医用臭氧气浴对体外培养液正常人表皮细胞影响的研究
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摘要
目的模拟臭氧气浴治疗,观察医用臭氧气体对体外培养液人表皮细胞的影响。方法取武汉大学同仁医院暨武汉市第三医院泌尿外科包皮环切术后废弃包皮,消化分离表皮细胞原代培养液后采用传代第3代细胞,在50 mg/L臭氧环境下进行实验干预。(1)观察原代和传代细胞,行Hoechst 33258染色观察细胞形态学变化与凋亡情况;(2)细胞活力检测分氧气组和臭氧组(n=5),采用细胞计数试剂盒8(CCK-8)及酶标仪检测无培养液基时气体直接干预细胞,在短期内(0、5、10、15、20、25、30 s)的细胞活力趋势;(3)酶生化检测分为空气组、氧气组和臭氧组(n=5),分别检测干预10、30、60 min各时间点的超氧化物歧化酶(SOD)活性水平、丙二醛含量、乳酸脱氢酶(LDH)及细胞内还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)的水平,并计算GSH/GSSG值。细胞活性吸光度值、酶生化组间两两比较及同组内多个时相点两两比较采用LSD-t检验。结果 (1)原代培养液的表皮细胞呈椭圆状或多角状贴壁,稳定后呈典型的"铺路石"样,Hoechst 33258荧光染色后正常细胞核呈弥散均匀的淡蓝色荧光,臭氧干预后,随持续时间延长,出现浓染致密的亮蓝色颗粒,表皮细胞逐渐呈核固缩、染色质浓集和凋亡小体等凋亡特征,干预60 min时部分表皮细胞出现成片脱落;(2)臭氧直接干预表皮细胞,随时间的增加细胞活力呈明显下降趋势,臭氧组干预30 s细胞吸光度值为(98.72±1.20)%,氧气组干预30 s细胞吸光度值为(22.70±3.78)%,两组比较差异有统计学意义(t=48.758,P<0.05);(3)臭氧对细胞酶生化影响:氧气组干预60 min与空气组比较,SOD、丙二醛、LDH值差异均无统计学意义(P>0.05);臭氧干预10 min,SOD、丙二醛、LDH值分别为(153.63±8.41)U/mg prot、(52.41±6.30)nmol/mg prot、(186.19±20.20)U/g prot,臭氧干预30 min SOD、丙二醛、LDH值分别为(84.31±7.23)U/mg prot、(79.09±6.98)nmol/mg prot、(221.22±20.79)U/g prot,两个时相点比较差异有统计学意义(t=13.972、-6.343、-2.703,P值均小于0.05);臭氧干预60 min,SOD、丙二醛、LDH值分别为(30.31±2.79)U/mg prot、(97.5±7.35)nmol/mg prot、(280.76±20.06)U/g prot,与臭氧干预30 min比较差异有统计学意义(t=15.569、-4.059、-4.608,P值均小于0.01);臭氧组与氧气组比较,SOD、丙二醛、LDH差异均有统计学意义(P<0.05);氧气组干预60 min与空气组比较,GSH、GSSG、GSH/GSSG值均差异无统计学意义(P值均大于0.05);臭氧组与氧气组比较,除臭氧组干预10 min GSH值与氧气组干预60 min比较差异无统计学意义以外(t=1.811,P>0.05),其余臭氧干预时相点GSH、GSSG、GSH/GSSG值差异均有统计学意义(P值均小于0.05)。臭氧干预10 min GSH、GSSG、GSH/GSSG值分别为(11.67±1.37)μmol/L、(1.83±0.18)μmol/L、6.48±1.28,臭氧干预30 min GSH、GSSG、GSH/GSSG值分别为(9.37±0.75)μmol/L、(1.59±0.2)μmol/L、6.00±1.23,两个时相点比较,GSH明显下降,差异有统计学意义(t=3.295,P=0.011),GSSG值与GSH/GSSG值差异均无统计学意义(t=1.98、0.605,P值均大于0.05);臭氧干预60 min GSH、GSSG、GSH/GSSG值分别为(8.34±1.16)μmol/L、(2.02±0.24)μmol/L、4.13±0.44,与臭氧干预30 min比较,GSH和GSSG差异无统计学意义(t=1.673、-3.08,P值均大于0.05),GSH/GSSG值明显下降,差异有统计学意义(t=3.216,P<0.05)。结论体外培养液的人表皮细胞对臭氧气体敏感,临床应用臭氧汽浴疗法处理皮肤创面,宜考虑控制治疗时间。
Objective To observe the effect of medical ozone gas on human epidermal cells in vitro by simulating ozone gas bath treatment. Methods Normal skin tissue was taken,and the primary culture of epidermal cells were digested and separated. The third generation cells were used for experimental intervention in 50 mg/L ozone environment.(1) Observe the primary and inherited cells,and observe cell morphological and apoptosis changes by Hoechst 33258 staining;( 2) Cell viability assay was divided into oxygen group and ozone group( n = 5),the cell counting kit-8( CCK-8) method and enzymatic marke were employed to detect the cell viability trend of cells directly interfered with cells in the absence of medium in short period of time(0,5,10,15,20,25,30 s);(3) Enzyme biochemical detection was divided into air group,oxygen group and ozone group( n = 5),the activity level of superoxide dismutase( SOD),malondialdehyde content,LDH leakage rate detection,and the indicators of enzyme biochemical after intervention 10,30,60 min were detected respectively. The levels of intracellular reduced glutathione(GSH) and oxidized glutathione( GSSG) were measured according to the intervention time of 10,30,60 min,and GSH/GSSG value was calculated. The overall comparison of cell viability was analyzed by repeated measures analysis of variance. The