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目的探讨神经元型一氧化氮合成酶(nNOS)抑制剂7-硝基吲唑(7-nitroindazole,7-NI)对氟致体外培养细胞生长、凋亡保护作用的机制。方法体外培养SH-SY5Y细胞,在培养液中加入不同浓度Na F(0、0.02、0.2、2.0、4.0 mmol/L),分别培养24、48、72 h,同时在各组细胞培养液中联合应用7-NI(10~(-4)、10~(-3)、10~(-2) mmol/L);于倒置显微镜下观察细胞生长,以CCK-8试剂盒方法检测细胞存活率,以比色法和硝酸还原酶法测定细胞培养液中NO含量及NOS活力,以流式细胞仪检测细胞凋亡情况。结果与对照组比较,氟浓度为0.2 mmol/L(低氟组)和2.0 mmol/L(高氟组)时,24、48、72 h后细胞存活率分别下降至(83.76±1.04)%,(70.27±1.20)%,(54.40±0.98)%和(52.17±1.07)%,(40.60±1.11)%,(29.50±1.40)%。10~(-4)mmol/L 7-NI可刺激细胞增殖,24 h细胞存活率为(105.96±3.44)%;10~(-3) mmol/L 7-NI处理细胞24 h后的细胞存活率为(100±2.30)%;10~(-2) mmol/L 7-NI可使细胞存活率下降,24 h细胞存活率为(88.64±4.75)%。10~(-4)、10~(-3)、10~(-2) mmol/L 7-NI处理细胞24 h后细胞培养液中NO含量、NOS活力均降低,分别为(1.156±0.356)、(0.958±0.074)、(0.672±0.188)μmol/L和(0.403±0.089)、(0.398±0.010)、(0.103±0.076)U/ml。高剂量(10~(-2) mmol/L)7-NI组的SH-SY5Y细胞凋亡指数升高至(8.7±2.3)%,高于对照组(P<0.05);低剂量(10~(-4)、10~(-3) mmol/L)7-NI组的凋亡指数分别为(0.6±0.4)%和(1.0±0.2)%,与对照组差异无统计学意义(P>0.05);低剂量7-NI组凋亡指数低于高剂量组,差异有统计学意义(P<0.05)。染氟24、48、72 h后细胞培养液中NO含量及NOS活力升高,低氟组的NO含量、NOS活力分别为(3.074±0.160)、(5.604±1.374)、(10.173±1.307)μmol/L和(0.743±0.122)、(0.959±0.054)、(1.446±0.12)U/ml,高氟组分别为(8.974±0.919)、(14.729±1.241)、(20.976±0.712)μmol/L和(1.086±0.024)、(1.728±0.130)、(2.583±0.192)U/ml,高于对照组[(1.320±0.280)、(2.420±0.658)、(2.867±0.662)μmol/L,(0.452±0.012)、(0.517±0.072)、(0.607±0.013)U/ml],差异有统计学意义(P<0.05)。与氟单独处理组相比,NaF联合应用7-NI后,各组NO含量及NOS活力均下降,差异有统计学意义(P<0.05)。与对照组相比,氟可使SH-SY5Y细胞凋亡指数升高,差异有统计学意义(P<0.01),并随着氟染毒剂量及时间的增加,细胞凋亡指数随之增加;联合应用7-NI后,各组细胞凋亡指数下降,差异有统计学意义(P<0.01)。氟可使SH-SY5Y细胞数量减少,形态变为圆形、不规则形,细胞存活率降低;低剂量(10~(-4)、10~(-3) mmol/L)7-NI组在相同视野下细胞数增加,高剂量(10~(-2) mmol/L)7-NI组细胞数明显减少,大部分细胞呈圆形或不规则形。结论氟中毒所致细胞凋亡可能与NOS活力和NO含量升高有关,7-NI可降低细胞凋亡指数。
Objective To investigate the mechanism of neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (7-NI) on the cell growth and apoptosis induced by fluoride in vitro. Methods SH-SY5Y cells were cultured in vitro, and different concentrations of NaF (0,0.02,0.2,2.0,4.0 mmol / L) were added to the culture medium for 24, 48, and 72 h, respectively, and combined with cell culture medium Application of 7-NI (10 -4, 10 -3, 10 -2 mmol / L); observed under inverted microscope cell growth, cell viability was detected by CCK-8 kit method, The content of NO and the activity of NOS in the cell culture medium were determined by colorimetric method and nitrate reductase method, and the apoptosis was detected by flow cytometry. Results Compared with the control group, the cell viability decreased to (83.76 ± 1.04)% at 24 h, 48 h and 72 h after treatment with 0.2 mmol / L (low fluoride) and 2.0 mmol / L (high fluoride) (70.27 ± 1.20)%, (54.40 ± 0.98)% and (52.17 ± 1.07)%, (40.60 ± 1.11)% and (29.50 ± 1.40)%, respectively. The cell viability was