【摘要】：目的探討神經(jīng)元型一氧化氮合成酶(nNOS)抑制劑7-硝基吲唑(7-nitroindazole,7-NI)對(duì)氟致體外培養(yǎng)細(xì)胞生長(zhǎng)、凋亡保護(hù)作用的機(jī)制。方法體外培養(yǎng)SH-SY5Y細(xì)胞,在培養(yǎng)液中加入不同濃度Na F(0、0.02、0.2、2.0、4.0 mmol/L),分別培養(yǎng)24、48、72 h,同時(shí)在各組細(xì)胞培養(yǎng)液中聯(lián)合應(yīng)用7-NI(10~(-4)、10~(-3)、10~(-2) mmol/L);于倒置顯微鏡下觀察細(xì)胞生長(zhǎng),以CCK-8試劑盒方法檢測(cè)細(xì)胞存活率,以比色法和硝酸還原酶法測(cè)定細(xì)胞培養(yǎng)液中NO含量及NOS活力,以流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況。結(jié)果與對(duì)照組比較,氟濃度為0.2 mmol/L(低氟組)和2.0 mmol/L(高氟組)時(shí),24、48、72 h后細(xì)胞存活率分別下降至(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可刺激細(xì)胞增殖,24 h細(xì)胞存活率為(105.96±3.44)%;10~(-3) mmol/L 7-NI處理細(xì)胞24 h后的細(xì)胞存活率為(100±2.30)%;10~(-2) mmol/L 7-NI可使細(xì)胞存活率下降,24 h細(xì)胞存活率為(88.64±4.75)%。10~(-4)、10~(-3)、10~(-2) mmol/L 7-NI處理細(xì)胞24 h后細(xì)胞培養(yǎng)液中NO含量、NOS活力均降低,分別為(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。高劑量(10~(-2) mmol/L)7-NI組的SH-SY5Y細(xì)胞凋亡指數(shù)升高至(8.7±2.3)%,高于對(duì)照組(P0.05);低劑量(10~(-4)、10~(-3) mmol/L)7-NI組的凋亡指數(shù)分別為(0.6±0.4)%和(1.0±0.2)%,與對(duì)照組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);低劑量7-NI組凋亡指數(shù)低于高劑量組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。染氟24、48、72 h后細(xì)胞培養(yǎng)液中NO含量及NOS活力升高,低氟組的NO含量、NOS活力分別為(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,高氟組分別為(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,高于對(duì)照組[(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],差異有統(tǒng)計(jì)學(xué)意義(P0.05)。與氟單獨(dú)處理組相比,NaF聯(lián)合應(yīng)用7-NI后,各組NO含量及NOS活力均下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。與對(duì)照組相比,氟可使SH-SY5Y細(xì)胞凋亡指數(shù)升高,差異有統(tǒng)計(jì)學(xué)意義(P0.01),并隨著氟染毒劑量及時(shí)間的增加,細(xì)胞凋亡指數(shù)隨之增加;聯(lián)合應(yīng)用7-NI后,各組細(xì)胞凋亡指數(shù)下降,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。氟可使SH-SY5Y細(xì)胞數(shù)量減少,形態(tài)變?yōu)閳A形、不規(guī)則形,細(xì)胞存活率降低;低劑量(10~(-4)、10~(-3) mmol/L)7-NI組在相同視野下細(xì)胞數(shù)增加,高劑量(10~(-2) mmol/L)7-NI組細(xì)胞數(shù)明顯減少,大部分細(xì)胞呈圓形或不規(guī)則形。結(jié)論氟中毒所致細(xì)胞凋亡可能與NOS活力和NO含量升高有關(guān),7-NI可降低細(xì)胞凋亡指數(shù)。
[Abstract]:Objective to investigate the protective mechanism of 7-nitroindazole-7-NI (7-NI), a inhibitor of neuronal nitric oxide synthase (nNOS), on the growth and apoptosis of cultured cells induced by fluoride in vitro. Methods SH-SY5Y cells were cultured in vitro with different concentrations of Na F (_ (0. 02) and 0.2 ~ (2) mmol/L) and cultured for 24 ~ (48) h for 72 h, respectively. 7-NI (10 ~ (-4) ~ (-3) ~ (-3) 10 ~ (-2) mmol/L) was used in each cell culture medium, cell growth was observed under inverted microscope, and cell survival rate was detected by CCK-8 assay. The content of NO and the activity of NOS were measured by colorimetry and nitrate reductase method, and apoptosis was detected by flow cytometry. Results compared with the control group, 姘熸祿搴︿負(fù)0.2 mmol/L(浣庢盁緇，

本文編號(hào)：2235365