人退变椎间盘髓核组织中 miR-146a的表达及对 NP细胞增殖、凋亡和炎性细胞因子的影响作用机制

目的　检测人退变椎间盘髓核组织中 miR-146a的表达变化，并探讨其可能的作用机制。方法　实时荧光定量 PCR (qRT-PCR) 检测人退变腰椎间盘髓核组织中 miR-146a表达。用脂多糖（LPS）刺激人髓核（nucleus pulposus，NP）细胞模拟椎间盘变性，qRT-PCR检测 NP细胞中 miR-146a的表达变化。将 miR-146a mimics或 miR-146a inhibitor转染至 NP细胞，然后 LPS刺激 NP细胞，观察 miR-146a对 NP细胞增殖活性、凋亡、 TLR4蛋白表达及 IL-1β,TNF-α和 IL-6等炎性因子水平的影响。结果　miR-146a在椎间盘退变（intervertebral disc degeneration, IVDD组）椎间盘髓核组织中表达（0.65±0.12）明显低于对照组（ 1.01±0.10），miR-146a在 LPS组 NP细胞中表达（ 0.29±0.11）明显低于阴性对照组（1.06±0.07），差异均有统计学意义（ t=11.856，10.229，均 P＜ 0.01）。与阴性对照组比较， LPS组上清液中的 IL-1β, TNF-α和 IL-6含量明显升高，差异均有统计学意义（ t=15.572~23.354，均 P＜ 0.001）。上调 NP细胞 miR-146a表达，能够提高 LPS刺激的 NP细胞的增殖活性（ t=4.436~7.995, 均 P＜ 0.05），降低其凋亡率（ t=9.255，P=0.001），降低 TLR4蛋白（ t=5.881，P=0.004）和 IL-1β，TNF-α和 IL-6等炎性细胞因子水平（ t=8.854~10.772，均 P＜ 0.01）；而下调 NP细胞 miR-146a表达则出现相反的结果（ t=2.977~6.777，均 P<0.05）。结论　miR-146a在退变椎间盘髓核组织中表达降低， miR-146a可能能够通过靶向 TLR4调控 NP细胞的增殖、凋亡和炎症反应，为椎间盘退变的生物治疗提供了新的方向。

Expression of miR-146a in the Nucleus Pulposus Tissue of Human Degenerative Intervertebral Disc and Its Effect Mechanism onthe Proliferation,Apoptosis and Inflammatory Cytokines of NP Cells

Objective　To detect the expression of miR-146a in nucleus pulposus of human degenerative intervertebral disc andexplore its possible mechanism. Methods　qRT-PCR was used to detect the expression of miR-146a in human degenerativelumbar disc nucleus pulposus.Human nucleus pulposus (NP) cells were stimulated with lipopolysaccharide (LPS) to simulateintervertebral disc degeneration. The expression of miR-146a in NP cells was detected by qRT-PCR.miR-146a mimics or miR-146a inhibitor were transfected into NP cells, and then LPS stimulated NP cells. The effects of miR-146a on proliferation,apoptosis, TLR4 protein expression and expression of IL-1β, TNF-α and IL-6 were observed. Results　 The expression ofmiR-146a in the intervertebral disc nucleus pulposus tissue of the (intervertebral disc degeneration IVDD) group (0.65±0.12)was significantly lower than that of the control group (1.01±0.10), and the expression of miR-146a in the NP cells of the LPSgroup (0.29±0.11) was significantly lower than that of the negative control group (1.06±0.07), the difference was statisticallysignificant (t=11.856, 10.229, all P<0.01). Compared with the negative control group, the levels of IL-1β, TNF-α and IL-6 inthe supernatant of the LPS group were significantly increased, and the differences were statistically significant (t=15.572~23.354,all P<0.001) .Up regulation of miR-146a expression in NP cells could increase the proliferation activity (t=4.436~7.995, allP<0.05), reduce the apoptosis rate (t=9.255，P=0.001), and decreased the levels of TLR4 protein (t=5.881，P=0.004) andinflammatory cytokines such as IL-1β, TNF-α and IL-6 (t=8.854~10.772，all P ＜ 0.01), while down regulating the expressionof miR-146a in NP cells had the opposite effect (t=2.977~6.777, all P<0.05). Conclusion　The expression of miR-146a innucleus pulposus of degenerative intervertebral disc was decreased. MiR-146a may be able to regulate the proliferation, apoptosisand inflammatory reaction of NP cells by targeting TLR4, which provides a new direction for the biological treatment ofintervertebral disc degeneration.