| BackgroundWith the development of the aviation industry, osteoporosis caused by weightlessness in space was pay more and more concern by scientists.The accumulated data of Space flight test have been summarized that loss of bone induced by weightlessness is mainly due to rare decrease in bone formation rather than increase in bone resorption. Osteoblasts is the important cells in the course of bone formation, development and growth. In the past studies focus mainly on the influence of micro-gravity on osteoblast differentiation, now the study of osteoporosis in weight loss gradually gathered in the exploration of cellular and molecular level with the development of Cytology. Under the conditions of microgravity, the changes in osteoblast structure of feelings and conduction of mechanical stress, in cell signal transduction and gene expression have made some progress.A large number of Study found that microgravity cause cell differentiation and maturation related substances (alkaline phosphatase, osteocalcin and collagen I) mRNA expression and protein synthesis decreased, Also resulted in osteoblast gene expression of COX-2 decreased and increased secretion of PGE2 and NO. Some studies also show that the cytoskeleton is very sensitive to the micro-gravity environment, microtubules and actin cytoskeleton rearrangement may be involved in the process of changes in cell morphology and function induced by micro-gravity.In recent years, NO as the physiological role of molecular information has also been taken attention by the people in the development of osteoporosis. Consensus has been reached is that the proliferation of osteoblasts and function of bone matrix protein secretion depend on the existence of a small amount of NO, but high concentration of NO inhibited both osteoblast proliferation and differentiation, Also significantly inhibited ALP activity and osteoblast synthesis of osteocalcin and other bone matrix proteins. NO is synthesized by NOS (nitric oxide synthase, NOS) with L-arginine as substrate, NOS as a local signaling molecule, different concentrations of NO generated by isoforms NOS have completely different physiological significance on osteoblast cells. Studies have shown that microgravity may promote NO/NOS system to take place a series of changes, such as NO concentration increase,and so on.This experiment on the basis of previous studies, after actin changed induced by simulated microgravity, detected the changes of NO/NOS system and signs of osteoblast products (alkaline phosphatase and osteocalcin) in osteoblasts, then to discuss the mechanism of osteoporosis leaded by weightlessness that whether the effects of microgravity induced changes in actin on NO/NOS signaling system lead to changes in gene expression affect the process of cell differentiation.ObjectiveThis study probed the mechanisms of bone loss in weightlessness on the cellular level, through the observation of actin cytoskeleton and detection of NO/NOS system and the products of osteoblast mark of Osteoblasts,to found the correlation between them, To provide new clues for the mechanism of osteoporosis included by Weightlessness.Subjects and Methods1.Cell lines and culture In this experiment, the mouse osteoblastic cell line MC3T3-E1 was provided by the Department of Anatomy, Southern Medical University. We adopted the third generation of cell after revived the same batch of frozen cells, in order to ensure the stability of cell characteristics. Cells culture with 10% fetal bovine serum (FBS) and low sugar DMEM culture medium contains double-antibody, cell culture medium was changed every two days, and cell was passaged every four days.2.1dentification of activity of osteoblast cell lineTo detect mineralization in vitro with Alizarin red staining of mineralized nodules.3.To build a model of osteoblasts in simulated microgravity, then group and interveneGroups were divided into normal gravity, simulated microgravity, simulated microgravity +0.5μg/ml cytochalasin B group,0.5μg/ml cytochalasin B group.4.Stainingstained to actin cytoskeleton with FITC labeled phalloidin, stained with DAPI for nuclei.5.measure the activity of ALPTo measure the activity of ALP in the culture medium of each group with p-nitrophenol method.6.Detect the expression of OCNTo detect the expression of OCN in the culture medium of each group using ELISA.7.determine the content of NOTo determine the concentration of NO in cell culture medium of each group adopting Nitrate reductase assay.8.measure the activity of NOS To measure the activity of NOS in cell lysates using the NOS typing kit.Result1.The growth of osteoblastsAfter MC3T3-E1 cells were revived and cultured,cells were triangle, diamond, and other irregular polygon shapes, transparency is good, In contrast microscope. Cells had rich cytoplasm, had synapses and extend outward, several protrusions extending out of the processes connected to adjacent cells, then arised the phenomenon of gathered the group (colony).There are Large and clear nucleus, were round or oval, with one or two nucleoli.2.Detection of mineralization in vitroThe last passage of cell grow for two weeks then the cells were appeared