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目的探讨发动相关蛋白1(Drp-1)在新生儿窒息后血清诱导人近曲肾小管上皮细胞(HK-2细胞)损伤中的作用及重组人促红细胞生成素(rhEPO)干预的影响。方法将培养的HK-2细胞分为对照组、窒息组和rhEPO干预组,以新生儿重度窒息(Apgar评分<4分)后24 h血清(20%体积比)作为攻击因素。光镜下观察其线粒体形态变化,活细胞计数试剂盒(CCK-8)法检测细胞活力,免疫组织化学检测其Drp-1表达,投射电子显微镜下观察其线粒体的形态变化。结果 1.HK-2细胞形态学变化:对照组及rhEPO干预组细胞生长良好,窒息组细胞发生凋亡相关改变。2.细胞活力变化:与对照组细胞活力(0.74±0.08)相比,窒息组细胞活力(0.41±0.06)明显降低,rhEPO干预组细胞活力(0.60±0.08)较窒息组好转,但未恢复至对照组水平(P<0.05)。3.免疫组织化学法测定Drp-1表达:与对照组(0.24±0.03)相比,窒息组细胞Drp-1表达(0.51±0.03)显著升高,rhEPO干预组(0.38±0.03)较窒息组降低,但未恢复至对照组水平(P<0.05)。4.线粒体形态变化:对照组和rhEPO干预组线粒体结构完整,可见线粒体嵴;窒息组线粒体嵴消失,膜肿胀,空泡变性。结论 rhEPO可减轻新生儿窒息后血清诱导的HK-2细胞凋亡,其机制可能是通过减少Drp-1的表达抑制线粒体的分裂而实现。
Objective To investigate the effect of Drp-1 on the injury of human proximal proximal tubular epithelial cells (HK-2) induced by neonatal asphyxia and the effect of recombinant human erythropoietin (rhEPO) on it. Methods The cultured HK-2 cells were divided into control group, asphyxia group and rhEPO intervention group. Serum (20% by volume) was used as the attacking factor after neonatal severe asphyxia (Apgar score <4) and 24 h. The morphological changes of mitochondria were observed under light microscope. The cell viability was detected by CCK-8 assay. The expression of Drp-1 was detected by immunohistochemistry. The morphological changes of mitochondria were observed under electron microscope. The morphological changes of HK-2 cells: The cells in control group and rhEPO intervention group grew well, and the changes of apoptosis in asphyxiated group were observed. The viability of asphyxia group was significantly lower than that of control group (0.74 ± 0.08) (0.41 ± 0.06), the viability of rhEPO-treated group (0.60 ± 0.08) was better than that of asphyxia group, but not restored to Control group level (P <0.05). Compared with the control group (0.24 ± 0.03), the expression of Drp-1 in the asphyxia group (0.51 ± 0.03) was significantly higher than that in the control group (0.38 ± 0.03), compared with the asphyxia group Decreased, but not restored to the control group (P <0.05). Mitochondrial morphological changes: control group and rhEPO intervention group mitochondrial structure is complete, visible mitochondrial cristae; mitochondrial cristae disappeared, membrane swelling, vacuolar degeneration. Conclusion rhEPO can reduce neonatal asphyxia serum-induced apoptosis of HK-2 cells, its mechanism may be through the reduction of the expression of Drp-1 mitochondrial mitosis to achieve.