硫化氢对顺铂诱导近端肾小管上皮细胞凋亡的干预研究  

Intervention of hydrogen sulfide on apoptosis of proximal renal tubular epithelial cells induced by cisplatin

作　　者：杨波[1] 倪倩 朱艳[1] 赵欢顺[1] 杨成[2] 段绍斌[3] 蒋云生[3] Bo Yang;Qian Ni;Yan Zhu;Huan-shun Zhao;Cheng Yang;Shao-bin Duan;Yun-sheng Jiang(Department of Nephrology,the First Affiliated Hospital,University of South China,Hengyang,Hunan 421001,China;Clinical Department,Changsha Medical College,Changsha,Hunan 410219,China;Department of Nephrology,Second Xiangya Hospital,Central South University,Changsha,Hunan 410011,China)

机构地区：[1]南华大学附属第一医院肾内科,湖南衡阳421001 [2]长沙医学院临床系,湖南长沙410219 [3]中南大学湘雅二医院肾内科,湖南长沙410011

出　　处：《中国现代医学杂志》2018年第25期20-25,共6页China Journal of Modern Medicine

基　　金：湖南省自然科学基金(No:13JJ3082)

摘　　要：目的探讨硫化氢H_2S对顺铂(DDP)诱导近端肾小管上皮细胞凋亡的干预作用。方法研究分为阴性对照组、DDP组、DDP+硫氢化钠NaHS组。DDP组:DDP浓度为0、1、2、5、10、20和40 mg/L;DDP+NaHS组:NaHS浓度分别为0、0.2、0.4、0.5、0.8、1.0和2.0 mmol/L,加DDP(20 mg/L)共同作用。噻唑蓝(MTT)实验:DDP组、DDP+NaHS组与人近端肾小管上皮细胞株(HK-2细胞)共同培养,24 h后测光密度(OD)值。NaH(0.5S和1.0 mmol/L)加DDP(20 mg/L)与HK-2细胞共同培养24 h。4,6-联脒-2-苯基吲哚(DAPI),Annexin V-FITC/PI染色通过显微镜观察各组细胞的形态学改变。Annexin V-FITC/PI流式细胞术检测各组细胞凋亡率。结果 MTT实验:0、1、2、5、10和20 mg/L DDP浓度的OD值分别为(0.270±0.064)、(0.229±0.022)、(0.198±0.024)、(0.189±0.069)、(0.188±0.030)和(0.165±0.012),OD值随DDP浓度上升逐渐下降,在20 mg/L低于阴性对照组(P<0.05)。20 mg/L DDP加浓度分别为0.2、0.4、0.5、0.8和1.0 mmol/L NaHS的OD值分别为(0.173±0.051)、(0.186±0.023)、(0.259±0.050)、(0.263±0.033)和(0.268±0.098),以上与浓度为20 mg/L DDP的OD值(0.153±0.017)比较,差异有统计学意义(P<0.05)。DAPI、Annexin VFITC/PI染色荧光显微镜显示,对照组HK-2细胞平均凋亡细胞数为(4.230±1.015),20 mg/L DDP影响下为(35.020±6.079),两者差异有统计学意义(P<0.05),DDP组高于阴性对照组。DDP+NaHS组在0.5和1.0 mmol/L的平均凋亡细胞分别为(22.550±2.912)和(9.780±2.063),差异有统计学意义(P<0.05),DDP+NaHS组低于DDP组。Annexin V-FITC/PI流式细胞术检测,阴性对照组HK-2细胞凋亡率(5.167±0.612)%,在20 mg/L DDP影响下,为(31.598±1.014)%,细胞凋亡率增加(P<0.05)。合用NaHS凋亡减少,在0.5和1.0 mmol/L的凋亡率为(18.375±2.239)%和(11.636±1.233)%,差异有统计学意义(P<0.05),DDP+NaHS组低于DDP组。结论 NaHS对DDP导致的近端肾小管上皮细胞的凋亡有保护作用。Objective To explore the intervention of hydrogen sulfide(H2S)on apoptosis of proximal renal tubular epithelial cells induced by cisplatin(DPP).Methods The HK-2 cells were randomly assigned into a control group,DPP treated groups(0,1,2,5,10,20 and 40 mg/L)and DDP(20 mg/L)+NaHS(0,0.2,0.4,0.5,0.8,1.0 and 2.0 mmol/L)treated groups.After the HK-2 cells were co-cultured with DPP or DPP+NaHS for 24 h,MTT assay was used to measure the OD value.Using 4’,6-diamidino-2-phenylindole(DAPI)and Annexin V-FITC/PI double staining the morphological changes of each group were observed under fluorescence microscope,and flow cytometry was used to detect the apoptosis rate of each group.Results According to MTT,the OD value was(0.270±0.064),(0.229±0.022),(0.198±0.024),(0.189±0.069),(0.188±0.030)and(0.165±0.012)respectively in the DDP groups with different concentrations(0,1,2,5,10,and 20 mg/L),DDP accelerated HK-2 cell apoptosis in a concentrationdependent manner,there was significant difference in the OD value between the 20 mg/L DDP group and the control group(P<0.05).Adding H2S(0.2,0.4,0.5,0.8,and 1.0 mmol/L)and DPP(20 mg/L)into the cells,the OD was(0.173±0.051),(0.186±0.023),(0.259±0.050),(0.263±0.033)and(0.268±0.098)respectively,with significant increases compared to(0.153±0.017)after adding 20 mg/L DDP alone(P<0.05),the protective effect of H2S was also in a concentration-dependent manner.Through the DAPI,Annexin V-FITC/PI staining,under the effect of 20 mg/L DDP,the average number of apoptotic cells in visual field of HK-2 cells was(35.020±6.079),while that in the normal negative control group was(4.230±1.015),there was statistical significance between them(P<0.05).Adding 0.5 and 1.0 mmol/L of NaHS could ameliorate apoptosis,the average number of apoptotic cells in visual field was(22.550±2.912)and(9.780±2.063),which were significantly lower than that of the DDP group(P<0.05).Flow cytometry revealed that 20 mg/L DDP incubated with HK-2 cells for 24 h led to apoptosis with the rate of(31.598±1.014)%which was

关 键 词：近端肾小管上皮细胞 硫化氢 顺铂 细胞凋亡 

分 类 号：R692.6[医药卫生—泌尿科学]