Apoptosis Of Humam Leukemia HL-60 Cells Induced By Cytochrome C

ObjectiveCytochrome C is used to act on lekemic clone HL-60 cells directly. The aim is to examine different effects of different concentrations of Cytochrome C inducing HL60 cells, and the relationship between apoptosic effect and cell cycle. Meanwhile, to investigate changes of intracellular calcium in apoptosis, to primarily study the mechanism of apoptosis of HL-60 induced by cytochrome C.Method(1) The effects of cytochrome C on HL-60 cells and the relationship between apoptosic effect and cell cycle were determined by flow cytometry(FCM). (2) DNA agarose gels eletrophoresis is used to clarify the apoptosic effect of cytochrome C on HL-60 cells. (3) The morphology and ultra-structure changes of cells were recognized with transmission electric microscopy. (4) The intracellular calcium was determined with the technique of spectrometry..Result1. Analysis of FCM(1) Different concentration of cytochrome C can induce proliferation apoptosis and necrosis of HL-60cells. Concentration of cytochrome C in proliferation and necrosis was <0.94 g/ml and >28.13g/ml, respectively. Concentration of cytochrome C in apoptosis was 5.63 g/ml-24.38 jig/ml. And the appropriate concentration of it was 9.38 g/ml-22.50 jig/ml.(2) If interactive time between cytochrome C with HL-60 cells is different, ratio apoptosis is different. The most appropriate interactive time was 24 hours because of the highest ratio of apoptosis in the experiment.(3) The apoptosis effect of Cytochrome C inducing HL-60 is closely related to cell cycle. With rise of apoptosic ratio contents of S phase and 62 phase decline accordingly. There shows a remarkable negative relationship beyween content of S phase and apoptosis ratio.2. Analysis of DNA agarose gel eletrophoresisDNA extracted from HL-60 cell interacted with 9.38g/ml~22.5g/ml cytochrome C reveals a characteristic ladder pattern.3. Morphologic and structural changes of HL-60 cells in apoptosis The morphologic changes of HL-60 cells in apoptosis exhibited a formation of apoptosic body under light microscope, such as contraction of cell membrane, irregulation of cell shape, condensation of cell nuclear and so on. In addition to these, the cell ultra-structural changes are also shown under transmission electron microscope,such as breaking of cell membrane, fragmentation of cell chromosone , condensation of cell plasma.4.Changes of the intracellular calcium during apoptosis(1) If time of interaction between various content cytochrome C and HL-60 is different, apoptosic ratio and intracellular calcium content are different.(2) Tntracellular calcium content of apoptosic group and contral group was 10.35+1.37(10"8g/l) and 7.26+ 1.08(10~8g/l) repectively. There is significant differencebetween two groups(p<0.01, n=12). It shows that thereis a significant increase of the intracellular calcium presenting in apoptosic HL-60 cells.ConclusionIn this study, we demonstrate that different concentration of cytochrome C can induce proliferation apoptosis and necrosis of HL-60 cells. Low dose and high dose of cytochrom C induce proliferation and necrosis respectively, and appropriate dose of cytochrome C induces apoptosis. The most appropriate interactive time between cytochrome C and HL-60 cells is 24 hours. The result indicates the relationship between dose-effect and time-effect of apoptosis induced by cytochome C. It implicated that threshold value of intensity and time of damage may be important in switching on and regulating of apoptosis and necrosis. The apoptosis effect ofcytochrome C inducing HL-60 is closely related to cell cycle. There is a remarkable negative relationship between content of S phase and apoptosis ratio. Maybe there is a specificity of S phase during apoptosis of HL60 cells induced by cytochrome C. Besides, the study testified that a significant increase of the intracellular calcium was in apoptotic HL-60 cells. It can be concluded t...