H_2O_2诱导建立HTR-8/SVneo胎盘滋养细胞氧化应激模型
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摘要
目的通过观察不同浓度过氧化氢(H_2O_2)对人胎盘滋养层细胞HTR-8/SVneo处理不同时间后的氧化应激状态,探讨建立H_2O_2诱导HTR-8/SVneo胎盘滋养细胞氧化损伤模型的最佳条件。方法 2017年11月至2018年2月于西安交通大学第一附属医院进行实验研究。将HTR-8/SVneo细胞实验组分别给予不同终浓度的H_2O_2(50、150、300、500μmol/L),同时设立对照组,相同实验条件下分别继续培养1、3、6、12h,随后对此进行细胞存活率、细胞形态学观察,对超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)、过氧化氢酶(CAT)测定,流式细胞仪分析活性氧自由基(ROS)水平和细胞凋亡的水平。结果与对照组比较,300μmol/L 3h经H_2O_2处理后可降低HTR-8/SVneo细胞的存活率(P=0.00<0.01),而且提高了LDH的释放,促使了SOD、CAT活性的下降和MDA含量的增加(P=0.00<0.01)。300μmol/L 3h经H_2O_2处理后增加了细胞的凋亡率(P=0.00<0.01)及细胞内ROS水平(P=0.00<0.01)。同时,300μmol/L 3h经H_2O_2处理后可明显促进细胞的形态学改变。结论通过H_2O_2可以成功建立HTR-8/SVneo氧化应激损伤细胞模型,最佳实验条件为300μmol/L H_2O_2处理3h。
Objective To investigate the optimum condition of establishing hydrogen peroxide(H_2O_2) induced oxidative injury model of placenta trophoblasts HTR-8/SVneo by observing the oxidative stress after treatment by different concentrations of H_2O_2 on human placenta trophoblast cell HTR-8/SVneo. Methods The experiment was carried out in the First Affiliated Hospital of Xi'an Jiaotong University from November 2017 to February 2018. A panel of concentration gradient of H_2O_2(50μmol/L, 150μmol/L, 300μmol/L, 500μmol/L)was given to the experimental group of HTR-8/SVneo cell, and control group was also set to culture for 1 h, 3 h, 6 h and 12 h under the same conditions. The survival rate of cells and morphology were observed. The levels of superoxide dismutase(SOD), malonic dialdehyde(MDA), lactate dehydrogenase(LDH) and catalase(CAT) were tested and flow-cytometry was used to analyze the levels of reactive oxygen species(ROS) and apoptosis. Results Our findings showed that 300μmol/L H_2O_2 treating for 3 hours could reduce the survival rate of HTR-8/SVneo(P=0.00<0.01), enhance the production of LDH, improve the decline of SOD and CAT and increase of MDA(P=0.00<0.01). Apoptosis rate and intracellular ROS level were also increased(P=0.00<0.01). Meanwhile, the morphological changes were remarkably promoted. Conclusion We conclude under the optimum condition, which is 300μmol/L H_2O_2 for 3 hours, H_2O_2-induced oxidative stress damage model in HTR-8/SVneo cells can be successfully established.
Objective To investigate the optimum condition of establishing hydrogen peroxide(H_2O_2) induced oxidative injury model of placenta trophoblasts HTR-8/SVneo by observing the oxidative stress after treatment by different concentrations of H_2O_2 on human placenta trophoblast cell HTR-8/SVneo. Methods The experiment was carried out in the First Affiliated Hospital of Xi'an Jiaotong University from November 2017 to February 2018. A panel of concentration gradient of H_2O_2(50μmol/L, 150μmol/L, 300μmol/L, 500μmol/L)was given to the experimental group of HTR-8/SVneo cell, and control group was also set to culture for 1 h, 3 h, 6 h and 12 h under the same conditions. The survival rate of cells and morphology were observed. The levels of superoxide dismutase(SOD), malonic dialdehyde(MDA), lactate dehydrogenase(LDH) and catalase(CAT) were tested and flow-cytometry was used to analyze the levels of reactive oxygen species(ROS) and apoptosis. Results Our findings showed that 300μmol/L H_2O_2 treating for 3 hours could reduce the survival rate of HTR-8/SVneo(P=0.00<0.01), enhance the production of LDH, improve the decline of SOD and CAT and increase of MDA(P=0.00<0.01). Apoptosis rate and intracellular ROS level were also increased(P=0.00<0.01). Meanwhile, the morphological changes were remarkably promoted. Conclusion We conclude under the optimum condition, which is 300μmol/L H_2O_2 for 3 hours, H_2O_2-induced oxidative stress damage model in HTR-8/SVneo cells can be successfully established.
