PDK1在内皮细胞向造血细胞转换中的作用研究  

The role of PDK1 in the transition of endothelial to hematopoietic cells

作　　者：孙晓璐 王乐[2] 袁卫平 王伟丽 Sun Xiaolu;Wang Le;Yuan Weiping;Wang Weili(Institute of Hematology ＆ Blood Diseases Hospital,CAMS ＆ PUMC,State Key Laboratory of Experimental Hematology,Tianjin 300020,China)

机构地区：[1]实验血液学国家重点实验室中国医学科学院、北京协和医学院血液病医院(血液学研究所),天津300020 [2]天津市第一中心医院、卫生部危重病急救医学重点实验室

出　　处：《中华血液学杂志》2018年第9期709-716,共8页Chinese Journal of Hematology

基　　金：国家自然科学基金（81600082、81770105）;中国医学科学院医学与健康科技创新工程（2016-12M-017、2017-12M-3-015）;北京协和医学院“协和青年基金”（3332016093）

摘　　要：目的研究磷酸肌醇依赖性激酶1（PDK1）在内皮细胞向造血细胞转化阶段对造血干细胞（HSC）发生的影响。方法应用Vec—Cre在内皮细胞中特异性敲除PDK1基因，取对照组PDK1^fl/fl、PDK1^fl/＋小鼠及敲除组Vec—Cre；PDK1^fl/fl小鼠胚胎的主动脉-性腺-中肾区（AGM区）细胞进行集落形成实验，检测PDK1基因对造血祖细胞功能的影响；取对照组和敲除组AGM区细胞行移植实验，检测PDK1对HSC功能的影响；取对照组和敲除组AGM区细胞，通过流式细胞术检测PDKl对能够向造血转化的CD31＋c—Kit^high细胞群比例、细胞周期及细胞凋亡的影响；分选对照组和敲除组AGM区CD31＋c—Kit^high细胞群，通过Real—time PCR检测PDKl对内皮向造血转换相关的转录因子（RUNX1、P2-RUNX1、GATA2）的影响。结果PDK1敲除后，造血祖细胞形成的克隆形态变小，数目减少[敲除组CFU—GM为（24±5）个／ee，对照组为（62±1）个／ee，P=0．001]；破坏了造血干细胞重建造血及多向分化的能力（敲除组移植5只，0只重建，对照组移植7只，5只重建，P=0．001）；AGM区CD31＋c—Kit^high比例降低[敲除组CD31＋c-Kit^high比例为（0．145±0．017）％，对照组比例为（0．385±0．04）％，P=0．001]；并且AGM区由内皮细胞向造血细胞转换的关键转录因子表达下降，但对CD31＋c—Kit^high细胞的增殖和凋亡无明显影响。结论在内皮细胞中特异敲除PDK1基因，导致具有向造血转化的内皮细胞群比例降低，影响了HSC的发生，破坏了HSC重建造血的能力。Objective To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC. Methods PDK1 was deleted specifically in endothelial cells expressing VEC （Vascular Endothelial Cadherin）. CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK^fl/fl, PDK^fl/＋ and Vec-Cre; PDK^fl/fl AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31 ＊c-Kit^high cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells. Results In contrast to the wild type group, the CFU from PDKl-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was （24±5）/ee in knockout group, and the control group was （62±1）/ee （P= 0.001）. PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells, hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group （P= 0.001）. The proportion of intra-vascular clusters in the AGM was decreased （the frequency of CD31＋c-Kit^high in the knockout group was （0.145±0.017）%, and the control group ratio was （0.385±0.040）% （P = 0.001）, but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster. Conclusion PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the ge

关 键 词：PDK1 早期造血 AGM区 造血干细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]