【作者】 陈亮；

【导师】 董文斌；

【作者基本信息】 泸州医学院 ， 儿科学， 2009， 硕士

【摘要】 目的:研究Na~+/H~+交换器-1(Na~+/H~+ exchanger 1,NHE1)在新生儿窒息后血清诱导人近曲肾小管上皮细胞(HK-2)损伤中的作用及机制。方法:(1)以HK-2为研究对象;(2)以200ml/l窒息后第一天的血清作为攻击因素;(3)第一阶段实验分为2组:对照组和窒息组,探讨窒息后血清诱导人近曲肾小管上皮细胞损伤时NHE1是否参与并发挥损伤作用;(4)第二阶段实验分为:对照组,窒息组,EIPA组,探讨利用EIPA阻断NHE1活性后在窒息后血清诱导人近曲肾小管上皮细胞损伤时,对HK-2细胞的是否具有保护作用及其机制;(5)检测指标:第一阶段实验采用免疫组织化学的方法检测HK-2细胞在损伤前后的NHE1表达含量的变化,;第二阶段实验通过倒置相差显微镜观察对照组,窒息组,EIPA组的各组细胞形态,四唑盐(MTT)比色法检测HK-2细胞活力变化,生物化学法检测培养液中LDH漏出率的变化。(6)统计学处理:所有数值以均数±标准差(x±S)表示,实验组组间进行LSD检验。P<0.05为差异有显著性。采用SPSS10.0统计软件完成。结果:(1)与对照组相比,窒息组细胞NHE1表达含量明显增加,细胞形态受损明显。(2)倒置相差显微镜下观察:对照组细胞贴壁良好,透明度大,折光性强,呈扁平的多角形,分裂相多,细胞排列紧密,连接成片,数量多;窒息组细胞生长缓慢,细胞数量比空白对照组明显减少,细胞形态发生改变,由典型的扁平多角形细胞变为不规则圆形或椭圆形,折光性减弱,轮廓增强,胞质中出现空泡,脂滴,颗粒样物质,细胞间空隙加大,连接松散,细胞间可见较多细胞碎片;(3)与对照组相比,窒息组LDH漏出率较明显升高,细胞活力(OD值)明显降低(P=0.000)。而与窒息组相比,EIPA(2μmol/l浓度)组, LDH漏出率明显降低,细胞活力(OD值)明显增高,差异具有统计学意义(P <0.05)。结论:新生儿窒息后血清诱导HK-2细胞NHE1的表达是导致HK-2细胞损伤的重要机制。更多还原

【Abstract】 Objective : To investigate the role of NHE1 in injury of human renal tubular cells(HK-2) induced by postasphyxial-serum in neonates.Methods (1)Human renal proximal tubular cell(HK-2)was used as the target cel1.(2) The serum of neonates in one day after asphyxia, whose concentration were 200ml/l (volume fraction),were applied as attacking factor .(3)The experiment was designed as control group,asphyxia group,which were used to research whether NHE1 play an important role in the injury of human renal tubular cell induced by postasphyxial- serum of neonate .(4)In the second experiment was designed as control group,asphyxia group,EIPA group,which were researched whether EIPA had the protective effect . (5) The following indicators were detectied : In the first experiment,the expression of NHE1 in the HK-2 were detected by immunohistochemical method.In the second experiment,the cell viability was measured by 3-(4,5-dimethy lthiazcl-2-y1)-2,5-diphenyl–tetazoliumbromide (MTT) methods.In the first experiment,the changes of morphology were observed under inverted microscope,and the cell viability was measureed by 3-(4,5-dimethy lthiazcl-2-y1)-2,5-diphenyl -tetazolium bromide(MTT) methods,and the leakage rate of lactate dehydrogenase(LDH) was determined by biochemical methods.(6) All parameters were expressed as the mean value±standard difference( x±S), statistical analysis were carried out by the use of the least significant difference.Difference was considered significance when the p.value was less than 0.05.The statistics work were finished by spss10.0 software.Results (1)Compared with control group,the expression of NHE1 in the HK-2 were increased significantly in asphyxia group.(2) Under inverted microscopy,HK-2 cells in control group were significantly increased,sticked to each other tightly and grew very quickly.Their adhesion were better,multyang applan,and refraction raised.The form and quantity of HK-2 cell was normal .Compared with control group,the changes in morphology of HK-2 were most serious and obvious in asphyxia group,the cells grew slowly,the mount decreased,form of the cells changed from typical multy-ang applan to off-normal round or ellipse.Refraction rate was decreased,and contour enhanced.The vacuolus,lipid droplet and granulation appeared in the kytoplasm.There was much cell debris in accrescent intercellular space.the cell viability (optical density,OD) were obviously decreased in the asphyxia group(P=0.000) ,the leakage rate of LDH were significantly increased in asphyxia group(P=0.000) ,statistical significance existed between control group and asphyxia group(P<0.05).But compared with asphyxia group, the changes in morphology of HK-2 were obviously improved in the EIPA groups ( 2μmol/l), the leakage rate of LDH were significantly decreased,statistical significance existed between EIPA group and asphyxia group(P<0.05); the cell viability were obviously increased,statistical significance existed between EIPA group and asphyxia group(P<0.05).Conclusion :The postasphyxial-serum could induced the increased the expression of NHE1,which could play an important role in injury of renal tubular cells induced by postasphyxial-serum in neonates.更多还原