中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2014年
4期
263-267
,共5页
蔡典其%杨春涛%陈凤娟%陈存特%陈珏铭%骆月妍%江伟炽%郑洁蓉%张辉
蔡典其%楊春濤%陳鳳娟%陳存特%陳玨銘%駱月妍%江偉熾%鄭潔蓉%張輝
채전기%양춘도%진봉연%진존특%진각명%락월연%강위치%정길용%장휘
糖尿病%硫化氢%角朊细胞%丙酮醛%皮肤溃疡
糖尿病%硫化氫%角朊細胞%丙酮醛%皮膚潰瘍
당뇨병%류화경%각원세포%병동철%피부궤양
Diabetes%H2S%Keratinocytes%Methylglyoxal%Skin ulcer
目的 观察硫化氢(H2S)对丙酮醛(MGO)诱导的人皮肤角质形成细胞(HaCaT)损伤的影响.方法 HaCaT细胞分为MGO组、对照组和MGO+NSHD(H2S供体)组.MGO组用200、400、600 μmol/LMGO处理HaCaT细胞48 h造成细胞损伤;对照组给予等体积培养基;MGO+NSHD组应用400 μmol/LMGO处理,48 h前用50、100、200μmol/LNSHD预处理1h.通过CCK-8检测各组细胞活性.HaCaT细胞经100 μmol/LNSHD预处理1h,用20 μmol/L的H2S荧光探针WSP-1染色结合荧光照相术检测NSHD预处理1h组和对照组培养基和细胞内的H2S含量.将HaCaT细胞分为MGO组、MGO+NSHD组、NSHD组和对照组.MGO组仅用400 μmol/L MGO处理48 h;MGO+NSHD组在400 μmol/L MGO处理48 h前用100 μmol/L NSHD预处理1 h;NSHD组的细胞仅用100 μmol/L NSHD处理1h;对照组则给予等体积培养基.Hoechst 33258核染色结合荧光照相术检测细胞凋亡.Rh123染色结合荧光照相术检测线粒体膜电位(MMP).结果 HaCaT细胞经200、400和600 μmol/L MGO处理48 h,细胞活性依次为0.325±0.023、0.224±0.009和0.095±0.102,均低于对照组0.415±0.031(F=37.866,P＜0.05),其中400 μmol/LMGO组细胞活性约为对照组的1/2,选用此浓度的MGO作为有效损伤浓度.100 μmol/L NSHD处理1h可使培养基和细胞内的H2S含量较对照组均增加.在400μmol/L MGO处理48 h前,分别用50、100和200 μnol/L NSHD预处理1h,细胞活性依次为0.235±0.028、0.314±0.017、0.346±0.020,均高于单独400μmol/L MGO处理组0.224±0.009(F=61.209,P＜0.05).400 μmol/L MGO处理48 h后细胞凋亡率高于对照组和NSHD组[(32.6±3.5)％比(5.1±1.2)％、(3.4±0.8)％,均P＜0.05],MMP低于对照组和NSHD组(28.5±2.9比46.1±3.8、48.6±4.3,均P＜0.05).MGO+ NSHD组细胞凋亡率(18.3±2.6)％,低于MGO组但高于对照组和NSHD组(均P＜0.05),MMP为38.9±3.2,高于MGO组但低于对照组和NSHD组(均P＜0.05).结论 NSHD能通过释放H2S拮抗MGO诱导的皮肤细胞损伤.
