各指标均无明显变化(P>0.05)。与Shh-BMSCs外泌体组比较,Shh-BMSCs外泌体+miR-133a inhibitorObjective To study the impact of sonic hedgehog(Shh)-bone marrow mesenchymal stem cells(BMSCs)exosomes regulating miR-133a on lipopolysaccharide(LPS)-induced apoptosis of spinal cord neurons.Methods Rat spinal cord neurons were cultured in vitro,and the effects of 0,100,300,500,700,900μg/mL LPS on cell viability were detected by CCK-8 assay,and the optimal concentration was screened.Then LPS-induced spinal cord neurons were treated with 0,20,40,80,120,and 160μg/mL of Shh-BMSCs exosomes,and the optimal concentration was screened by the same method.Rat spinal cord neurons were randomly grouped into control group,model group,Shh-BMSCs exosome group,miR-133a inhibitor negative control group,miR-133a inhibitor group,and Shh-BMSCs exosome+miR-133a inhibitor group.Except for the control group,the other groups were induced by LPS to induce the SCI cell model,and then after grouping,the apoptosis of each group was detected by flow cytometry;immunofluorescence staining was used to detect the length of cell axon in each group;enzyme-linked immunosorbent assay(ELISA)method was used to detect the release levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-10 in each group;real-time fluorescence quantitative PCR was used to detect the expression of miR-133a and Shh mRNA in cells in each group;and Western blot was used to detect the expression of Shh and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammatory signal-related proteins in cells in each group.Results Compared with the control group,the spinal cord neuron cell axonal length,IL-10 release level,miR-133a expression,Shh mRNA and protein expression were significantly decreased in the model group(P<0.05),the apoptosis rate,TNF-αand IL-1βrelease levels,NLRP3,ASC,and caspase-1 protein expression were significantly increased(P<0.05).Compared with the model group,the spinal cord neuron cell axonal length,IL-10 release level,miR-133a expression,Shh mRNA and protein expression were increased in the Shh-BMSCs exosome group(P<0.0

关 键 词：Shh-BMSCs外泌体 脂多糖 脊髓神经元 凋亡 

分 类 号：R745.4[医药卫生—神经病学与精神病学]