Impacts Of Simulated Microgravity On Proliferration Of K562 Cells

BackgroudSpace flight led to multiple body systems change, which includes a series of changes such as blood system, cardiovascular, vestibular, bone, muscle, immune, endocrine etc. With the development of space technology, people take more and more attention to these changes and impact brought by space flight. In fact hematological changes caused by the space have attracted the attention of space medical scientists long ago. They named the phenomenon which showd plasma volume reducing, red blood cell decreasinng, shaped red blood cells incraseing as "space anemia". Researches show that the "space anemia" will increase with the flight time. So these changes would become an irreversible pathological condition? Early scientists have only observed on the blood indices. However, Only know the indices such as the decraesing number of red blood cells, increaseing of shaped red blood cells are not enough. Now in recent years scientists have started to study and explore the mechanisms of "space anemia", in order to resolve and prevent these negative factors in space flight, and promote the further development of space science.Because that space experiments require huge costs, and the experimental conditions can not control very well, there are many laboratories at home and abroad started using microgravity bioreactor to simulate a variety of microgravity environment experiments. NASA invented the Rotray Cell Culture System (RCCS) is a highly repetitive, complex, three-dimensional culture system in vitro. It can create a low shear, high mass transfer efficiency and nuique micro-gavity environment. We can develop three-dimensional tissues by it in ordinary laboratories. Currently, it has become recognized as the Cultivation device simulating microgravity environment.After reading literature, we found that many studies had shown that microgravity environment simulated by RCCS can inhibit the proliferation and differentiation of cells such as CD34+ cells and K562cells. Cell proliferation and differentiation is a complex prcess, which is subject to the regulation of a number of factors, such as signal transduction pathways, cytokines, transcription factors and cancer genes. These factors act together on cells. Recently, the mechanisms of that microgravity affects on cell proliferation and differentiation need further discussion and study. K562 cell lline is a human leukemia cell. It can differentiate into teminal erythroid, megakaryocyte and macrophage cell lines when induced by some factors, so people often treat it as a model to study cell proliferation and differentiation.Proliferation is a prerequisite for differentiation. Cell proliferation is related to activation of intracellular signal transduction pathway and the exprssion of genes associated. According to researches at home and abroad, we already know that the signaling pathways related to cell proliferation are PI3K/Akt, pRb/E2F, MAPKs and so on.Mammlian MAPKs are divided into three groups, and ERK1/2 plays an important role in both erythroid proliferation and differentiation. Now ERK pathway promotes cell proliferation has been widely confirmed. Experiments show that ERK1/2 can regulate a series of cell cycle regulatory proteins such as c-myc, c-fos, P21C1P1, cyclinD1, CyclinD3, ect. Inhibition of ERK1/2 activity can block CD34+ erythroid progenitor cells'proliferation and differentiation, and induced the apoptosis.Erythropoietin is a complex physiological process. It is regulated by a lot of genes especially erythroid hematopoitic growth factors and transcription factor. Hemin is iron protoporphyrin IX, which as an important component of blood proteins can provide oxygen carrying capacity. It is an inducer of erythroid differentiation, and it can induce the hemoglobin synthesis of erythroid progenitor cells and human leukemia K562 cells. Like stem/progenitor cells, K562 cells have multilineage differentiation potential. It's differentiation depends on a series of specific transcription factors, the trend of changes of which have determined the differentiation direction. When given an external stimulus(such as hemin), K562 cells'directed differentiation has begun to "start". As a member of GATA family, Transcription factor GATA-1 plays a key role in the process of erythroid differentiation and maturation. GATA-1 gene has two promoters, that are the red line promoter in distal and the testis promoter in proximal. There are GATA-1 binding sites at upstream of promoters, which constitutes a strong GATA-1 gene promoter. This strong promoter is necessary for getting fully activation and combining with its own erythroid nuclear protein. At