| BackgroundSpace flight can lead to multiple body systems change, which includes blood system, digestive system, genital system, cardiovascular system, immune system, skeletal system and so on. The effects of space environment on human body have become a hot research topic in scientific field. Growth and development of life on earth are in 1 g gravity environment. Microgravity also known as zero weight, is one of the main characteristics of the space environment, the research of the effects of microgravity environment on the astronaut health, discuss its mechanism and make effective control measures have become an urgent work. Total hemoglobin, red blood cell count and red blood cell volume decline have attracted the attention of space medical researchers, they named it "spaceflight anemia". The cause of this phenomenon is not yet clear, but studies show that this may be caused by microgravity affect hematopoietic stem cell differentiation and apoptosis.The generational process of erythrocyte is complex. First, hematopoieticstem cells develope into progenitor cells, and then progenitor cells differentiate into erythrocyte. All kinds of cytokines in the process of erythropoiesis need to work together to achieve a stable physical conditions. The GATA and Ets of transcription factors play a key role in erythroid differentiation. The GATA family of transcription factors is zinc finger structure, GATA-1 and GATA-2 widely exist in the erythron, megakaryocytes, mast cells and T cells, GATA-3 is seldom or not expressed in erythron. GATA-1 is unique to hematopoietic system and necessary for growth and development of normal red blood cells. GATA-1 is mainly expressed in late mature erythrocytes and with the increase of expression K562 cells gradually mature. GATA-1 can specificity activate a variety of specific erythron gene and induce erythroid differentiation. GATA-2 is mainly expressed in multipotent progenitor cells and has important significance to maintain and proliferation of hematopoietic cells. Overexpression of GATA-2 suppresses erythroid differentiation and promotes megakaryocytes differentiation. The Ets family is one of the largest family in the transcription factors. In the hematopoietic system Ets-1 plays an important role and is involved in the regulation of lymphopoiesis and the development of natural killer cells. Ets-1 acts as an inhibitor of erythroid differentiation by increase the expression of GATA-2. Ets-1 and GATA-2 synergy regulate erythroid differentiation. So, change of any one transcription factor expression may result in disorder of erythroid differentiation.Cytoskeleton is a cross-connected network of fibrous polymer composed of microfilament, microtubule and intermediate filament and various regulatory proteins which links with special structures on the cell membrane, such as focal adhesion and intercellular connections and so on, interacts with nucleus support system. Microtubule is composed of alpha and beta tubulin, exists in the normal form of beta dimers, can maintain cell morphology, auxiliary material transport and play an important role in chromosome disjunction of eukaryocyte. The chemical composition of intermediate filament is complicated. Vimentin is the main component of intermediate filament, which interacts with the matrix of the nucleus to regulate gene expression and cell differentiation. Microfilament is a solid shape of fiber, mainly exists in the form of polymer (F-actin), is the main driving force of the migration, play an important role in the process of cell movement.Cytoskeleton is considered to be the structure of cell transduction stress strain simulation and external force. The study found that cell integral feeling gravity transmit signals by physical and chemical pathways, the intersection of the two pathways is cytoskeleton. Cytoskeleton is a very active system which can change between polymers and free monomer. Because of cytoskeleton is a dynamic structure, it is play an important role in microgravity perception. The abnormal expression of skeleton protein can damage cytoskeleton which affects the normal cell morphology and function. Proteins on the cell membrane feeling the changes of cytoskeleton caused by microgravity can activate a series of downstream signal transduction pathways such as ERK, p38 and JNK, which influences the nuclear transcription factors and genes such as jun, c-fos and basic life process such as energy metabolism of cell, material synthesis and information transfer and change the process of cell proliferation, differentiation and apoptosis. The study found that the mechanism of stem cells differentiation associated with the changes of cytoskeleton. Therefore, this experiment was designed to study the effects of simulated microgravity on K562 