| BackgroundThe abnormal deposition of a collagen material in bone marrow with rayeloid metaplasia is known as myelofibrosos(MF). It has been described as a independent disorder for more than 100 years, but the machine of MF is still unknown , also the optimal forms of treatment have not yet been defined.The hypothesis of a clonal expansion of hematopoietic stem celland reactive fibroblast proliferation was first supported by Kaha, and a lot of scholars accept this hypothesis. Megakaryocytes play a very important role in MF, it can excretion a series of cytokines, these cytokines can induce the proliferation of bone marrow fibroblast and the collagen synthesis. But the most important cytokines are platelet derived growth factor(PDGF) and transforming growth factor-P (TGF-P).MF patients always died from marrow failure but not the clonal expansion of hematopoietic stem cell. We successful treated two primary myelofibrosis patients with interforen- a (IFN- a ) and 1,25-(OH)2 VitD3 , and the parallel individual cases can been seen.The effect of IFN-a treat MF is not been confirmed, and the machine of IFN-a treat MF is unclear, too.1. methods1. 1 Primary normal human bone marrow fibroblasts culture: aspirate 5ml bone marrow from non-malignancy hematologic patients, separate mono- nucleus cells. The culture medium consisted PRMI 1640 and 20% fetal calf serum(FCS). Cells were incubated at 37"C, 5% C02 condition, replaced with fresh medium every 3 to 4 days.1. 2 Transfer of culture: digest the primary normal human bonemarrow fibroblasts with 0. 25% Trypsin, cells were seeded at7the density of lX104/ml, replaced with fresh medium every 3 days.1. 3 MTT: Cells were seeded into 96-well plates at a final density of 4X10Vml, and were exposed to IFN- a 2b(10-10000u/ml) , TGF-P ,(0. 01-lng/ml) and PDGF-AB(l-50ng/ml) .control was with cell alone and total volume was 200 ul. And every concentration was with 6 duplicate well. Cells were pulsed with MTT 20 ul/well after incubated 96 hours. After incubated another 4 hours, cells were added anhydrous alcohol 150ul/well and at a wavelength of 490 nm absorbance readings were taken on a spectrophotometer.1.4 Flow cytometry (FCM) to measure cell cycle: cells culture with PRMI 1640 for 24 hours , then changed with PRMI 1640 and 20% PCS, added IFN- a 2b(1000u/ml), TGF- P , (0. Ing/ml) and PDGF-AB(25ng/ml), culture for anther 24 hours. Collect cells, added DNA-PREP LPR and DNA-PREP Stain, then measured with FCM.1.5 Statistics :The results were analyzed with SPSS10. 0.2. Results2. 1 IFN- a 2b effect the proliferation of normal human bonemarrow f ibroblasts: MTT showed IFN- a 2b at the concentration of 10 and lOOu/ml stimulated fibroblast growth, but 1000 and lOOOOu/ml suppressed . After added lOOOu/ml IFN-a 2b for 24 hours, FCM showed PrI decreased 16.9%, P=0. 012.2. 2 IFN- a 2b influence the effect of the proliferation of normal human bone marrow f ibroblasts induced by PDGF-AB: MTT showed PDGF-AB stimulated fibroblast growth in a dose-dependent fashion. After added 25ng/ml PDGF-AB for 24 hours, FCM showed s% increased 65. 7%, P=0. 003. If added lOOOu/ml IFN-a 2b, MTT showed mean value decreased 7.62%(P=0.001) compared with PDGF group, FCM showed S% of IFN- a 2b+PDGF-AB group decreased 19. 6% compared with PDGF group.2. 3 IFN- a 2b influence the effect of the proliferation of normal human bone marrow f ibroblasts induced by TGF-P i: MTT showed TGF- P j stimulated fibroblast growth at the concentration of 0.01-lng/ml. If added lOOOu/ml IFN- a 2b, MTT showed mean value decreased 37. 97%(P<0. 01) compared with TGF- P , group, and decreased 10. 37%(P=0. 028) compared with IFN-a 2b group; FCM showed PrI and G2% of IFN- a 2b+ TGF- P , group decreased 19. 26%(P=0. 052) and 63. 44%(P=000. 031) compared with TGF- P , group, and d% decreased 8.66%(P=0.031) compared with IFN-a2b group.92. 4 IFN- a 2b influence the effect of the proliferation of normal human bone marrow fibroblasts induced by TGF- P i and PDGF-AB:MTT showed... |
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