引文
[1]Wang Q,Zeng F,Sun Y,et al.Etk interaction with PFKFB4 modulates chemoresistance of small-cell lung cancer by regulating autophagy[J].Clinical Cancer Research,2018,24(4):950-962.
[2]Lim J,Choi A,Kim H W,et al.Constrained adherable area of nanotopographic surfaces promotes cell migration through the regulation of focal adhesion via focal adhesion kinase/Rac1 activation[J].ACS Applied Materials Interfaces,2018,10(17):14331-14341.
[3]Wang Y,Rezey A C,Wang R,et al.Role and regulation of Abelson tyrosine kinase in Crk-associated substrate/profilin-1 interaction and airway smooth muscle contraction[J].Respiratory Research,2018,19(1):4.
[4]Spassov D S,Ruiz-Saenz A,Piple A,et al.A Dimerization function in the intrinsically disordered N-Terminal region of src[J].Cell Reports,2018,25(2):449-463.e4.
[5]Goňi G M,Epifano C,Boskovic J,et al.Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes[J].Proceedings National Academy Sciences,2014,111(31):E3177-E3186.
[6]Zhang Y,Zhou J,Li M,et al.MicroRNA-184 promotes apoptosis of trophoblast cells via targeting WIG1 and induces early spontaneous abortion[J].Cell Death Disease,2019,10(3):223.
[7]Wojsiat J,Prandelli C,Laskowska-Kaszub K,et al.Oxidative stress and aberrant cell cycle in Alzheimer’s disease lymphocytes:diagnostic prospects[J].Journal Alzheimer's Disease,2015,46(2):329-350.
[8]Turunc Bayrakdar E,Uyanikgil Y,Kanit L,et al.Nicotinamide treatment reduces the levels of oxidative stress,apoptosis,and PARP-1 activity in Aβ(1-42)-induced rat model of Alzheimer’s disease[J].Free Radical Research,2014,48(2):146-158.
[9]Li L,Yu A Q.The functional role of peroxiredoxin 3 in reactive oxygen species,apoptosis,and chemoresistance of cancer cells[J].Journal Cancer Research Clinical Oncology,2015,141(12):2071-2077.
[10]Amri F,Ghouili I,Amri M,et al.Neuroglobin protects astroglial cells from hydrogen peroxide-induced oxidative stress and apoptotic cell death[J].Journal Neurochemistry,2017,140(1):151-169.
[11]Ido Y,Duranton A,Lan F,et al.Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes[J].PLoS One,2015,10(2):e0115341.
[12]Geyikoglu F,Koc K,Colak S,et al.Propolis and its combination with boric acid protect against ischemia/reperfusion-induced acute kidney injury by inhibiting oxidative stress,inflammation,DNA damage,and apoptosis in rats[J].Biol Trace Elem Res,2019,[Epub ahead of print].doi:10.1007/s12011-019-1649-2.
[13]Domingueti C P,Dusse L M S A,das Gra?asCarvalho M,et al.Diabetes mellitus:the linkage between oxidative stress,inflammation,hypercoagulability and vascular complications[J].Journal Diabetes Its Complications,2016,30(4):738-745.
[14]Narasimhulu C A,Fernandez-Ruiz I,Selvarajan K,et al.Atherosclerosis-do we know enough already to prevent it?[J].Current Opinion Pharmacology,2016,27:92-102.
