| Hypoxia is pathophysiologic response of many diseases in human. But until hypoxia inducible factor-1 was found, we can further understand about passage of hypoxia. Investigations about hypoxia have become current hot issue in related fields. Now investigations about hypoxia mainly focus on tumors and cardiovascular diseases, few works in leukemia were done. We investigate the effect of cobalt chloride which mimicks hypoxia and induce differentiation of acute myeloid leukemia cells on cytomorphology, NBT reduction test and mature-related cell surface differentiation antigen CD11b by FCM in our experimentation. Results show leukemia cells begain to change in morphology after treated by indicated concentration of CoCl2 for six days, such as condensed chromatin, decreased nuclei/cytoplasm ratio with smaller nuclei. NBT reduction OD control is 0.2367±0.0191；CoCl2 is 0.403±0.0169in NB4 cell. Control is 0.2063±0.0176；CoCl2 is 0.5653±0.0447in kasumi-1 (P<0.01). CD11b-positive cells were measured in NB4 cell, CoCl2 is 48.67% ,and 33.56% in kasumi-1. This show that hypoxia can induce differentiation of leukemia cells. Comparing CoCl2 with ATRA, CD11b-positive cells were measured in NB4 cell, CoCl2 is 40.1%, ATRA is 73% (P<0.01). The rate of differentiation of CoCl2 is lower than that of ATRA. But CoCl2 can increase the rate of differentiation of ATRA (P<0.01). HIF1 is regarded as a important mediator during hypoxia induce differentiation of leukemia. We examined the HIF1 by Western blot and found it increase quickly, it maybe play an important role in hypoxia. We found that hypoxia inhibits the proliferation of leumemia cells by via programmed cell death and through growth arrest. We measured cell cycle by FCM, the results show that the cells in G1 phase increased and cells in S phase decreased in CoCl2. It pointed out that hypoxia can induce cell cycle arrest in G1/S phase, This is one of reasons which can inhibit the proliferation of cells. Agents of chemotherapy inhibit the proliferation of tumor cell through different machines. Now therapy of leukemia lie on chemotherapy. We found that CoCl2 can inhibit the proliferation of leumemia cell, can it increase the effection of chemotherapy ? The investigations about this have not be reported. The enhancing effect of CoCl2 on the chemosensitivity of NB4 cell was assessed by MTT colorometry and 3H-thymidine incorporation. FCM was used to examine the intracellular collection of drug (DAM, represented by its Dau fluorescent intensity). OD is 0.7380±0.089in DAM, and is 0.5233±0.065 in DAM and CoCl2, there is significant statistical difference between two groups. 3H-thymidine incorporation is 4756.88±96.23 in DAM group, and 2799.89 ±35.20in DAM and CoCl2 group. there is significant statistical difference between two groups. This illustrated that CoCl2 can increase the inhibited effection of DAM on DNA replication. Examination of intracellular collection of DAM shows that there is no significant difference between two groups. CoCl2 did not affect the drug collection inside leukemia cell, and the toxicity of DAM did not increase, CoCl2 is a good chemosensitive modulator. The investigation about hypoxia provided a signal mechanism for leukemia cell differentiation and leukemogenesis, and identify some novel drug targets, has a broad clinical potentials. |
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