血红素氧合酶1对TNF-α诱导的人退变椎间盘髓核细胞凋亡的作用研究  

Effect of heme oxygenase 1 on the apoptosis of human degenerated nucleus pulposus cells induced by tumor necrosis factor α

作　　者：侯孝忠 徐林飞 沈皆亮[3] 胡侦明[3] 

机构地区：[1]河南省省立医院骨科,郑州450000 [2]河南省胸科医院骨科,郑州450003 [3]重庆医科大学附属第一医院骨科,重庆400010

出　　处：《中国修复重建外科杂志》2018年第1期69-74,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金面上项目(81372003)~~

摘　　要：目的探讨血红素氧合酶1(heme oxygenase 1,HO-1)对TNF-α诱导的人退变椎间盘髓核细胞凋亡的作用及其可能的分子机制。方法取腰椎间盘突出症患者自愿捐赠的椎间盘组织,体外培养人退变髓核细胞并传代,取第3代细胞进行实验。采用细胞计数试剂盒8测定不同浓度(10、20、50、100和200 ng/mL)TNF-α对髓核细胞活性的影响,筛选最佳刺激浓度进行下一步实验。将髓核细胞分成4组,分别采用单纯基础培养液(对照组)、TNF-α刺激(TNF-α组)、TNF-α和CoPP 10μmol/L刺激(CoPP组)、TNF-α和ZnPP 15μmol/L刺激(ZnPP组)培养,24 h后采用Hoechst染色以及流式细胞仪检测细胞凋亡;Western blot检测凋亡相关蛋白活化型Caspase-3(cleaved Caspase-3)、上皮膜蛋白1(epithelial membrane protein 1,EMP-1)以及HO-1、p-P65的表达。为进一步探讨HO-1对髓核细胞凋亡的潜在分子机制,取髓核细胞分别采用TNF-α刺激(TNF-α刺激组)以及TNF-α和吡咯烷二硫基甲酸(pyrrolidine dithiocarbamate,PDTC)5μmol/L刺激(TNF-α+PDTC刺激组)培养24 h,采用流式细胞仪检测髓核细胞凋亡率。结果经检测确定TNF-α最佳抑制浓度为100 ng/mL。Hoechst染色示,对照组和CoPP组凋亡细胞较少;TNF-α组和ZnPP组可见凋亡样改变细胞核,其中ZnPP组最显著。流式细胞仪检测示,与对照组相比,TNF-α组、CoPP组及ZnPP组细胞凋亡率均显著提高(P<0.05);与TNF-α组相比,CoPP组细胞凋亡率显著降低(P<0.05),而ZnPP组显著提高(P<0.05)。Western blot检测示,与对照组相比,TNF-α组HO-1蛋白表达降低,cleaved Caspase-3、EMP-1及p-P65蛋白表达升高(P<0.05)。与TNF-α组相比,CoPP组HO-1蛋白表达明显升高,cleaved Caspase-3、EMP-1、p-P65蛋白表达明显降低(P<0.05);而ZnPP组HO-1蛋白表达明显降低(P<0.05),cleaved Caspase-3、EMP-1蛋白表达增高(P<0.05),p-P65蛋白表达无明显变化(P>0.05)。与TNF-α刺激组相比,TNF-α+PDTC刺激组细胞凋亡率明显降低(t=3.076,P=0.031)。结论 HObjective To investigate the effect of heme oxygenase 1（HO-1） on the apoptosis of human degenerated nucleus pulposus（NP） cells induced by tumor necrosis factor α（TNF-α）, and explore its possible molecular mechanism. Methods The intervertebral disc tissues were derived from patients with lumbar intervertebral disc herniation. Then, the NP cells were cultured in vitro and the third generation of NP cells were used for subsequent experiments. Cell counting kit 8（CCK-8） method was used to observe the proliferative effect of TNF-α on the NP cells in vitro at the concentration of 10, 20, 50, 100, and 200 ng/mL. The most apropriate concentration was selected according to the result of CCK-8. The NP cells were cultured with basal medium（control group）, TNF-α（TNF-α group）, TNF-α and CoPP 10 μmol/L（CoPP group）, and TNF-α and ZnPP 15 μmol/L（ZnPP group）, respectively. After cultured, the cell poptosis was detected by Hoechst staining and flow cytometry; the expression of cleaved Caspase-3, epithelial membrane protein 1（EMP-1）, HO-1, and p-P65 proteins were detected by Western blot. In order to further explore the potential molecular mechanisms of HO-1 for cell apoptosis, the NP cells were cultured with TNF-α（TNF-α stimulated group）, TNF-α and pyrrolidine dithiocarbamate（PDTC） 5 μmol/L（TNF-α＋PDTC stimulated group）, respectively. Then the cell apoptosis rate was measured by flow cytometry at 24 hours after cultured. Results The optimal concentration of TNF-α was 100 ng/mL. Hoechst staining showed that a few apoptotic cells could be observed in control group and CoPP group; the apoptosis-like nucleis were observed in TNF-α group and ZnPP group, which was the most significant in ZnPP group.Flow cytometry showed that the cell apoptosis rates of TNF-α group, CoPP group, and ZnPP group were significantly increased when compared with the control group（P0.05）. Compared with TNF-α group, the cell apoptosis rate in CoPP group decreased（P0.05）, while in ZnPP gro

关 键 词：血红素氧合酶1 TNF-Α 髓核细胞 细胞凋亡 

分 类 号：R681.5[医药卫生—骨科学]