低氧环境对椎间盘自发性吸收的影响及其作用机制

目的探讨低氧环境对椎间盘自发性吸收的影响及其作用机制。方法取SPF级成年日本大耳兔9只,雌雄不限,平均体质量2 kg。将兔处死后取脊柱髓核组织,经消化、分离、培养后获得传代髓核细胞,将生长良好的髓核细胞制成细胞悬液。根据不同时效的低氧环境将细胞分为5组对照组(常氧浓度下培养6 h)、低氧6 h组(2%O2浓度下培养6 h)、低氧12 h组(2%O2浓度下培养12 h)、低氧24 h组(2%O2浓度下培养24 h)和低氧48 h组(2%O2浓度下培养48 h)。采用实时聚合酶链反应法检测缺氧诱导因子(HIF)-1α、3型酸敏感离子通道(ASIC3)及水通道蛋白3(AQP3)mRNA表达水平,采用流式细胞仪检测各组髓核细胞凋亡情况。采用SPSS 24.0软件对数据进行分析。结果与对照组比较,低氧各组细胞凋亡率均明显升高,差异均有统计学意义(均P<0.01);与低氧12、24和48 h组比较,低氧6 h组细胞凋亡率最高,差异均有统计学意义(均P<0.01)。与对照组比较,低氧各组细胞HIF-1α和ASIC3 mRNA表达水平均明显上升,AQP3 mRNA表达水平均明显下降,差异均有统计学意义(均P<0.01);与低氧12、24和48 h组比较,低氧6 h组HIF-1α和ASIC3 mRNA表达水平最高,差异均有统计学意义(均P<0.01)。结论短时间低氧环境可以促进髓核细胞凋亡,从而加速椎间盘突出组织自发性吸收进程,其机制可能与HIF-1α和ASIC3的表达增加及AQP3的表达下降有关。

Effect of hypoxic environment on spontaneous regression of intervertebral disc and the mechanism of action

Objective To explore the effects of hypoxic environment on spontaneous regression of intervertebral disc and the mechanism of action.Methods The rabbit nucleus pulposus cells were treated under hypoxic environment for different periods and divided into 5 groups:control group(cultured for 6 h under normoxic concentration),hypoxia 6 h group(cultured at 2%O2 concentration for 6 h),hypoxia 12 h group(cultured at 2%O2 concentration for 12 h),hypoxia 24 h group(cultured at 2%O2 concentration for 24 h),hypoxia 48 h group(cultured at 2%O2 concentration for 48 h).Flow cytometry was used to detect apoptosis rate and the expression of hypoxia indueible factors-1α(HIF-1α),acid-sensing ion channel 3(ASIC3)and aquaporin 3(AQP3)mRNA were measured by using real-time PCR.The apoptosis of nucleus pulposus cells was measured by flow cytometry.Results The percentage of apoptotic cells in the hypoxic groups were higher than that in the control group(P<0.01).Compared with the hypoxic group of 12,24 and 48 h,the apoptotic rate was the highest in the hypoxic 6 h group(P<0.01).Compared with the control group,HIF-1αand ASIC3 were increased expression in the hypoxia groups(P<0.01)and the expression of AQP3 were significantly decreased(P<0.01).Compared with the hypoxic group of 12,24 and 48 h,the expression of HIF-1αand ASIC3 mRNA in the hypoxic 6 h group was the highest(P<0.01).Conclusion Short-term hypoxic environment can promote apoptosis of nucleus pulposus cells and accelerate the spontaneous absorption process of intervertebral disc protrusion.The mechanism may be related to the increased expression of HIF-1αand ASIC3 and the decreased expression of AQP3.