促红细胞生成素在斑马鱼胚胎发育中抑制肾脏细胞凋亡  

Renal protective and anti-apoptotic functions of erythropoietin in zebrafish embryonic development

作　　者：折剑青[1] 娄博文 吴岳[1] 袁祖贻[1] SHE Jian-qing;LOU Bo-wen;WU Yue;YUAN Zu-yi(Department of Cardiology,The First Affiliated Hospital of Xi＇ an Jiaotong University,Xi＇ an 710048,China)

机构地区：[1]西安交通大学第一附属医院心血管内科,陕西西安710048

出　　处：《中国病理生理杂志》2018年第6期1067-1074,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81570406);西安交通大学中央高校基本科研业务费资助项目(No.1191329724)

摘　　要：目的:研究促红细胞生成素(Epo)在斑马鱼早期胚胎肾脏细胞发育中的凋亡和坏死方面起到的作用。方法:使用吗啉反义寡核苷酸技术通过胚胎注射使Epo及其受体(EpoR)的表达下调,并用real-time PCR及血红蛋白染色进行基因下调的有效性验证。在胚胎受精48 h观察肾脏形态并进行TUNEL凋亡染色,使用流式细胞术分析Annexin V凋亡染色,并用Western blot进行Akt磷酸化验证。结果:Epo及EpoR基因表达下调的斑马鱼胚胎在受精48 h可见明显肾小球囊样改变,且肾小管颈部缩短消失。TUNEL及Annexin V染色结果提示受精48 h后,Epo及EpoR基因表达下调的斑马鱼胚胎中肾脏凋亡细胞增多,但EpoR基因下调的斑马鱼胚胎内主要表现为晚期凋亡细胞及坏死细胞增多。Western blot实验结果提示Epo及EpoR基因表达下调的斑马鱼胚胎较对照组Akt磷酸化减少(P<0.05)。结论:促红细胞生成素可引起Akt磷酸化。促红细胞生成素通过保护肾脏细胞免于凋亡和坏死而在斑马鱼胚胎肾脏发育中发挥重要作用。AIM： To analyze zebrafish embryos and to identify erythropoietin（ Epo） as an active renal antiapoptotic factor in an Epo receptor（ EpoR）-dependent manner. METHODS： For transient knockdown of Epo and EpoR in renal Tg（ wt1b： EGFP） zebrafish reporter lines,the morpholino antisense oligonucleotide technique was used. Morphant zebrafish embryos were phenotypically analyzed using fluorescence microscopy. Apoptosis was determined by TUNEL assay and Annexin V staining. Western blot was used to identify Akt phosphorylation in Epo and EpoR knockdown zebrafish. RESULTS： Epo and EpoR zebrafish morphants exhibited pathophysiological changes within the pronephros,adversely affecting pronephric structure. Zebrafish embryos upon silencing of Epoa and EpoR showed a significant increase in the apoptosis within the zebrafish pronephros,consequently leading to renal pathophysiological effects. Decreased p-Akt was identified in Epo and EpoR knockdown zebrafish embryos. CONCLUSION： Epo is an essential regulator of renal development and function by interacting with its receptor EpoR and thereby repressing apoptosis,mechanistically by promoting Akt phosphorylation.

关 键 词：促红细胞生成素 斑马鱼 胚胎发育 肾脏 细胞凋亡 

分 类 号：R692[医药卫生—泌尿科学] R363[医药卫生—外科学]