内皮细胞靶向的可溶性Notch配体hD1R蛋白对急性髓系白血病细胞增殖的影响

OBJECTIVE: To evaluate the effects of endothelial cell-targeted soluble Notch ligand hD1R protein on the proliferation of acute myeloid leukemia (AML) cells. METHODS: The expression levels of Notch1, Notch2, Notch3, Notch4, Hes1 in bone marrow CD34(+) cells from 24 cases of untreated AML (AML group) and 9 healthy controls (control group) were determined by real time quantitative polymerase chain reaction (PCR). Recombinant hD1R protein was first induced and purified. Bone marrow CD34(+) cells were co-cultured on human umbilical vein endothelial cells (HUVEC) supplemented with a cocktail containing 5 types of human cytokines (5GF) and soluble hD1R. The cultured cells were tested under different culture conditions including PBS group (PBS replaces HUVEC), hD1R group, 5GF group, GSI group (hD1R plus GSI). Proliferation and apoptosis of cultured cells were also analyzed. Real time quantitative PCR was used to test the expression levels of Hes1 and Bcl-2 in cultured cells. RESULTS: The expression levels of Notch1 and Hes1 in primary AML patients were significantly lower, and Notch4 expression was higher compared to the control group (P<0.05). Cell counting showed a remarkable decrease of AML cells number in the culture with hD1R compared with that in the PBS group[ (0.74±0.13) ×10(5)vs (2.16±0.21) ×10(5), P<0.01]. FACS analysis showed that the percentage of AML cells was (18.48±2.51) % in apoptosis, which was higher than that of control cells (3.19±0.58) % after co-culture with hD1R. AML cells in the hD1R group underwent significantly increased apoptosis compared with those in the PBS one (P<0.05). Moreover, apoptosis of AML cells in the GSI group was lower than that in the hD1R one (P<0.05). Apoptosis in the PBS group also decreased compared with that in the 5GF one (P<0.05). Finally, hD1R up-regulated Hes1 expression and inhibited Bcl-2 one in the AML cells. CONCLUSION: hD1R effectively activated Notch signaling and down-regulated Bcl-2 mRNA in AML cells, which lead to cell apoptosis.