目的 觀察硫化氫(H2S)對丙酮醛(MGO)誘導的人皮膚角質形成細胞(HaCaT)損傷的影響.方法 HaCaT細胞分為MGO組、對照組和MGO+NSHD(H2S供體)組.MGO組用200、400、600 μmol/LMGO處理HaCaT細胞48 h造成細胞損傷;對照組給予等體積培養基;MGO+NSHD組應用400 μmol/LMGO處理,48 h前用50、100、200μmol/LNSHD預處理1h.通過CCK-8檢測各組細胞活性.HaCaT細胞經100 μmol/LNSHD預處理1h,用20 μmol/L的H2S熒光探針WSP-1染色結閤熒光照相術檢測NSHD預處理1h組和對照組培養基和細胞內的H2S含量.將HaCaT細胞分為MGO組、MGO+NSHD組、NSHD組和對照組.MGO組僅用400 μmol/L MGO處理48 h;MGO+NSHD組在400 μmol/L MGO處理48 h前用100 μmol/L NSHD預處理1 h;NSHD組的細胞僅用100 μmol/L NSHD處理1h;對照組則給予等體積培養基.Hoechst 33258覈染色結閤熒光照相術檢測細胞凋亡.Rh123染色結閤熒光照相術檢測線粒體膜電位(MMP).結果 HaCaT細胞經200、400和600 μmol/L MGO處理48 h,細胞活性依次為0.325±0.023、0.224±0.009和0.095±0.102,均低于對照組0.415±0.031(F=37.866,P＜0.05),其中400 μmol/LMGO組細胞活性約為對照組的1/2,選用此濃度的MGO作為有效損傷濃度.100 μmol/L NSHD處理1h可使培養基和細胞內的H2S含量較對照組均增加.在400μmol/L MGO處理48 h前,分彆用50、100和200 μnol/L NSHD預處理1h,細胞活性依次為0.235±0.028、0.314±0.017、0.346±0.020,均高于單獨400μmol/L MGO處理組0.224±0.009(F=61.209,P＜0.05).400 μmol/L MGO處理48 h後細胞凋亡率高于對照組和NSHD組[(32.6±3.5)％比(5.1±1.2)％、(3.4±0.8)％,均P＜0.05],MMP低于對照組和NSHD組(28.5±2.9比46.1±3.8、48.6±4.3,均P＜0.05).MGO+ NSHD組細胞凋亡率(18.3±2.6)％,低于MGO組但高于對照組和NSHD組(均P＜0.05),MMP為38.9±3.2,高于MGO組但低于對照組和NSHD組(均P＜0.05).結論 NSHD能通過釋放H2S拮抗MGO誘導的皮膚細胞損傷.
목적 관찰류화경(H2S)대병동철(MGO)유도적인피부각질형성세포(HaCaT)손상적영향.방법 HaCaT세포분위MGO조、대조조화MGO+NSHD(H2S공체)조.MGO조용200、400、600 μmol/LMGO처리HaCaT세포48 h조성세포손상;대조조급여등체적배양기;MGO+NSHD조응용400 μmol/LMGO처리,48 h전용50、100、200μmol/LNSHD예처리1h.통과CCK-8검측각조세포활성.HaCaT세포경100 μmol/LNSHD예처리1h,용20 μmol/L적H2S형광탐침WSP-1염색결합형광조상술검측NSHD예처리1h조화대조조배양기화세포내적H2S함량.장HaCaT세포분위MGO조、MGO+NSHD조、NSHD조화대조조.MGO조부용400 μmol/L MGO처리48 h;MGO+NSHD조재400 μmol/L MGO처리48 h전용100 μmol/L NSHD예처리1 h;NSHD조적세포부용100 μmol/L NSHD처리1h;대조조칙급여등체적배양기.Hoechst 33258핵염색결합형광조상술검측세포조망.Rh123염색결합형광조상술검측선립체막전위(MMP).결과 HaCaT세포경200、400화600 μmol/L MGO처리48 h,세포활성의차위0.325±0.023、0.224±0.009화0.095±0.102,균저우대조조0.415±0.031(F=37.866,P＜0.05),기중400 μmol/LMGO조세포활성약위대조조적1/2,선용차농도적MGO작위유효손상농도.100 μmol/L NSHD처리1h가사배양기화세포내적H2S함량교대조조균증가.재400μmol/L MGO처리48 h전,분별용50、100화200 μnol/L NSHD예처리1h,세포활성의차위0.235±0.028、0.314±0.017、0.346±0.020,균고우단독400μmol/L MGO처리조0.224±0.009(F=61.209,P＜0.05).400 μmol/L MGO처리48 h후세포조망솔고우대조조화NSHD조[(32.6±3.5)％비(5.1±1.2)％、(3.4±0.8)％,균P＜0.05],MMP저우대조조화NSHD조(28.5±2.9비46.1±3.8、48.6±4.3,균P＜0.05).MGO+ NSHD조세포조망솔(18.3±2.6)％,저우MGO조단고우대조조화NSHD조(균P＜0.05),MMP위38.9±3.2,고우MGO조단저우대조조화NSHD조(균P＜0.05).결론 NSHD능통과석방H2S길항MGO유도적피부세포손상.