LSD-t test was used to compare the two groups and multiple time points in the same group of enzyme biochemical. Results(1) The original cultured epidermal cells showed oval or multi-angle adherent walls, and showed typical of "paving stones " after stabilization. After fluorescence staining of Hoechst 33258, the normal nucleus showed diffuse and uniform pale blue fluorescence. And after ozone interference,with the prolonged of the duration,the dense bright blue particles appeared,epidermal cells gradually undergo nuclear condensation,chromatin condensation and apoptotic bodies,and partial epidermal cells appeared to fall off after 60 min of intervention.( 2) Ozone directly interfered with the epidermal cells,and the cell activity showed a significant decline with the increase of time. Intervened by Ozone for 30 s,the cell absorbance value in the ozone group was(98. 72 ±1. 20) %,and intervened by oxygen for 30 s,the cell absorbance value in the oxygen group was(22. 70 ±3. 78) %,the difference was statistically significant( t = 48. 758,P < 0. 05).( 3) Biochemical effects of ozone on cell enzymes: there were no significant differences in SOD,malondialdehyde,and LDH values between the oxygen group and the air group intervention for 60 min( with P values below 0. 05). Intervened by ozone for 10 min,the values of SOD,malondialdehyde and LDH were( 153. 63 ± 8. 41) U/mg prot,(52. 41 ± 6. 30) nmol/mg prot and(186. 19 ± 20. 20) U/g prot,intervened by Ozone for 30 min,the values of SOD,malondialdehyde and LDH were(84. 31 ± 7. 23) U/mg prot,(79. 09 ± 6. 98) nmol/mg prot and(221. 22 ± 20. 79) U/g prot,the differences was statistically significant( t = 13. 972,-6. 343,-2. 703; with P values below 0. 05). Intervened by Ozone for 60 min,the values of SOD,malondialdehyde and LDH were( 30. 31 ± 2. 79) U/mg prot,( 97. 5 ± 7. 35) nmol/mg prot and( 280. 76 ±20. 06) U/g prot,compared with the intervention for 30 min,the difference was statistically significant( t =15. 569,-4. 059,-4. 608; with P value below 0. 01). Comparison between ozone group and oxygen group,the difference of the SOD,malondialdehyde and LDH values were statistically significant( with P values below 0. 05). There were no significant differences in GSH,GSSG,GSH/GSSG values between the air group and the oxygen group( with P values above 0. 05). Between ozone group and oxygen group,therewere no significant differences of the GSH value between the ozone group intervention for 10 min and the oxygen group intervention for 60 min( t = 1. 811,P > 0. 05). The differences of GSH,GSSG and GSH/GSSG values in the other times intervened by ozone were statistically significant( with P values below0. 05). Intervened by ozone for 10 min, the values of GSH, GSSG, GSH/GSSG were( 11. 67 ±1. 37) μmol/L,(1. 83 ± 0. 18) μmol/L,6. 48 ± 1. 28,intervened by ozone for 30 min,the values of GSH,GSSG,GSH/GSSG were( 9. 37 ± 0. 75) μmol/L,( 1. 59 ± 0. 2) μmol/L and 6. 00 ± 1. 23. The value of GSH decreased obviously,the difference was statistically significant( t = 3. 295,P = 0. 011),and the difference of the values of GSSG and GSH/GSSG was no statistically significant( t = 1. 98,0. 605; with P values above 0. 05). Intervened by ozone for 60 min,the values of GSH,GSSG,GSH/GSSG were(8. 34 ± 1. 16) μmol/L,(2. 