撤除粒-巨噬细胞集落刺激因子对TF-1细胞生长的影响

Effects of cytokine withdrawal on the proliferation of TF-1 cells

采用MTT实验测定撤除细胞因子后TF 1细胞的增殖活性 ,经流式细胞仪、透射电镜观察撤除细胞因子对TF 1细胞凋亡的作用及其形态学变化。采用荧光微孔板读数仪测定凋亡过程中Caspase 3活性的改变。结果发现 ,撤除细胞因子后 4 8h ,细胞存活率为正常培养时的 4 8 8% ;电镜显示TF 1细胞出现了凋亡典型的变化。细胞凋亡率在撤除因子后 2 4h、4 8h时分别为 2 1 2 3%和 71 36 % ,TF 1细胞被阻滞在G0 G1 期。细胞内Caspase 3活性在撤除因子后 12h、36h时明显升高 ,荧光度分别为 6 32 1和 1782 3。结果表明 ,撤除因子后可导致TF 1细胞凋亡 ,Caspase 3活化参与了此凋亡过程。另外通过MTT实验观察到一种新型造血刺激因子粒 -巨噬细胞集落刺激因子 (GM CSF) 白细胞介素 3(IL 3)融合蛋白可维持TF

MTT assay was used to measure the proliferation of TF 1 cells that were dependent on growth factors GM CSF, IL 3 and EPO upon cytokine deprivation. The apoptosis induced by cytokine withdrawal was observed with flow cytometer and E.M. microscopy. Because the level of Caspase 3 activity was directly proportional to the fluorescence signal density, it's changes were detected by a fluorescent microplate reader. Cytokine withdrawal elicited typical apoptotic morphologic changes. After the deprivation of cytokine for 48hrs., the viability rate of TF 1 cells was 48.8% compared with that of the normal cells. The apoptosis rate induced by cytokine deprivation was 21.23% and 71.36% at 24h, 48h respectively. TF 1 cells were blocked in G 0/G 1 cycle phase. 12hrs and 36hrs after deprivation of cytokine, Caspase 3 activity in TF 1 cells remarkably increased and the fluorescence density was 6321 and 17823 respectively. These results indicated that apoptosis of TF 1 cells can be induced by cytokine withdrawal and Caspase 3 activation participates in this process. In addition, we found a new type of hematopoietic growth factor,GM CSF/IL 3 fusion protein that can keep the survival and promote the proliferation of TF 1 cells.