The Research Of The Effect Of Simulated Microgravity To The Morphology And The Function Of Bone Mesenchymal Stem Cells Directing By The"Circumference Philosophical" Paradigm

Bone marrow mesenchymal stem cells (BMSCs) are considered as the most optimal stem cells which will be soon introduced in the clinical treatment because of its own advantages. The research for stem cells belongs to the field of regenerative medicine which has an open prospect. At present there are more and more scholars and experts have been engaged to stem cell research in the world. However, the low efficiency and high risk of stem cell in the clinical use restricts its widely using in clinical application. It is a problem for the researchers that how to improve the stem cell differentiation potential from itself and then to be used in clinical efficiently and safely.In recent years, many researchers developed the research means to space field, with the rapid development of aerospace cause. And they gradually realized that the microgravity have a huge impact on the physiological functions of organisms. Then, the role of microgravity in cell level had also caused the extensive attention for the researchers. Previous studies observed that many kinds of cells turn round after simulated microgravity (SMG). The classic side "something inside, must be take on some form outside", so this change of the morphology must effect the function of cells. In the guidance of "Circumference philosophical" paradigm, we speculated that the simulated microgravity provide the bone marrow mesenchymal stem cells with a kind of balance, harmony environment which can promote its return to the original state. Thus it is more beneficial for stem cells to maintain their own characteristics. In order to prove this speculation, this study research the effect of the simulated microgravity on the morphology and the proliferation, multipotential differentiation, migration capacities of bone mesenchymal stem cells. This study set out from the stem cell itself and provided a novel method for obtaining higher-quality stem cells with an improved capacity for differentiation and self-renewal. This study first treat the simulated microgravity as a novel method to enhance the capacities of proliferation and multipotential differentiation of stem cells under the guidance of "Circumference philosophical" paradigm and further provide a new idea for the stem cell using in clinical as soon as possible.Part1The effect of simulated microgravity on the morphologyl of BMSCs1. ObjectiveStudy the changes of the shape and the cytoskeleton of BMSCs followed by the simulated microgravity.2. MethodAfter isolation and cell culture of BMSCs, this study chose the3rd to6th passage cells for testing their surface makers with Flow Cytometry. Then the BMSCs were divided into normal gravity (NG) group, simulated microgravity (SMG) groups (48h、72h、120h). Phase-contrast microscopic analyzed the change of the morphology of BMSCs; the change of cytoskeleton was observed by FITC-Phalloidin fluorescence staining. 3. ResultsFlow cytometry results showed that these cells were CD44and CD90positive (the ratios were94.74%and93.57%respectively) and CD45and CD34negative. Phase-contrast microscopic found the longer of SMG stimulation duration, the more close to1of the ratio of the cells’ width/length and the most significant change of the shape occurred after SMG72h, but difference was found rarely at longer duration. Phalloidin fluorescence staining showed SMG induced transient changes of cytoskeleton, decreased the skeleton tension, induced the modification of some bundles and then made the shape of cells from spindle to round. However partial restoration of F-actin occurred in longer SMG duration.4. ConclusionBMSCs exposed to microgravity become morphologically changed from spindle shape to round shape. This shape change achieved through the cytoskeleton adjustment under SMG condition.Part2The effect of simulated microgravity on the function of BMSCs1. ObjectiveTo study the changes of the proliferation, multipotential differentiation, migration capacities of shape changed BMSCs followed by the simulated microgravity.2. MethodBMSCs were divided into two groups:normal gravity (NG) group and simulated microgravity (SMG) group (72h). To explore2.1. The proliferation capability:①BrdU incorporation assay detected the BrdU positive rate of two groups cells.②The growth curves of two groups cells were evaluated by cell counting after cultured the cells0day,3days and7days.③Flow Cytometry analyzed the apoptotic rate.2.2. Multipotential differentiation capability:①Quantitative PCR and Western Blotting analyzed the expression of pluripotency marker OCT4.②Endothelium oriented differentiation of two groups BMSCs in normal gravity:The expression level of VWF and CD31were analyzed by immunofluore scent and Western Blotting; Adipogenic differentiation of BMSCs:Oil red O solution staining was applied to examine the efficiency of adipogenic induction. Western Blotting was used to analyzed the expression level of PPARy2; Neuron oriented differentiation of BMSCs:Immunofluorescent and Western Blotting were used to analyzed the expression level of MAP2and NF-H.㏕hen the changes of multipotential differentiation capability in SMG condition were explored: The BMSCs were induced in the SMG condition after cultured in SMG72h. After induction, the changes of above related indexes were redetected.2.3. Migration capability:Cell scratch experiment detected the scratch width of two groups cell after scratch0h,24h,48h,72h. Western Blotting was used to analyzed the expression level of CXCR4.3. Results3.1. The proliferation capability:①Flow Cytometry showed the percent of BrdU+cell was higher in SMG group than in NG group.②The quantity of BMSCs under SMG condition was obviously higher than that under NG condition after cultured3days and7days.③Flow Cytometry results showed there was little difference of the quantity of apoptotic BMSCs among different groups.3.2. Multipotential differentiation capability:①Analysis of Quantitative PCR and Western Blotting detected that cells maintained in SMG could up-regulate OCT4and most obvious change was found in SMG72h.②The result of endothelium oriented differentiation in normal gravity showed:the induced cells in the SMG group expressed higher VWF and CD31than that seen in the NG group; The result of adipogenic differentiation showed:the positive rate of Oil red O staining indicated in the SMG group (47%) were higher than the NG group (18%). Western Blotting showed the expression of PPARy2increased significantly under SMG condition; The result of neuron oriented differentiation showed:MAP2and NF-H were shown lower expression in the differentiated cells in the NG group than that in the SMG group.③The change of multipotential differentiation capability in SMG condition:when the two groups BMSCs were induced under the NG condition, the OCT4was little expression. But after the cells were induced under the SMG condition, they nearly have no Red-oil O positive cell (2%) and only expressed little PPARy2. In addition, OCT4expressed more in this group than the two groups before. After cells were induced to neuron in the SMG condition, they expressed little MAP2(0.8%) and NF-H(1.5%) but expressed more OCT4.3.3. Migration capability:Cell scratch experiment showed compared with the NG group, the change of the scratch width in SMG group had no statistical significance after the cells were scratched0h,24h,48h,72h. Western Blotting results showed the difference of the expression level of CXCR4in two groups was no significance.4. Conclusion4.1. The simulated microgravity could potentiate proliferation of BMSCs and no inducing apoptosis.4.2. SMG could enhance the multipotential differentiation capacity of bone mesenchymal stem cells, promote BMSCs to differentiate into endothelium-like cells, adipocytes and neuron-like cells and this increase of multi-potential differentiation capacity may be related to simulated microgravity promote BMSC better maintain its undifferentiated state.4.3. There is no evidence showed that the simulated microgravity could effect on the migration capability of BMSCs.