不同浓度甲基丙烯酸甲酯对SD大鼠乳鼠脊髓背根神经元的影响

摘要

Objective To explore the effect of bone cement monomer methyl methacrylate (MMA) on SD rat spinal cord dorsal root ganglion neurons in vitro.Methods The concentration range and trends of MMA monomer released from bone cement samples made up and eluted in PBS once per 10 min by simulating vertebralplasty in vitro were determined by headspace solid phase microextraction and gas chromatography.Neonatal rat dorsal root ganglions were extracted under the microscope operation,and cells were digested and purified by the 0.25％ trypsin.Neonatal rat dorsal root ganglion cells (DRGs) were cultured in DMEM-F12 medium in vitro,and identified by immunofluorescence dyeing with neuron specificity enolization enzymes (NSE).At the 7th day,DRGs were cultivated in medium contained with MMA and were divided into 6 groups on the basis of concentration of MMA,including of control group and MMA group (10-3,10-2,10-1,1,10 g/L).Every group was divided into two subgroups.One subgroup was dealed with trypan blue staining to evaluate the survival rate after 10 min cultivation;other group was dealed with flow cytometry detection test by Annexin-V method to analyze cell apoptosis induced by MMA monomer after 60 min cultivation.Results A large number of monomer were released when the bone cement was implanted.90％ monomers could be released within 10 main,then the releasing process was almost consistent and the released quantity trend to stabilization with the elongation of time.The maximum concentration range of MMA monomer within 60 min was (0.102 5 ± 0.006 8) g/L.The different concentration of MMA (0,10-3,10-2 g/L) had no statistical significances that cell survival rate was (97.00 ± 1.87)％,(95.00 ± 2.24) ％ (P =0.380),(92.20 ± 2.77) ％ (P =0.070),respectively.Monomer concentration of 10-1 g/L had obvious cytotoxicity,the cell survival rate was (89.40 ± 5.18)％ (P ＜ 0.05).Cell survival rate was (86.20 ±4.32) ％ (P ＜ 0.05) when monomer concentration was up to 1 g/L.Cell survival rate was (77.60 ± 3.78) ％ (P ＜0.05) when monomer concentration was up to 10 g/L.MMA monomer could induce apoptosis of DRGs that the apoptosis rate of blank control group was (22.6 ± 5.03)％.The different concentration of MMA (10-3,10-2,10-1,1,10 g/L) induced cell apoptosis that apoptosis rate was (38.3 ± 2.19) ％,(40.5 ± 1.07) ％,(44.1 ± 3.45) ％ and (48.0±l.81)％,(54.82±2.72)％,respectively (P＜0.05).Conclusion MMA monomer released from percutaneous vertebroplasty or percutaneous kyphobroplasty had certainly toxicity to sensory nerve cells in centruns.The toxiceffect of MMA monomer was significant to the rapid analgesia mechanism.%目的 体外实验探索骨水泥单体甲基丙烯酸甲酯(methyl methacrylate,MMA)对斯普拉格大鼠(Sprague Dawley,SD)乳鼠脊髓背根神经元的影响.方法 体外模拟椎体成形术制备骨水泥球,采用气相色谱法测定MMA单体释放的浓度范围及变化趋势;显微操作下提取乳鼠背根神经节并体外培养乳鼠脊髓背根神经细胞(dorsal root ganglion neurons,DRGs),用神经元特异性烯醇化酶(neuron specificity enolization enzymes,NSE)免疫荧光化学染色鉴定DRGs;根据体外实验模拟椎体成形术测得的MMA释放浓度范围,DRGs加入含有不同浓度MMA的培养基,设为正常对照组,10-3,10-2,10-1,1和10 g/L MMA组;采用台盼蓝染色法测定神经元存活率,采用Annexin V法进行流式细胞检测,分析MMA单体诱导的DRGs细胞凋亡情况.结果 骨水泥在植入后前10min内释放出大量单体,约占总释放量的90％以上,随着时间延长,单体释放总量基本保持平稳.60 min内MMA单体最大浓度范围为(0.1025±0.0068) g/L.DRGs加入MMA单体作用后,10-3和10-2 g/L组的细胞存活率分别为(95.00±2.24)％、(92.20±2.77)％,与对照组(97.00±1.87)％比较,差异无统计学意义(P值分别0.380和0.070);10-1、1、10 g/L细胞存活率分别为(89.40±5.18)％、(86.20±4.32)％、(77.60±3.78)％,与对照组比较,差异有统计学意义(P均＜0.05).MMA单体诱导的DRGs的凋亡,MMA单体10-3、10-2、10-1、1、10 g/L均能够诱导细胞的凋亡,凋亡率分别为(38.3±2.2)％、(40.5±1.1)％、(44.1±3.5)％、(48.0±1.8)％、(54.8±2.7)％,与空白对照组凋亡率(22.6±5.0)％比较,差异有统计学意义(P均＜0.05).结论 椎体成形术中存在MMA单体的释放,并且对椎体内感觉神经细胞存在毒性,因此MMA单体释放对椎体成形术的快速止痛有一定意义.