enhanced by 10 -4 mmol / L 7-NI and the survival rate at 24 h was (105.96 ± 3.44)%. The survival rate of cells treated with 10 -3 mmol / L 7-NI for 24 h The rate of (100 ± 2.30)%, 10 -2 mmol / L 7-NI decreased the cell viability and the cell viability at 24 h was (88.64 ± 4.75)%. The content of NO and the activity of NOS in the culture medium of 10 -4, 10 -3, 10 -2 mmol / L 7-NI for 24 h were (1.156 ± 0.356) , (0.958 ± 0.074), (0.672 ± 0.188) μmol / L and (0.403 ± 0.089), (0.398 ± 0.010) and (0.103 ± 0.076) U / ml. The apoptotic index of SH-SY5Y cells in high dose (10-2 mmol / L) 7-NI group increased to (8.7 ± 2.3)%, higher than that in control group (P <0.05) The apoptotic index in 7-NI group was (0.6 ± 0.4)% and (1.0 ± 0.2)%, respectively, which was not significantly different from that in control group (P> 0.05). The apoptotic index of low dose 7-NI group was lower than that of high dose group, the difference was statistically significant (P <0.05). After 24, 48 and 72 h of fluorine exposure, the content of NO and the activity of NOS in the cell culture fluid increased. The content of NO and the activity of NOS in low fluoride group were (3.074 ± 0.160), (5.604 ± 1.374), (10.173 ± 1.307) μmol / (0.743 ± 0.122), (0.959 ± 0.054) and (1.446 ± 0.12) U / ml in high fluoride group and (8.974 ± 0.919), (14.729 ± 1.241) and (20.976 ± 0.712) μmol / (1.086 ± 0.024), (1.728 ± 0.130) and (2.583 ± 0.192) U / ml higher than those in the control group [(1.320 ± 0.280), (2.420 ± 0.658), (2.867 ± 0.662) μmol / 0.012), (0.517 ± 0.072) and (0.607 ± 0.013) U / ml, respectively, with significant difference (P <0.05). Compared with fluorocarbon alone treatment group, NO content and NOS activity decreased after NaF combined with 7-NI, the difference was statistically significant (P <0.05). Compared with the control group, fluoride could increase the apoptotic index of SH-SY5Y cells, the difference was statistically significant (P <0.01). With the dose and time of fluorine exposure increasing, the apoptosis index increased. After combined application of 7-NI, the apoptosis index in each group decreased, the difference was statistically significant (P <0.01). Fluorine could reduce the number of SH-SY5Y cells and change the shape of the cells into round and irregular shape and decrease the cell viability. In the low dose (10 ~ (-4), 10 ~ (-3) mmol / L) 7-NI group, Under the same field of view, the number of cells increased and the number of 7-NI cells in high dose (10 ~ (-2) mmol / L) group decreased significantly. Most cells were round or irregular. Conclusion The apoptosis induced by fluorosis may be related to the increase of NOS activity and NO content. 7-NI can reduce the apoptosis index.