cells differentiation mechanism through detecting K562 cells differentiation, its related transcription factors and the expression of cytoskeleton.Researchers are now through two ways to obtain microgravity for studying microgravity related life science, the first one is microgravity exists in space and another is ground simulated microgravity. Due to fewer opportunities, long experiment period and huge cost space research cannot be widely applied in the experiment. Ground microgravity study mainly used mouse tail hanging model and microgravity simulator for a variety of experiments. Rotary Cell Culture System (RCCS) is a kind of recognized ground microgravity simulator which can be used to do research on microgravity. By the study, we explore possible mechanism of erythroid differentiation of K562 cells by using RCCS4. As hematopoietic stem cell is not easy to obtain and needs a long period to culture, K562 cell lines have been widely used in erythroid differentiation model. K562 cell lines have multilineage differentiation potential. Hemin is a common erythroid differentiation inducer, which can induce production of hemoglobin in erythroid progenitor cells and K562 cells.ObjectThis experiment was designed to study the effects of simulated microgravity on erythroid differentiation of K562 cells to further explore the possible mechanism of "spaceflight anemia"MethodsRCCS-4 was used to generate the simulated microgravity environment. Human erythroleukemia cell line K562 was chosen as the hematopoietic progenitor cell model. The experiment were divided into two parts, in the first part we used hemin induction erythroid differentiation of K562 cells, the experiment were divided into the normal gravity (NG) group, hemin induced (hemin) group, simulated microgravity (SMG) group and simulated microgravity hemin induced (SMG+hemin) group. The benzidine staining was used to observe the expression of hemoglobin in K562 cells cultured for 48h. The mRNA levels of K562 cells cultured for 6h,12h,24h and 48h respectively of three transcriptional factors (GATA-1, GATA-2, Ets-1), which played very important role during the process of erythroid differentiation were detected by Reverse Transcriptase-quantitative PCR (RT-qPCR). In the second part, the experiment was divided into group NG and SMG. The immunofluorescence was performed to show the expression of cytoskeleton (F-actin, β-tubulin and Vimentin) of K562 cells cultured for 24h. The gene expression levels of cytoskeleton were detected by RT-qPCR of K562 cells cultured for 6h,12h,24h and 48h respectively. The protein levels were detected by western blot of K562 cells cultured for 12h and 24h. The experiment was repeated by three times and without any correlation between the different time points. Statistical analysis was performed using SPSS Statistics Software 20.0. Measurement data were performed by mean value and standard deviation (x±s). Data of two groups were compared by two samples t test and one-way ANOVA. A two-tailed P value of less than 0.05 was considered statistically significant.Results(1)The benzidine-positive percentage of group hemin was significantly higher than that of group NG, and group SMG+hemin was significantly lower than that of group hemin when cultured for 48h.(2) Hemin treatment of K562 cells resulted in up-regulated GATA-1 mRNA expression after 12h and down-regulated mRNA expressions of GATA-2 after 12h, Ets-1 after 6h. Simulated microgravity treatment of the cells resulted in down-regulated GATA-1 mRNA expression after 24h and up-regulated mRNA expressions of GATA-2 after 12h, Ets-1 after 24h. Compared with cells of group Hemin, the expression of GATA-1 mRNA in cells of group SMG+Hemin was down-regulated, the expression of Ets-1 mRNA was upregulated and the expression of GATA-2 mRNA has no obvious change.(3)The fluorescence intensity of skeleton proteins of group SMG were decreased than that of group NG when cultured for 24h.(4) Gene levels of cytoskeleton (F-actin, P-tubulin and Vimentin) in cells of group SMG were significantly lower than that of group NG when respectively cultured for 12h and 24h, but no obvious change when respectively cultured for 6h and 48h.(5) Protein levels of cytoskeleton (F-actin, β-tubulin and Vimentin) in cells of group SMG were significantly lower than that of group NG when respectively cultured for 12h and 24h.ConclusionWe found that simulated microgravity can inhibit erythroid differentiation of K562 cells. The mechanism may be that cytoskeleton is gravity sensitive molecular. And under microgravity condition, cytoskeleton expression down-regulated and damaged, which change downstream transcription factors (GATA-1, GATA-2, Ets-1) related to erythroid differentiation. |
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