【作者】 李延红；

【导师】 皇甫超申； 金家岩；

【作者基本信息】 河南大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 目的:探讨低剂量亚硝酸钠（NaNO₂）预处理对过氧化损伤鼠肾上腺嗜铬细胞瘤（PC12）细胞的保护作用;同时观察其诱导PC12细胞分化的作用。方法:分别以系列浓度的NaNO₂作用PC12细胞不同时间,采用四甲基偶氮唑蓝（MTT）法检测NaNO₂对PC12细胞活力的影响,研究剂量-效应和时间-效应关系,寻找最低促增殖效应剂量和最佳促增殖效应时间。过氧化损伤PC12细胞模型制作采用400 mmol·L^-1乙醇、45 mmol·L^-1 NaNO₂、1.1 mmol·L^-1过氧化氢（H₂O₂）处理2 h。细胞增殖指标采用MTT、活细胞计数法、Hoechst33258荧光单染计数有丝分裂指数;细胞凋亡检测采用Hoechst33258染色计数细胞凋亡率、流式细胞术（FITC-annexinⅤ/ PI）双染法和Hoechst 33258/PI活细胞染色法;比色法测定超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活力和谷胱甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）含量。Western-blotting检测相关蛋白表达。瑞氏染色观察PC12细胞分化状态。结果:MTT结果显示,低剂量NaNO₂促进细胞增殖,高剂量抑制其增殖,剂量-效应关系呈典型的倒U型曲线。NaNO₂最低促增殖剂量为（促增殖效应高于对照组的15%）0.14 mmol·L^-1,最佳促增殖效应时间点出现在第24 h;400 mmol·L^-1乙醇、45 mmol·L^-1的NaNO₂、1.1 mmol·L^-1 H₂O₂作用PC12细胞2 h,流式细胞术结果显示,细胞凋亡率分别为（50.63±2.14）%、（52.06±8.32）%、（53.6±8.51）%,用0.14 mmol·L^-1 NaNO₂预处理细胞24 h,再分别用上述氧化剂作用2 h,细胞凋亡率分别下降为（19.71±3.19）%、（19.65±2.17）%、（18.23±4.19）%差异具有显著性（P﹤0.05）;用0.14 mmol·L^-1 NaNO₂和一氧化氮清除剂（c-PTIO）预处理细胞24 h,再用上述氧化剂作用2 h,细胞凋亡率则分别为（32.28±7.31）%、（38.63±5.08）%、（30.75±3.81）%。Hoechst 33258染色、Hoechst 33258/PI活细胞染色计数细胞死亡率结果与其相似。用400 mmol·L^-1乙醇作用PC12细胞2 h,SOD、CAT酶活性,GSH-Px、MDA含量分别为（20.95±1.68） U/mg. Prot、（11.45±0.93） U/mg. Prot、（41.17±2.72）μmol/mg. Prot、（98.38±11.5）μmol/mg .Prot,用NaNO₂预处理细胞再用乙醇作用,SOD、CAT酶活性和GSH-Px、MDA含量分别为（62.38±2.98）U/mg. Prot、（19.29±0.27） U/mg. Prot、（62.59±1.41）μmol/mg.Prot、（49.38±2.93）μmol/mg.Prot;用NaNO₂+c-PTIO预处理细胞24 h,再用乙醇作用2 h,SOD、CAT酶活性和GSH-Px、MDA含量分别为（40.23±1.57）U/mg.Prot、（15.98±1.9） U/mg.Prot、（51.21±1.52）μmol/mg.Prot、（73.58±14.7）μmol/mg.Prot。用NaNO₂预处理再用乙醇作用组,与单纯乙醇组相比,Bax、Caspase-9、Caspase-3蛋白表达量降低,Bcl-2和HIF^-1α蛋白表达量升高（P﹤0.05）;c-PTIO可以部分逆转这种现象。45 mmol·L^-1 NaNO₂和1.1 mmol·L^-1 H₂O₂处理细胞,上述各指标变化情况与乙醇组一致。瑞氏染色结果显示,0.14 mmol·L^-1 NaNO₂作用PC12细胞48 h,明显促进PC12细胞突起生长,缺氧诱导因子(HIF^-1α)、血管内皮生长因子（VEGF）表达量增加,作用效果类似神经生长因子（NGF）。c-PTIO可以部分抑制这种现象。结论:1.低剂量NaNO₂促进PC12细胞增殖,高剂量抑制其增殖,剂量-效应关系呈典型的倒U型曲线。2.低剂量NaNO₂预处理能够使PC12细胞抗氧化酶活性增强、脂质过氧化水平降低、HIF^-1α的表达增加,对过氧化损伤的细胞有保护作用。3.低剂量NaNO₂可以促进PC12细胞分化。更多还原

