CAJ | 학술논문

目的研究宫内生理性低氧微环境对哺乳动物胚胎早期肾脏发育的影响.方法采用大鼠胚胎肾脏体外培养的方法建立肾脏发育模型,应用低氧培养箱模拟胚胎发育早期宫内生理性低氧微环境,免疫荧光染色技术标记胚肾输尿管芽分枝和后肾间充质向上皮转分化过程,激光共聚焦显微镜观察胚肾发育形态学变化;原位末端转移酶标记技术(TUNEL法)检测胚肾细胞凋亡的变化,胸腺嘧啶核苷类似物(EdU)掺入法检测胚肾细胞增殖的变化,实时荧光定量PCR法检测胚肾发育相关基因表达的变化.结果体外培养条件下低氧(3％～5％O2)微环境显著抑制胚肾发育,输尿管芽分枝和肾小球数目均减少(P＜0.05).低氧(5％O2)条件下胚肾细胞的凋亡程度较常氧下显著减轻,细胞增殖则受到了显著抑制(P＜0.05).低氧增强胚肾后肾间充质Six2阳性干细胞的自我更新,上调Six2、Wt-1等发育重要相关基因的表达,下调Wnt9b和Wnt4等诱导分化相关基因的表达.结论宫内生理性低氧微环境可能通过影响哺乳动物胚肾发育相关信号通路抑制胚肾干细胞的增殖和分化,从而促进胚肾干细胞维持自我更新.
목적 연구궁내생이성저양미배경대포유동물배태조기신장발육적영향.방법 채용대서배태신장체외배양적방법건립신장발육모형,응용저양배양상모의배태발육조기궁내생이성저양미배경,면역형광염색기술표기배신수뇨관아분지화후신간충질향상피전분화과정,격광공취초현미경관찰배신발육형태학변화;원위말단전이매표기기술(TUNEL법)검측배신세포조망적변화,흉선밀정핵감유사물(EdU)참입법검측배신세포증식적변화,실시형광정량PCR법검측배신발육상관기인표체적변화.결과 체외배양조건하저양(3％～5％O2)미배경현저억제배신발육,수뇨관아분지화신소구수목균감소(P＜0.05).저양(5％O2)조건하배신세포적조망정도교상양하현저감경,세포증식칙수도료현저억제(P＜0.05).저양증강배신후신간충질Six2양성간세포적자아경신,상조Six2、Wt-1등발육중요상관기인적표체,하조Wnt9b화Wnt4등유도분화상관기인적표체.결론 궁내생이성저양미배경가능통과영향포유동물배신발육상관신호통로억제배신간세포적증식화분화,종이촉진배신간세포유지자아경신.
Objective To explore the effects of physiological intrauterine hypoxia on the mammal kidney development in ex vivo model.Methods Kidney rudiments were micro-dissected from embryonic day 13.5 SD rat embryos.They were pooled and assigned randomly to control group (normoxia,21％ O2) and experimental groups(hypoxia,3％-5％ O2).For conventional culture,the rudiments were placed on 0.4 μm pore-size polycarbonate filters at the bottom of a well insert in a six well plate and incubated at 5％ CO2 at 37℃.The hypoxia micro-environment was created in an incubator through injecting N2 Immunostaining was carried out to label the structure of developing kidney and pictures were taken by a laser confocal microscope.TdT-mediated dUTP nick end labeling was used to detect cell apoptosis while EdU (5-Ethynyl-2'-deoxyuridine) was added to the medium for cell proliferation detection during kidney development.After culture kidneys of both groups were processed and the changes of genes expression were measured by real time PCR.Results Kidney development was significantly suppressed under hypoxia condition with decreased ureteric buds as well as nephrons compared with that under normoxia condition (P＜0.05).Hypoxia inhibited the proliferation of cultured kidneys while reduced the apoptosis of their cells (P ＜ 0.05).Six2-positive progenitors were better maintained within kidneys cultured under low O2 (P ＜ 0.05).Moreover,genes regulating metanephric mesenchymal to epithelial transformation such as Wnt9b and Wnt4 were down-regulated while genes functioning to maintain metanephric mesenchymal progenitors such as Six2,Wt-1 and Pax2 were up-regulated in hypoxia.Conclusion Physiological intrauterine hypoxia as an important microenvironment factor may suppress mammal kidney development through inhibiting the proliferation and differentiation of embryonic kidney progenitors while maintain their undifferentiated status and self-renewal,thus contributing to the balance between differentiation and maintenance of a mesenchymal progenitor cell pool which determines the final number of nephrons in adult kidneys.