[15]Hernández J A,López-Sánchez R C,Rendón-Ramírez A.Lipids and oxidative stress associated with ethanol-induced neurological damage[J].Oxidative Medicine Cellular Longevity,2016,2016:1543809.
[16]Hwang H J,Jung S H,Lee H C,et al.Identification of novel therapeutic targets in the secretome of ionizing radiation-induced senescent tumor cells[J].Oncology Reports,2016,35(2):841-850.
[17]Bremer L,Schramm C,Tiegs G.Immunology of hepatic diseases during pregnancy[C]//Seminars in immunopathology[J].Springer Berlin Heidelberg,2016,38(6):669-685.
[18]Menon R.Oxidative stress damage as a detrimental factor in preterm birth pathology[J].Frontiers Immunology,2014,5:567.
[19]Wu F,Tian F J,Lin Y.Oxidative stress in placenta:health and diseases[J].Bio Med Research International,2015,2015:293271.
[20]Wu F,Tian F J,Lin Y,et al.Oxidative stress:placenta function and dysfunction[J].American Journal Reproductive Immunology,2016,76(4):258-271.
[21]Zhang S,Regnault T,Barker P,et al.Placental adaptations in growth restriction[J].Nutrients,2015,7(1):360-389.
[22]Truong V L,Jun M,Jeong W S.Role of resveratrol in regulation of cellular defense systems against oxidative stress[J].Biofactors,2018,44(1):36-49.
[23]Yu H,Deng W,Zhang D,et al.Antioxidant defenses of Onychostoma macrolepis in response to thermal stress:insight from mRNA expression and activity of superoxide dismutase and catalase[J].Fish Shellfish Immunology,2017,66:50-61.
[24]Fago A,Jensen F B.Hypoxia tolerance,nitric oxide,and nitrite:lessons from extreme animals[J].Physiology,2015,30(2):116-126.
[25]Soltan M Y,Sumarni U,Assaf C,et al.Key Role of Reactive Oxygen Species(ROS) in Indirubin Derivative-Induced Cell Death in Cutaneous T-Cell Lymphoma Cells[J].International Journal Molecular Sciences,2019,20(5):1158.
[26]Chen J C,Hsieh M C,Lin S H,et al.Coronarin D induces reactive oxygen species-mediated cell death in human nasopharyngeal cancer cells through inhibition of p38 MAPK and activation of JNK[J].Oncotarget,2017,8(64):108006.
[2]Lim J,Choi A,Kim H W,et al.Constrained adherable area of nanotopographic surfaces promotes cell migration through the regulation of focal adhesion via focal adhesion kinase/Rac1 activation[J].ACS Applied Materials Interfaces,2018,10(17):14331-14341.
[3]Wang Y,Rezey A C,Wang R,et al.Role and regulation of Abelson tyrosine kinase in Crk-associated substrate/profilin-1 interaction and airway smooth muscle contraction[J].Respiratory Research,2018,19(1):4.
[4]Spassov D S,Ruiz-Saenz A,Piple A,et al.A Dimerization function in the intrinsically disordered N-Terminal region of src[J].Cell Reports,2018,25(2):449-463.e4.
[5]Goňi G M,Epifano C,Boskovic J,et al.Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes[J].Proceedings National Academy Sciences,2014,111(31):E3177-E3186.
[6]Zhang Y,Zhou J,Li M,et al.MicroRNA-184 promotes apoptosis of trophoblast cells via targeting WIG1 and induces early spontaneous abortion[J].Cell Death Disease,2019,10(3):223.
[7]Wojsiat J,Prandelli C,Laskowska-Kaszub K,et al.Oxidative stress and aberrant cell cycle in Alzheimer’s disease lymphocytes:diagnostic prospects[J].Journal Alzheimer's Disease,2015,46(2):329-350.
[8]Turunc Bayrakdar E,Uyanikgil Y,Kanit L,et al.Nicotinamide treatment reduces the levels of oxidative stress,apoptosis,and PARP-1 activity in Aβ(1-42)-induced rat model of Alzheimer’s disease[J].Free Radical Research,2014,48(2):146-158.
