高渗应激下ERK信号转导通路在兔髓核细胞凋亡中的作用

Effect of extracellular signal-regulated kinase on apoptosis of nucleus pulposus cells by hypertonic stress

[目的]观察高渗透压对体外培养的兔髓核细胞凋亡的影响及细胞外信号调节激酶(ERK1/2)信号转导通路在此过程中的作用。[方法]用不同的渗透压梯度以及ERK1/2特异性抑制剂PD 98 059(50μmol/L)分不同作用时间处理髓核细胞;流式细胞仪检测髓核细胞在不同处理组的凋亡情况,蛋白质免疫印迹技术检测各组ERK1/2和p-ERK1/2蛋白的表达情况;免疫荧光标记技术检测p-ERK1/2蛋白在髓核细胞内的分布情况。[结果]高渗透压(600 mOsm)培养基中兔髓核细胞凋亡显著增加,与对照组相比差异有统计学意义(P=0.013),并呈时间依赖性;PD98059预处理组与相应时间段同渗透压组相比,细胞凋亡进一步显著地增加(P=0.035);400 mOsm组及其抑制组在各时间段细胞凋亡均不明显;600 mOsm高渗透压显著激活p-ERK1/2的表达,并且在3 h组表达水平最高,与对照组比较差异显著(P=0.027),而PD98059几乎阻断p-ERK1/2的表达;免疫荧光标记检测到p-ERK1/2在600 mOsm高渗组的细胞胞浆和胞核中均有分布。[结论]高渗透压(600 mOsm)应激下兔髓核细胞凋亡显著增加,而且激活的ERK1/2信号转导通路具有抵抗髓核细胞凋亡的作用;并且髓核细胞能够适应轻度增加的渗透压(400mOsm)环境。

[ Objective ] To observe the apoptosis of primary culture rabbit nucleus pulposus cells in hypertonic environment and the effect of extracellular signal - regulated kinase/ERK signal pathways in this process. [ Methods ] After treatment with different osmotic pressure gradients and time, flow cytometer/FCM was used to detect the apoptosis, Western blotting technology was used to analyse the expression of phospho - ERK1/2, and immunofluorescence was done to observe the distribution of phos- pho - ERK1/2 in the nucleus pulposus cells treated with hypertonic mediums. [ Results ] Total 600 mOsm hypertonic mediums powerfully induced nucleus pulposus cells apoptosis compared with control groups （P = 0. 013 ）, and that appeared time depend- ence. However, pretreatment groups with PD98059 （50 p^M） further increased apoptosis （P = 0. 035） . Apoptosis was not ob- vious in 400 mOsm hypertonic mediums with or without PD98059. 600 mOsm hypertonic mediums markedly increased the expres- sion of phospho - ERK1/2 compared with control groups （P = 0. 027 ）, and the expression level was highest after 3 hours. While PD98059 （50 I^M） almost blocked expression of phospho -ERK1/2. The results of immunofluorescence showed that phospho - ERK1/2 dispersed throughout cytoplasm and nucleus. [ Conclusion] In hypertonic mediums （600mOsm） nucleus pulposus cells apoptosis obviously increased and anti - apoptosis of nucleus pulposus cells was related to activation of ERK1/2, moreover nucleus pulposus ceils could adapt low- grade hypertonic environment （400mOsm） .