缺氧损伤对人脐静脉内皮细胞抗氧化能力、Ca^(2+)依赖的三磷腺苷酶活性、Ca^(2+)浓度及线粒体膜电位的影响

Effect of hypoxia on antioxidative capacity,Ca^(2+)adenosine triphosphate enzyme activity,Ca^(2+)concentration and mitochondiral membrane potential in ECV304 cells

目的探讨缺氧损伤对离体培养的人脐静脉内皮细胞(ECV304)抗氧化能力、Ca^(2+)依赖的三磷腺苷酶(Ca^(2+)-ATPase)活性、线粒体膜电位(MMP)和细胞内Ca^(2+)浓度等的影响。方法将ECV304细胞分为正常组和缺氧组。正常组用无血清培养基培养,缺氧组用连二亚硫酸钠(Na2S2O4)孵育ECV304细胞5 h,建立缺氧损伤模型。倒置显微镜下观察2组细胞形态的变化,细胞计数试剂盒(CCK-8)检测细胞活性,流式细胞仪测定细胞周期、细胞凋亡、细胞内Ca^(2+)浓度及MMP的变化,化学比色法检测超氧化物歧化酶(SOD)活性、谷胱甘肽(GSH)和丙二醛(MDA)含量、一氧化氮(NO)分泌量及Ca^(2+)-ATPase活性。结果正常组细胞贴壁良好,细胞饱满、排列紧密,透明度好;缺氧组细胞出现皱缩,细胞间排列疏松、紊乱。缺氧组细胞吸光度值为0.91±0.12,低于正常组的1.30±0.25(P<0.05)。缺氧组G0/G1期细胞所占比例为(88.53±1.36)%,高于正常组的(81.27±0.25)%(P<0.05);S期细胞所占比例为(5.17±0.21)%,低于正常组的(6.07±0.25)%(P<0.05);缺氧组细胞凋亡率为(19.75±0.81)%,高于正常组的(9.83±1.77)%(P<0.05)。正常组MDA、GSH含量,一氧化氮(NO)分泌量,SOD、Ca^(2+)-ATPase活性分别为(0.40±0.23)、(3.85±0.18)、(14.21±0.39)μmol·L-1、(64.61±0.05)×103U·L-1、(18.90±1.09)U·mgprot-1;缺氧组以上各指标分别为(1.65±0.24)、(3.34±0.13)、(78.70±5.29)μmol·L-1、(35.33±0.04)×103U·L-1、(5.80±0.31)U·mgprot-1;与正常组相比,缺氧组细胞SOD活性、GSH含量及Ca^(2+)-ATPase活性均降低,MDA含量增加,NO分泌量升高(P<0.05)。缺氧组细胞内Ca^(2+)荧光强度为(572.82±49.40)%,高于正常组的(505.97±19.40)%(P<0.05)。缺氧组细胞MMP为(182.07±6.00)%,低于正常组的(217.60±16.40)%(P<0.05)。结论 Na2S2O4作用于ECV304细胞引起细胞缺氧损伤,其机制可能与MMP降低、细胞内Ca^(2+)浓度升高和细胞抗氧化能力及Ca^(2+)-ATPase活性减弱相关。

Objective To observe the effect of hypoxia on antioxidant capacity, Ca2 ＋ adenosine triphosphate enzyme （ Ca2 ＋ -ATPase） activity, Ca2 ＋ concentration and mitochondiral membrane potential （MMP） of ECV304 ceils cultured in vitro. Methods ECV3（M cells were divided into normal group which cultured in serum-free culture medium and hypoxia group which were cultured in Na2S2O4 for 5 hours to establish hypoxic injury model. Then the change of ceil morphology in the two groups was observed by inverted microscope; the cytoactive was evaluated by cell counting kit-8 （ CCK-8 ） ; the cellcycle, apopto- sis, intracellular Ca2＋ concentration and MMP were assayed by flow cytometry;the activity of superoxide dismutase （SOD） and Ca2 ＋ -ATPase and the content of glutathione （ GSH）, malondialdehyde （ MDA）, nitric oxide （NO） secretion were measured by chemical colorimetry method. Results The cells in normal group were well adherent,full of luster, closely spaced, and highly pellucidity;while the ceils in hypoxia group appeared shrunken, and cell arranged loosely, disorder. The value of absorance in hypoxia group and normal group was 0.91 ±0.12,1.30 ±0.25 respectively;the value of OD in hypoxia group was significantly lower than that in normal group （ P 〈 0.05 ）. The proportion of G0/G1 and S phase cells in normal group was （ 81.27 ± 0. 25 ） %, （6.07 ± 0.25 ） % ; which in hypoxia group was （ 88.53 ± 1.36 ） %, （ 5.17 ± 0.21 ） % ; the proportion of G0/G1 phase cells was significantly higher and the S phase cells was significantly lower than that in normal group （ P 〈 0.05 ）. The cell apoptosis rate in hypoxia group [ （ 19.75 ±0. 81 ） % ] was significantly higher than that in normal group [ （ 9. 83 ± 1.77） % ] （P 〈 0.05 ）. The content of MDA and GSH, the content of NO secretion, the activity of SOD and Ca2 ＋ -ATPase in normal group was （0.40 ±0.23） ,（3.85 ±0. 18） ,（14.21 ±0.39） μmol · L-1 ,（64.61 ±0.05） × 10 ^3 U ·L-1 ,（18.90 ± 1.09） U · mgprot - 1 respectively; the above indexes in hypoxia group was （ 1.65 ± 0.24 ）, （ 3.34 ±0. 13 ）, （ 78.70 ± 5.29 ） μmol · L- 1, （35.33 ± 0.04） × 10^3 U · L-1 , （5.80 ± 0. 31 ） U · mgprot -1 respectively. Compared with normal group, the content of MDA, and content of NO secretion were increased（ P 〈 0. 05 ） ;the activity of SOD, Ca2＋ -ATPase and the content of GSH were decreased （ P 〈 0. 05 ） in hypoxia group. The fluorescence intensity of intracellular Ca2＋ in hypoxia group [ （ 572. 82 ± 49.40） % ] was significantly higher than that in normal group [ （505.97 ± 19.40） % ] （ P 〈 0.05）. The MMP in hypoxia group [ （ 182.07 ± 6.00） % ] was significantly lower than that in normal group [ （217.60 ± 16.40） % ] （P 〈 0.05）. Conclusion The hypoxia injury of ECV304 cells being induced by Na2S2O4 is related with the decreasing of MMP and Ca2＋ - ATPase activity, and the increasing of the intracellular concentration of Ca2＋ and antioxidant capacity.