PDK1在内皮细胞向造血细胞转换中的作用研究

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 PDK1(fl/fl), PDK1(fl/+) and Vec-Cre; PDK1(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 PDK1-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 generation and function of HSC.