02 ± 0. 24) μmol/L and 4. 13 ± 0. 44,compared with the intervention for30 min,the difference was statistically significant,the difference of the values of GSH and GSSG was no statistically significant( t = 1. 673,-3. 08; with P values above 0. 05),and the value of GSH/GSSG decreased significantly,the difference was statistically significant( t = 3. 216,P < 0. 05). Conclusion Human epidermal cells cultrued in vitro are sensitive to ozone gas,and clinical application of ozone gas bath therapy to reat skin wounds should be considered to control treatment time.
Objective To observe the effect of medical ozone gas on human epidermal cells in vitro by simulating ozone gas bath treatment. Methods Normal skin tissue was taken,and the primary culture of epidermal cells were digested and separated. The third generation cells were used for experimental intervention in 50 mg/L ozone environment.(1) Observe the primary and inherited cells,and observe cell morphological and apoptosis changes by Hoechst 33258 staining;( 2) Cell viability assay was divided into oxygen group and ozone group( n = 5),the cell counting kit-8( CCK-8) method and enzymatic marke were employed to detect the cell viability trend of cells directly interfered with cells in the absence of medium in short period of time(0,5,10,15,20,25,30 s);(3) Enzyme biochemical detection was divided into air group,oxygen group and ozone group( n = 5),the activity level of superoxide dismutase( SOD),malondialdehyde content,LDH leakage rate detection,and the indicators of enzyme biochemical after intervention 10,30,60 min were detected respectively. The levels of intracellular reduced glutathione(GSH) and oxidized glutathione( GSSG) were measured according to the intervention time of 10,30,60 min,and GSH/GSSG value was calculated. The overall comparison of cell viability was analyzed by repeated measures analysis of variance. The LSD-t test was used to compare the two groups and multiple time points in the same group of enzyme biochemical. Results(1) The original cultured epidermal cells showed oval or multi-angle adherent walls, and showed typical of "paving stones " after stabilization. After fluorescence staining of Hoechst 33258, the normal nucleus showed diffuse and uniform pale blue fluorescence. And after ozone interference,with the prolonged of the duration,the dense bright blue particles appeared,epidermal cells gradually undergo nuclear condensation,chromatin condensation and apoptotic bodies,and partial epidermal cells appeared to fall off after 60 min of intervention.( 2) Ozone directly interfered with the epidermal cells,and the cell activity showed a significant decline with the increase of time. Intervened by Ozone for 30 s,the cell absorbance value in the ozone group was(98. 72 ±1. 20) %,and intervened by oxygen for 30 s,the cell absorbance value in the oxygen group was(22. 70 ±3. 78) %,the difference was statistically significant( t = 48. 758,P < 0. 05).( 3) Biochemical effects of ozone on cell enzymes: there were no significant differences in SOD,malondialdehyde,and LDH values between the oxygen group and the air group intervention for 60 min( with P values below 0. 05). Intervened by ozone for 10 min,the values of SOD,malondialdehyde and LDH were( 153. 63 ± 8. 