the beginning of primitive cells'erythroid differentiation, the distal promoter of GATA-1 is activated, and the total GATA1 expression increase, then stimulate cells to erythroid differentiation.Our experiment used RCCS-1 simulated microgravity to culture K562 cells, and observed the changes in the training process. It can be divided into two parts. The first part we observed its proliferation, and changes of the signal pathway which related the proliferation; The second part we added the effect of microgravity on the differentation of K562 cells:PartⅠImpacts of simulated microgravity on proliferration of K562 cells and related mechanismObjective:Astronauts would get "space anemia"in space flight condition. This part of expriment based on the use of RCCS simulated microgravity environment, cultured K562 cells in the ground, study of the influence of cell proliferation in microgravity environment and its mechanism.Metods:We used Rotary Cell Culture System-1(RCCS-1), which invented by The National Aeronautics and Space Administration(NASA)to simulate microgravity environment, and cultured human leukemia K562 cells to study the proliferation.1) K562 cells in cultureCultured K562 cells with RPMI1640 containing 10% FBS in the cultured cell-based incubator. passage once per 2-3 d. Cells in logarithmic growth phase are used in experiments, and adjust the cell concentration to 1×105/mL;2) K562 cells cultured in RCCS-1 simulated microgravityUsed RCCS-1(Synthecon, USA) to simulate microgravity environment. K562 cells were cultured in 50mL high aspect container in the cell incubator, adjustd the speed to 10～12rpm;3) Cell countCollected K562 cells cultured in RCCS-1 and normal gravity condition, and then counted the cells with Pakistan's cattle count board in the optical microscope;4) The cell cycle analysisCollected K562 cells cultured in RCCS-1 and normal gravity condition in the same time, then detected cell cycle by flow cytometry;5) Western bloting detection of ERK1/2 expressionCollected K562 cells cultured in RCCS-1 at different time points, extracted proteins with phosphorylation of extraction kit, then detected the extracellular signal-regulated protein kinasel/2(ERK1/2) and its phosphorylation by Western bloting.Results:1) The proliferation speed of K562 cells cultured in simulated microgravity environment was significantly lower than the control group; Simulated microgravity environment inhibited cell cycle progression, that was stalled in the G0/G1 phase;2) In simulated microgravity environment, phosphorylation exprssion of ERK1/2 of K562 cells gradually decreased with time.Conclusion:Simulated microgravity environment inhibited K562 cell proliferation, cell cycle was arrest at G0/G1 phase, which may be caused by the reduction of ERK1/2 phosphorylation level.PartⅡImpacts of simulated microgravity on differentation of K562 cellsObjective:Space flight would cause "space anemia", the core of which is the decline in quantity and quality of red blood cells. This part of the experiment still based on the use of RCCS simulated microgravity environment, cultured K562 cells in the ground. Study of the influence of cell differentation in microgravity environment and its mechanism.Methods:We used RCCS-1 to simulate microgravity environment, and cultured human leukemia K562 cells. Used hemin to induce its erythroid differentiation.1) K562 cell culture and RCCS-1 culture as before, plus hemin as a inducer; Benizidine staining2) Collected K562 cells cultured in RCCS-1 and normal gravity condition at different time points, benzidin stained cells, and counted benzidine positive cells;3) Western bloting detection of GATA-1 expressionCollected K562 cells cultured in RCCS-1 and normal gravity condition in the same time, extracted proteins and then detected experssion of GATA-1 by Western bloting.Results:1) Hemin can induce erythroid differentiation of K562 cells. Cultured under the same conditions, the longer hemin stimulated the more erythroid differentiation; But in the same time, hemin-induced erythroid differentiation in microgravity condition significantly reduced compared to the control group;2) Detected by Western bloting, the expression of GATA-1 in both groups increased with time; and hemin-induced K562 cells GATA-1 expression has no significant differences between simulated microgravity group and the control group in 24 hours.Conclusion:Simulated microgravity inhibited hemin-induced K562 cell differentiation, the decresased production of red blood cells confirmeded in the weightless environment "space anemia" would happen. Microgravity did not affect the expression of GATA-1, suggesting that inhibition of K562 cell differentiation may be independent of GATA-1.