【Abstract】 Objective: To study the cytoprotective role of Low-dose sodium nitrite preconditioning against the peroxide-induced damage and to observe the differentiation of sodium nitrite in PC12 cells.Methods: PC12 cells were treated with different concentrations of sodium nitrite for different times, the cell viability was measured by MTT to find the minimum sodium nitrite dose and the optimum time which could promote the cell proliferation . The PC12 cells were treated with 400 mmol·L^-1 ethanol, 45 mmol·L^-1 NaNO₂ and 1.1 mmol·L^-1H₂O₂ for 2h to establish the peroxide-induced damaged cell model.The proliferation of PC12 cell was detected by MTT, cell counting and Hochest33258 staining. FITC-annexinⅤ/PI flow cytometry , Hoechst33258 staining and Hoechst 33258/PI staining were used to detect the cells apoptosis. Colorimetric technique was performed to measure the SOD, CAT and GSH-Px , MDA. Western-blotting to detect the expression of the apoptosis-related protein. Wright’s staining to observe the differentiation of PC12 cell.Results: Dose-responses results showed that NaNO₂ on PC12 cells proliferation was typical inverted U-shaped curve.The concentration of the minimum stimulatory response was 0.14 mmol·L^-1（more than 15% of the control group）and the maximum stimulatory response at the 24 h. The PC12 cells were treated with 400 mmol·L^-1 ethanol, 45 mmol·L^-1 NaNO₂ and 1.1 mmol·L^-1 H₂O₂ for 2 h, Flow cytometry assay show the apoptosis rate of the cells were （50.63±2.14）%, （52.06±8.32）%, （53.6±8.51）% respectively. The PC12 cells were pretreated with 0.14 mmol·L^-1 NaNO₂ for 24 h then treated with the oxygenants above-metioned for 2 h, the apoptosis rate of the cells were decreased as （19.71±3.19）%, （19.65±2.17）%, （18.23±4.19）% respectively（P﹤0.05）. While NaNO₂ and nitric oxide specific scavenger c-PTIO combined to treat the cells for 24 h, then treated with the oxygenants above-metioned for 2 h, the apoptosis rate of the cells were （32.28±7.31）%, （38.63±5.08）%, （30.75±3.81）% respectively. The results of Hoechst33258 staining, Hoechst 33258/PI staining and cell counting are same as flow cytometry. The PC12 cells were treated with 400 mmol·L^-1 ethanol, the activities of SOD, CAT, GSH-Px and the levels of MDA were （20.95±1.68）U/mg.Prot, （11.45±0.93） U/mg.Prot, （41.17±2.72）μmol/mg.Prot, （98.38±11.5）μmol/mg.Prot respectively. The PC12 cells were pretreated with NaNO₂ then treated with ethanol, the activities of SOD, CAT and the levels of GSH-Px, MDA were （92.38±2.98） U/mg. Prot, （19.29±0.27 ） U/mg.Prot, （62.59±1.41）μmol/mg.Prot, （49.38±2.93）μmol/mg.Prot. While NaNO₂ and nitric oxide specific scavenger c-PTIO combined to treat the cells for 24 h, then treated with the oxygenants above-metioned for 2 h, the activities of SOD, CAT and the levels of GSH-Px, MDA were （50.23±1.57） U/mg.Prot, （15.98±1.9） U/mg.Prot, （51.21±1.52）μmol/mg.Prot, （73.58±14.7）μmol/mg.Prot. Western blotting showed that in ethanol treated cells pretreated with NaNO₂, the expression of Bax, Caspase-9, Caspase-3 were decreased, HIF-1αand Bcl-2 were increased（P﹤0.05）. Add to c-PTIO could reverse this phenomenon. The results of 45 mmol·L^-1 NaNO₂ and1.1 mmol·L^-1 H₂O₂ were same as ethanol group. The PC12 cells were treated with 0.14 mmol·L^-1 NaNO₂ for 48h, the results of wright’s staining showed that NaNO₂ have the same effects of NGF, including prolong the prominency of the cells, and increase the expressions of HIF-1 and VEGF .Conclusions:1. Dose-responses results showed that NaNO₂ on PC12 cells proliferation was typical inverted U-shaped curve.2. The PC12 cells were pretreated with low dose of NaNO₂ could increase their anti-oxidant activities and expressions of HIF-1α, resist the apoptosis induced by ethanol, high dose of NaNO₂ and H₂O₂. The mechanism might be related with the reduction of NaNO₂ to NO and the increased expressions of HIF-1α.3. NaNO₂ could promote the differentiation of PC12 cells.更多还原