41) U/mg prot,(52. 41 ± 6. 30) nmol/mg prot and(186. 19 ± 20. 20) U/g prot,intervened by Ozone for 30 min,the values of SOD,malondialdehyde and LDH were(84. 31 ± 7. 23) U/mg prot,(79. 09 ± 6. 98) nmol/mg prot and(221. 22 ± 20. 79) U/g prot,the differences was statistically significant( t = 13. 972,-6. 343,-2. 703; with P values below 0. 05). Intervened by Ozone for 60 min,the values of SOD,malondialdehyde and LDH were( 30. 31 ± 2. 79) U/mg prot,( 97. 5 ± 7. 35) nmol/mg prot and( 280. 76 ±20. 06) U/g prot,compared with the intervention for 30 min,the difference was statistically significant( t =15. 569,-4. 059,-4. 608; with P value below 0. 01). Comparison between ozone group and oxygen group,the difference of the SOD,malondialdehyde and LDH values were statistically significant( with P values below 0. 05). There were no significant differences in GSH,GSSG,GSH/GSSG values between the air group and the oxygen group( with P values above 0. 05). Between ozone group and oxygen group,therewere no significant differences of the GSH value between the ozone group intervention for 10 min and the oxygen group intervention for 60 min( t = 1. 811,P > 0. 05). The differences of GSH,GSSG and GSH/GSSG values in the other times intervened by ozone were statistically significant( with P values below0. 05). Intervened by ozone for 10 min, the values of GSH, GSSG, GSH/GSSG were( 11. 67 ±1. 37) μmol/L,(1. 83 ± 0. 18) μmol/L,6. 48 ± 1. 28,intervened by ozone for 30 min,the values of GSH,GSSG,GSH/GSSG were( 9. 37 ± 0. 75) μmol/L,( 1. 59 ± 0. 2) μmol/L and 6. 00 ± 1. 23. The value of GSH decreased obviously,the difference was statistically significant( t = 3. 295,P = 0. 011),and the difference of the values of GSSG and GSH/GSSG was no statistically significant( t = 1. 98,0. 605; with P values above 0. 05). Intervened by ozone for 60 min,the values of GSH,GSSG,GSH/GSSG were(8. 34 ± 1. 16) μmol/L,(2. 02 ± 0. 24) μmol/L and 4. 13 ± 0. 44,compared with the intervention for30 min,the difference was statistically significant,the difference of the values of GSH and GSSG was no statistically significant( t = 1. 673,-3. 08; with P values above 0. 05),and the value of GSH/GSSG decreased significantly,the difference was statistically significant( t = 3. 216,P < 0. 05). Conclusion Human epidermal cells cultrued in vitro are sensitive to ozone gas,and clinical application of ozone gas bath therapy to reat skin wounds should be considered to control treatment time.
引文
[1]Valacchi G,Pagnin E,Corbacho AM,et al.In vivo ozone exposure induces antioxidant/stress-related responses in murine lung and skin[J].Free Radic Biol Med,2004,36(5):673-681.
[2]Valacchi G,Fortino V,Bocci V.The dual action of ozone on the skin[J].Br J Dermatol,2005,153(6):1096-1100.
[3]Iriti M,Faoro F.Oxidative Stress,the Paradigm of Ozone Toxicity in Plants and Animals[J].Water Air Soil Pollution,2008,187:285-301.
[4]Lootens L,Brusselaers N,Beele H,et al.Keratinocytes in the treatment of severe burn injury:an update[J].Int Wound J,2013,10(1):6-12.
[5]赵耀华,熊伟,袁东亮,等.臭氧水冲洗在感染创面治疗中的应用[J/CD].中华损伤与修复杂志(电子版),2017,12(6):437-440.
[6]Degli Agosti I,Ginelli E,Mazzacane B,et al.Effectiveness of a Short-Term Treatment of Oxygen-Ozone Therapy into Healing in a Posttraumatic Wound[J].Case Rep Med,2016,2016:9528572.
