视神经萎缩症蛋白在新生儿窒息后血清诱导HK-2凋亡中的作用及rhEPO干预的影响

Roles of optic atrophy in human renal tubular cells (HK-2) injured by postasphyxial-serum in neonates and effects of recombinant human erythropoietin on it

目的探讨视神经萎缩症蛋白(OPA1)在新生儿窒息后血清诱导人近曲肾小管上皮细胞(HK-2)凋亡中的表达变化及重组人促红细胞生成素(rhEPO)干预的影响。方法将HK-2分为对照组、窒息组和rhEPO组,以新生儿重度窒息后24 h血清作为攻击因素,光镜及投射电子显微镜下观察HK-2线粒体形态变化,CCK-8法检测HK-2细胞活力,免疫组化检测HK-2的OPA1表达。结果对照组及rhEPO组HK-2生长良好,窒息组HK-2发生凋亡相关改变。与对照组HK-2的细胞活力(0.74±0.08)相比,窒息组细胞活力(0.41±0.06)明显降低,而rhEPO组细胞活力(0.60±0.08)则较窒息组提高,差异均有统计学意义(P均<0.05)。与对照组HK-2的OPA1表达(0.47±0.02)相比,窒息组细胞OPA1表达(0.21±0.02)显著降低,而rhEPO组(0.36±0.02)较窒息组有所升高,差异均有统计学意义(P均<0.05)。对照组和rhEPO组HK-2的线粒体结构完整,可见线粒体嵴;窒息组HK-2的线粒体嵴消失,膜肿胀,空泡变性。结论 rhEPO可能通过增加OPA1表达而抑制线粒体分裂,减轻新生儿窒息后血清诱导HK-2细胞凋亡。

Objective To investigate the role of optic atrophy（OPA1）in human renal tubular cells（HK-2）injured by postasphyxial-serum in neonates and effects of recombinant human erythropoietin on it.Methods The experiment was designed as control group,asphyxia group and rhEPO-treated group.The serum of neonates at 24 hours after asphyxia（Apgar Scoring less than 4）was applied as attacking factor.The morphological change of HK-2 cells was observed under inverted microscope,the cell viability was measured by CCK-8 methods,the expression of OPA1 in cytoplast was detected by using of immunohistochemical method,the morphological change of mitochondria was observed under transmission electron microscope（TEM）.Results Under inverted microscopy,HK-2 cells in control group grew very quickly.The morphological change of HK-2 was more serious in asphyxia group than that in control group.The morphological change of HK-2 was improved after the treatment of rhEPO.The cell viability（OD）was decreased in the asphyxia group（0.41 ± 0.06,P 〈 0.05）.After the treatment of rhEPO,the cell viability（OD）was obviously increased（0.60 ± 0.08,P 〈 0.05）.The expression of OPA1（OD）was significantly increased in asphyxia group（0.21 ± 0.02,P 〈 0.05）.After the treatment of rhEPO,the expression of OPA1 was obviously decreased（0.36 ± 0.02,P 〈 0.05）.Under TEM,mitochondria in asphyxia group appeared extensive swelling and vacuolar degeneration with less matrix and obscure or vanished mitochondria cristae;but in control and rhEPO group,mitochondrial structure was integrated,with uniform matrix and visible mitochondria cristae.Conclusions RhEPO could relieve the injury of renal tubular cells induced by postasphyxial-serum in neonate.The pretreatment with rhEPO could increase the expression of OPA1,then improve the morphological change of mitochondria induced by postasphyxial-serum.