胎鼠不同造血部位的形态学和组织化学研究

Morphological and histochemical study of fetal mice in multiple hematopoietic sites

关键词:  组织学  形态学  造血干细胞  造血部位  胎鼠

Key words:  morphology  histology  hematopoietic stem cell  hematopoietic sites  fetal mice

摘要:

目的 观察胚胎11.5d(embryonic 11.5d, E11.5)不同造血部位的解剖结构和造血干细胞(hematopoietic stem cell, HSC)相关基因的转录水平、定位和形态,探讨不同造血部位HSC的起源、迁移、扩增和发育机制。方法运用尼氏(Nissl)染色、苏木精-伊红(htoxylin eosin, H-E)染色和免疫荧光技术(immunofluorescence technique, IF)观察E11.5小鼠胎盘(placenta, PL)、卵黄囊(yolk sac, YS)、主动脉-性腺-中肾(aorta-gonad-mesonephros, AGM)区、胎肝(fetal liver, FL)和头部(head, Hd)的形态学和组织学变化,以及运用实时定量基因扩增荧光检测系统(real-time quantitative, PCR, detecting system, QPCR)检测HSC相关基因的转录水平。结果 Nissl和H-E染色结果显示E11.5小鼠AGM区背主动脉、PL血管迷路、YS血管、Hd脑血管和FL血窦富含血细胞；CD34、CD45、CD133、c-kit、Sca-1和Runx1在不同造血部位均有表达(P＜0.05),其中PL表达水平显著高于其他造血部位(P＜0.05)；IF结果显示CD34在不同造血部位均有表达,YS血管和PL迷路血管呈强阳性；AGM区背主动脉和泌尿生殖嵴内皮细胞、Hd脑血管周围可见CD45呈弱阳性,而FL则显示CD45为阴性。c-kit和CD133在YS血管和PL迷路血管同为强阳性。结论 HSC产生关键时期,胚鼠PL和YS中HSC发育最旺盛。永久造血起源于小鼠胚内AGM区,而不同造血部位HSC的迁移与血管内皮细胞密切相关,HSC可能通过AGM区血管内皮迁移进入血液循环,首先到达 PL的血管迷路,然后定植于FL,扩增或维持HSC。

Abstract:

Objective To observe the anatomical structure and related gene expression in multiple hematopoietic sites of fetal mice at embryonic 11.5 d (E11.5). Methods The morphology and histology were observed by using Nissl staining, Hematoxylin and Eosin (HE) staining, and immunofluorescence technique; and the mRNA expression of hematopoietic stem cell (HSC) related genes was detected by Real-time quantitative PCR (RT-qPCR) at different hematopoietic sites, including the placenta (PL), yolk sac (YS), aorta-gonad-mesonephros (AGM) region, head (Hd) and fetal liver (FL) of fetal mice at E11.5. Results Nissl and HE staining indicated that placenta labyrinthica, blood vessel of yolk sac, dorsal aorta of AGM, the cerebrovascular head and fetal liver blood sinus contained abundant amount of blood cells. CD34, CD45, CD133, c-kit, sca-1 and Runx1 were expressed significantly at different hematopoietic sites (P＜0.05), and compared with other hematopoietic sites, the expression of HSC marker was significantly high in PL(P＜0.05). Immunofluorescence technique demonstrated that CD34⁺ HSC was expressed at different hematopoietic sites, and strongly positive in placenta vascular labyrinth and blood vessels of yolk sac. Additional HSC marker CD45 was lower expressed at dorsal aorta and urogenital ridges of AGM region, head cerebrovascular region, but CD45- cells were present in the fetal liver. Besides, vascular labyrinth of placenta and blood vessel of yolk sac was highly expressed with c-kit and CD133. Conclusion This study demonstrates that HSCs are the most active in embryonic mice PL and YS during a key period of HSC emergence. Definitive hematopoiesis originates from AGM region, and migration of HSCs in multiple hematopoietic sites is closely to the endothelial cells. HSCs may migrate through the vascular endothelial, the first niche for expansion of blood stem cells is the placenta's vascular labyrinth, HSCs engraft in FL, then amplify or maintain.