re-layered growth, the cells in center were intensived can calcify to form a visible white dots scattered in the tilting. Alizarin red method stained mineralized nodules, calcium deposition area was red.3.Changes in cell cytoskeletonTo detect changes in cell cytoskeleton structure of four groups in the same time with Actin fluorescent labeling. There were structural changes in microfilament Cells after treatment with cytochalasin B or rotation, the results show that the cytoskeleton to microgravity and relaxin are very sensitive. After cultured in simulated microgravity by rotating, actin cytoskeleton of MC3T3-E1 cells appeared partial depolymerization and the decline in stress fibers, stress fibers are short and thin, scattered in the cytoplasm, and randomly oriented. The extent of actin depolymerization in the control group treated with 0.5μg/ml cytochalasin B is similar with simulated microgravity group, microfilament was varying thickness and direction. Simulated microgravity+0.5μg/ml cytochalasin B group is even more significant changes in training groups, depolymerization of actin filaments was significant, scaffolding proteins dispersed, cytoskeleton network structure disappeared, the nucleus area is not resolved. Cells actin filaments arranged in an order, uniform thickness, to the clear in normal gravity control group.4.The activity of ALP in each groupMC3T3-E1 cells secreted the highest activity of ALP in normal weight group, it is 6.2867±0.3980 kim units/100ml, And compared with the other three groups,the difference were statistically significant (P<0.05).The activity of ALP was 2.9333±0.7805 kim units/100ml in Simulated microgravity+0.5μg/ml cytochalasin B group, it is Slightly lower than the simulated microgravity group, the difference was statistically significant (P<0.05). The activity of ALP in simulated microgravity group and 0.5μg/ml cytochalasin B group was 4.8900±0.6802 kim units/100ml and 5.6933±0.1266 kim units/100ml respectively, no significant difference between the two groups (P> 0.05).5.Expression of OCN in each groupMC3T3-E1 cells express the amount of OCN was 2.70816±0.53271pg/ml in simulated microgravity group, it is in middle of normal gravity group and simulated microgravity +0.5μg/ml cytochalasin B group, the differences between the three groups was statistically significant (P<0.05). The highest expression of OCN was 14.23681±2.85252pg/ml in normal gravity group, the lowest expression of OCN was 1.50685±0.40666 pg/ml in simulated microgravity +0.5μg/ml cytochalasin B group. The expression of OCN was 3.84456±1.23788pg/ml in 0.5μg/ml cytochalasin B group, and compared with simulated microgravity group,there was no significant difference (P> 0.05), and results were unity with alkaline phosphatase.6.Activity of iNOS in each groupThe activity of iNOS was 16.6043±1.3292U/ml in simulated microgravity group, it was higher than normal gravity group, the value was 5.8183±0.3196 U/ml, But lower than the simulated microgravity +0.5μg/ml cytochalasin B group, the value was 19.6990±0.5582 U/ml, And the differences were statistically significant (P<0.05). The activity of iNOS was 15.3760±2.8211 U/ml in 0.5μg/ml cytochalasin B group, and compared with the simulated microgravity group,there was no significant difference (P> 0.05).7.Activity of eNOS in each groupThe activity of eNOS was 7.1510±0.6780 U/ml in normal gravity group, it was the highest in all group, and compared with the other three groups, differences were statistically significant (P<0.05). The activity of eNOS was 1.0987±0.4776 U/ ml in simulated microgravity +0.5μg/ml cytochalasin B group, tit was slightly lower than the simulated microgravity group, the difference was statistically significant (P <0.05). The activity of eNOS in simulated microgravity group and 0.5μg/ml cytochalasin B group separately was 2.3613±0.2946 U/ml and 2.4633±0.4603 U/ ml, no significant difference between the two groups compared (P> 0.05).8.Concentration of NO in each groupWe could detecte the expression of NO in the culture medium of experimental group and control group. (Figure 2) shows the concentration of NO was 109.2593±4.8996μmol/L in simulated microgravity group, significantly higher than normal gravity group,the value was 81.4813±2.4494μmol/L, but lower than the simulated microgravity +0.5μg/ml cytochalasin B group,was 129.9383±9.9865μmol/L, the differences were statistically significant (P<0.5). The concentration of NO in 0.5μg/ml cytochalasin B group was 100.0003±8.0721μmol/L, and compared with simulated microgravity group,there was no significant difference (P> 0.05).Conclusion1.MC3T3-E1 osteoblast cell lines have the same biological characteristics with the primary cultured osteoblasts of rat.2.Microgravity has a similar effect that can destruct cytoskeleton structure of bone with cytochalasin B.3.As depolymerization of the osteoblast cytoskeleton deepening, the activity of osteoblast ruduct more obviously, so the activity of ALP and the expression of osteocalcin protein decreased more significantly.4.The change of osteoblast actin cytoskeleton can further control NO/NOS signal system. |
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