Objective To investigate the effects of hydrogen sulfide (H2S) on methylglyoxal (MGO)-induced human skin keratinocytes (HaCaT cells) injury.Methods HaCaT cells were assigned to control group,MGO group and MGO+NSHD(H2S donor) group.The MGO group was treated with 200,400 and 600 μmol/L MGO for 48 h to induce cell injury.Control group was treated with an aliquot of plain culture medium.In the MGO +NSHD group,before treatment with 400 μmol/L MGO for 48 h,the cells were preconditioned with 50,100 and 200 μmol/L NSHD for 1 h to examine the protective effects of H2S according to cell viability as measured by cell counting kit 8.After treatment with 100 μmol/L NSHD for 1 h,the levels of H2S in medium and cells of NSHD and control groups were assayed with 20 μmol/L WSP-1,the H2S fluorescent probe,for photofluorography.HaCaT cells were assigned to control group,MGO group,NSHD group and MGO+NSHD group.The MGO group was treated with 400 μmol/L MGO for 48 h to induce cell injury.Control group was treated with an aliquot of plain culture medium.In the MGO+NSHD group,before treatment with 400 μmol/L MGO for 48 h,the cells were preconditioned with 100 μmol/L NSHD,NSHD group was treated with only 100 μmol/L NSHD for 1 h.Cellular apoptosis and mitochondrial membrane potential (MMP) were observed by Hoechst 33258 and Rh123 staining followed by photofluorography respectively.Results Treatment with 200,400 and 600 μmol/L MGO for 48 h,were attenuated HaCaT cell viability 0.325±0.023,0.224±0.009 and 0.095±0.102 compared with control group 0.415±0.031 (F=37.866,P ＜ 0.05).Both in medium and in cells of NSHD group,the content of H2S was enhanced after treatment with 100 μmol/L NSHD for 1 h compared with control group.The viability in 400 μmol/L MGO treatment for 48 h group was halved compared with control group,and therefore 400 μmol/L was deemed the optimal concentration for MGO treatment.Before treatment with 400 μtmol/L MGO for 48 h,the cells were preconditioned with 50,100 and 200 μmol/L NSHD for 1 h,with the viability being 0.235±0.028,0.314± 0.017 and 0.346±0.020 respectively,There were higher than that in the individual 400 μmol/L MGO group (F=61.209,P ＜ 0.05).Compared with control and NSHD groups,The treatment with 400 μmol/L MGO for 48 h increased the apoptotic rate from (5.1±1.2)％,(3.4±0.8)％ to (32.6±3.5)％,and decreased the MMP from 46.1±3.8,48.6±4.3 to 28.5±2.9 (all P＜0.05).Prior to treatment with 400 μmol/L MGO for 48 h in the presence of preconditioning with 100 μmol/L NSHD for 1 h,the apoptotic rate was decreased to (18.3±2.6)％ and the MMP was increased to 38.9±3.2.The apoptotic rate in MGO+NSHD group was lower than that in MGO group but higher than those in control group and NSHD group.The MMP in MGO+NSHD group was higher than that in MGO group but lower than those in control group and NSHD group (all P＜0.05).Conclusion NSHD protects human skin keratinocytes from MGO-induced injury via release of H2S.