[9]Li L,Yu A Q.The functional role of peroxiredoxin 3 in reactive oxygen species,apoptosis,and chemoresistance of cancer cells[J].Journal Cancer Research Clinical Oncology,2015,141(12):2071-2077.
[10]Amri F,Ghouili I,Amri M,et al.Neuroglobin protects astroglial cells from hydrogen peroxide-induced oxidative stress and apoptotic cell death[J].Journal Neurochemistry,2017,140(1):151-169.
[11]Ido Y,Duranton A,Lan F,et al.Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes[J].PLoS One,2015,10(2):e0115341.
[12]Geyikoglu F,Koc K,Colak S,et al.Propolis and its combination with boric acid protect against ischemia/reperfusion-induced acute kidney injury by inhibiting oxidative stress,inflammation,DNA damage,and apoptosis in rats[J].Biol Trace Elem Res,2019,[Epub ahead of print].doi:10.1007/s12011-019-1649-2.
[13]Domingueti C P,Dusse L M S A,das Gra?asCarvalho M,et al.Diabetes mellitus:the linkage between oxidative stress,inflammation,hypercoagulability and vascular complications[J].Journal Diabetes Its Complications,2016,30(4):738-745.
[14]Narasimhulu C A,Fernandez-Ruiz I,Selvarajan K,et al.Atherosclerosis-do we know enough already to prevent it?[J].Current Opinion Pharmacology,2016,27:92-102.
[15]Hernández J A,López-Sánchez R C,Rendón-Ramírez A.Lipids and oxidative stress associated with ethanol-induced neurological damage[J].Oxidative Medicine Cellular Longevity,2016,2016:1543809.
[16]Hwang H J,Jung S H,Lee H C,et al.Identification of novel therapeutic targets in the secretome of ionizing radiation-induced senescent tumor cells[J].Oncology Reports,2016,35(2):841-850.
[17]Bremer L,Schramm C,Tiegs G.Immunology of hepatic diseases during pregnancy[C]//Seminars in immunopathology[J].Springer Berlin Heidelberg,2016,38(6):669-685.
[18]Menon R.Oxidative stress damage as a detrimental factor in preterm birth pathology[J].Frontiers Immunology,2014,5:567.
[19]Wu F,Tian F J,Lin Y.Oxidative stress in placenta:health and diseases[J].Bio Med Research International,2015,2015:293271.
[20]Wu F,Tian F J,Lin Y,et al.Oxidative stress:placenta function and dysfunction[J].American Journal Reproductive Immunology,2016,76(4):258-271.
[21]Zhang S,Regnault T,Barker P,et al.Placental adaptations in growth restriction[J].Nutrients,2015,7(1):360-389.
[22]Truong V L,Jun M,Jeong W S.Role of resveratrol in regulation of cellular defense systems against oxidative stress[J].Biofactors,2018,44(1):36-49.
[23]Yu H,Deng W,Zhang D,et al.Antioxidant defenses of Onychostoma macrolepis in response to thermal stress:insight from mRNA expression and activity of superoxide dismutase and catalase[J].Fish Shellfish Immunology,2017,66:50-61.
[24]Fago A,Jensen F B.Hypoxia tolerance,nitric oxide,and nitrite:lessons from extreme animals[J].Physiology,2015,30(2):116-126.
[25]Soltan M Y,Sumarni U,Assaf C,et al.Key Role of Reactive Oxygen Species(ROS) in Indirubin Derivative-Induced Cell Death in Cutaneous T-Cell Lymphoma Cells[J].International Journal Molecular Sciences,2019,20(5):1158.
[26]Chen J C,Hsieh M C,Lin S H,et al.Coronarin D induces reactive oxygen species-mediated cell death in human nasopharyngeal cancer cells through inhibition of p38 MAPK and activation of JNK[J].Oncotarget,2017,8(64):108006.