Induction Of Platelet Formation From Megakaryocyte By Aspirin

Objective:we chosed to use the immortalized human megakaryoblastic cell line, Meg-01 as a model system. The human megakaryoblastic cell line, Meg-01 could be induced to differentiate into mature megakaryocytes upon addition of TPA as a model system to study megakaryocytes. We exposed megakaryocytes to aspirin and detected the changes of the cell to make clear of the essential role of aspirin on megakaryocyte development and platele release.Methods:Meg-01 cells were grown in RPMI 1640 medium supplemented with 10% FCS at 37°C in a humidified atmosphere of 5%CO2. Meg-01 cells were studied by placing 3 x l05cells/mL in Falcon 3013 plastic flasks containing 2 mL of culture medium supplemented with 10% FCS containing TPA. The cells were cultured with 10-7M/L TPA for 72 h. After incubation for three days, the slides were gently washed with warm phosphate-buffered saline (PBS) for three times . The cells were cultured with aspirin for 24h and were collected after 24h in vitro. TUNEL was used to detect the apoptosis. Western blot was used to detect the expression of caspase3 and Bcl-2. RT-PCR was used to detect the expression of GPIIIamRNA and GPIIb mRNA.Results:1. 10-7mol/LTPA could bring about differentiation and maturation of a human megakaryoblastic cell line (Meg-01 ) for three days in vitro.2. In contrast to megakaryocytes only treated with TPA, The morphology of the megakaryocytes treated with aspirin changed dramatically and there were a population of platelet-sized particles.3. The mature MKs exhibited clear signs of apoptosis. In contrast to megakaryocytes only treated with TPA,We noted that the mature megakaryocytes treated with 12.5,25mg/L aspirin displayed a strong staining in the nucleus.The mature megakaryocytes treated with 1.25,2.5,5mg/L asipirin displayed a weak, but detectable staining in the nucleus.4. The six groups all expressed activated caspase3 and Bcl-2 proteins by Western blot after cultured with aspirin. The percentage of expression of activated caspase3 increased from the first group only treated with TPA to the sixth group treated with 25mg/L aspirin.The percentage of expression of Bcl-2 decreased from the first group only treated with TPA to the sixth group treated with 25mg/L aspirin.5. The six groups all expressed GPIIIamRNA and GPIIb mRNA by RT-PCR after cultured with aspirin. The percentage of GPIIIamRNA and GPIIb mRNA was not different from the first group only treated with TPA to the sixth group treated with 25mg/L aspirin.Conclusions:1. 10-7mol/LTPA could bring about differentiation and maturation of a human megakaryoblastic cell line (Meg-01) in vitro.2. Aspirin did not increase expression of GPIIIa and GPIIb.3.Caspase3 related to proplatelet formation and platelet release from megakaryocyte induced by aspirin.