[7]尚玉茹,申传安,柴家科,等.低浓度胰蛋白酶消化法优化大鼠角质形成细胞的传代培养液[J].中华烧伤杂志,2014,30(2):179-181.
[8]Elvis AM,Ekta JS.Ozone therapy:A clinical review[J].J Nat Sci Biol Med,2011,2(1):66-70.
[9]González-Guevara E,Martínez-Lazcano JC,Custodio V,et al.Exposure to ozone induces a systemic inflammatory response:possible source of the neurological alterations induced by this gas[J].Inhal Toxicol,2014,26(8):485-491.
[10]Valacchi G,Sticozzi C,Belmonte G,et al.Vitamin C Compound Mixtures Prevent Ozone-Induced Oxidative Damage in Human Keratinocytes as Initial Assessment of Pollution Protection[J].PLos One,2015,10(8):e0131097.
[11]Sagai M,Bocci V.Mechanisms of Action Involved in Ozone Therapy:Is healing induced via a mild oxidative stress?[J].Med Gas Res,2011,1:29.
[12]Singer AJ,Clark RA.Cutaneous wound healing[J].N Engl J Med,1999,341(10):738-746.
[13]郭小伟,张家平,黄跃生.表皮细胞迁移与创面愈合[J/CD].中华损伤与修复杂志(电子版),2016,11(3):186-190.
[14]Mccarthy JT,Pelle E,Dong K,et al.Effects of ozone in normal human epidermal keratinocytes[J].Exp Dermatol,2013,22(5):360-361.
[15]李韶辉,余革,陈国勤,等.不同浓度臭氧硬膜外注射对实验兔脊髓的CT和病理学影响[J].中华生物医学工程杂志,2014,20(3):209-211.
[16]Aquilano K,Baldelli S,Ciriolo MR.Glutathione:new roles in redox signaling for an old antioxidant[J].Front Pharmacol,2014,5:196.
[17]Kong Y,Trabucco SE,Hong Z.Oxidative Stress,Mitochondrial Dysfunction and the Mitochondria Theory of Aging[J].Interdiscip Top Gerontol,2014,39:86-107.
[18]Zhang H,Forman HJ.Glutathione synthesis and its role in redox signaling[J].Semin Cell Dev Biol,2012,23(7):722-728.
[19]Ghezzi P.Regulation of protein function by glutathionylation[J].Free Radic Res,2005,39(6):573-580.
[20]Aquilano K,Baldelli S,Ciriolo MR.Glutathione:new roles in redox signaling for an old antioxidant[J].Front Pharmacol,2014,5:196.
[21]Bocci V,Borrelli E,Travagli V,et al.The ozone paradox:ozone is a strong oxidant as well as a medical drug[J].Med Res Rev,2009,29(4):646-682.
[22]赵超莉,叶子青,阮琼芳,等.臭氧气浴对大鼠深Ⅱ度烧伤创面愈合及细胞因子表达的影响[J].中华实验外科杂志,2017,34(12):2084-2086.
[23]Bocci Velio.Ozone-A new medical drug[M].Netherlands:Springer,2005:20-28,75-83,198-208.
[24]Gazzeri R,Galarza M,Neroni M,et al.Fulminating septicemia secondary to oxygen-ozone therapy for lumbar disc herniation:case report[J].Spine(Phila Pa 1976),2007,32(3):E121-123.
[25]Eming SA,Martin P,Tomic-Canic M.Wound repair and regeneration:mechanisms,signaling,and translation[J].Sci Transl Med,2014,6(265):265sr6.
[2]Valacchi G,Fortino V,Bocci V.The dual action of ozone on the skin[J].Br J Dermatol,2005,153(6):1096-1100.
[3]Iriti M,Faoro F.Oxidative Stress,the Paradigm of Ozone Toxicity in Plants and Animals[J].Water Air Soil Pollution,2008,187:285-301.
[4]Lootens L,Brusselaers N,Beele H,et al.Keratinocytes in the treatment of severe burn injury:an update[J].Int Wound J,2013,10(1):6-12.
[5]赵耀华,熊伟,袁东亮,等.臭氧水冲洗在感染创面治疗中的应用[J/CD].中华损伤与修复杂志(电子版),2017,12(6):437-440.
[6]Degli Agosti I,Ginelli E,Mazzacane B,et al.Effectiveness of a Short-Term Treatment of Oxygen-Ozone Therapy into Healing in a Posttraumatic Wound[J].Case Rep Med,2016,2016:9528572.
[7]尚玉茹,申传安,柴家科,等.低浓度胰蛋白酶消化法优化大鼠角质形成细胞的传代培养液[J].中华烧伤杂志,2014,30(2):179-181.
[8]Elvis AM,Ekta JS.Ozone therapy:A clinical review[J].J Nat Sci Biol Med,2011,2(1):66-70.
[9]González-Guevara E,Martínez-Lazcano JC,Custodio V,et al.Exposure to ozone induces a systemic inflammatory response:possible source of the neurological alterations induced by this gas[J].Inhal Toxicol,2014,26(8):485-491.
[10]Valacchi G,Sticozzi C,Belmonte G,et al.Vitamin C Compound Mixtures Prevent Ozone-Induced Oxidative Damage in Human Keratinocytes as Initial Assessment of Pollution Protection[J].PLos One,2015,10(8):e0131097.
[11]Sagai M,Bocci V.Mechanisms of Action Involved in Ozone Therapy:Is healing induced via a mild oxidative stress?[J].Med Gas Res,2011,1:29.
[12]Singer AJ,Clark RA.Cutaneous wound healing[J].N Engl J Med,1999,341(10):738-746.
[13]郭小伟,张家平,黄跃生.表皮细胞迁移与创面愈合[J/CD].中华损伤与修复杂志(电子版),2016,11(3):186-190.
[14]Mccarthy JT,Pelle E,Dong K,et al.Effects of ozone in normal human epidermal keratinocytes[J].Exp Dermatol,2013,22(5):360-361.
[15]李韶辉,余革,陈国勤,等.不同浓度臭氧硬膜外注射对实验兔脊髓的CT和病理学影响[J].中华生物医学工程杂志,2014,20(3):209-211.
[16]Aquilano K,Baldelli S,Ciriolo MR.Glutathione:new roles in redox signaling for an old antioxidant[J].Front Pharmacol,2014,5:196.
[17]Kong Y,Trabucco SE,Hong Z.Oxidative Stress,Mitochondrial Dysfunction and the Mitochondria Theory of Aging[J].Interdiscip Top Gerontol,2014,39:86-107.
[18]Zhang H,Forman HJ.Glutathione synthesis and its role in redox signaling[J].Semin Cell Dev Biol,2012,23(7):722-728.
[19]Ghezzi P.Regulation of protein function by glutathionylation[J].Free Radic Res,2005,39(6):573-580.
[20]Aquilano K,Baldelli S,Ciriolo MR.Glutathione:new roles in redox signaling for an old antioxidant[J].Front Pharmacol,2014,5:196.
[21]Bocci V,Borrelli E,Travagli V,et al.The ozone paradox:ozone is a strong oxidant as well as a medical drug[J].Med Res Rev,2009,29(4):646-682.
[22]赵超莉,叶子青,阮琼芳,等.臭氧气浴对大鼠深Ⅱ度烧伤创面愈合及细胞因子表达的影响[J].中华实验外科杂志,2017,34(12):2084-2086.
[23]Bocci Velio.Ozone-A new medical drug[M].Netherlands:Springer,2005:20-28,75-83,198-208.
[24]Gazzeri R,Galarza M,Neroni M,et al.Fulminating septicemia secondary to oxygen-ozone therapy for lumbar disc herniation:case report[J].Spine(Phila Pa 1976),2007,32(3):E121-123.
[25]Eming SA,Martin P,Tomic-Canic M.Wound repair and regeneration:mechanisms,signaling,and translation[J].Sci Transl Med,2014,6(265):265sr6.