| BackgroundMicrogravity, known as zero gravity relative to the the1g gravity environment of earth’s surface in terms of a "zero weight" state, but also is the environment in which humans working and living in space. The human long time in microgravity can cause a variety of pathophysiological changes, including osteoporosis, muscle atrophy, and immune function decline and so on, and osteoporosis is one of the most common and serious complications. According to reports, the space fligh every month on average2%of bone loss, the equivalent of postmenopausal bone loss in women, and bone loss persists after the return to the ground, which seriously affect the pilot’s health. Existing research at home and abroad that the pathogenesis of osteoporosis induced by microgravity is space flight led to the shape and structure of osteoblasts (OB) change significantly, the cells show of concentrated nuclear or nuclear dispersion, due to the action of gravity osteoclasts cell activation caused by increased bone resorption, lack of integrity and function of osteoblasts[1]. Existing drugs and physical therapy failed to play the desired therapeutic effect. To explore other methods of prevention and treatment of weightlessness osteoporosis, the simulated microgravity bone metabolism and the study of cell signaling pathways has become the one of hotspot in the field of aerospace medicine at home and abroad.In1990Issemann[2] such as the first to discover a kind of can be fatty acid-like compound peroxidase enzyme body proliferative agents (peroxisome proliferators, PP) activation, and was named for the PP activated receptor(peroxisome prolifemtor-activated receptor, PPAR). PPAR is a kind of widely involved in glucose and lipid, regulating fat storage of nuclear transcription factor gene expression, belonging to the nuclear receptor superfamily members. Due to gene promoter and spliced in different ways, in amphibians, rodents and humans PPARs, there are three subtypes, namely PPAR a, PPARβ (also called PPARδ or NUC-1) and PPARy. In recent years, studies have found that peroxisome proliferator-activated receptor y (PPARy) with activator protein21(AP21), signal transducer and activator of transcription1(STAT1) Fos protein family members δFosB protein, cell cycle vitamin D and other factors and the Wnt signaling pathway regulating cell differentiation, growth and apoptosis with the synergistic effect, including the process of differentiation of bone marrow mesenchymal stem cells (BMSCs). Bone marrow mesenchymal stem cells (BMSCs), with multiple differentiation potential, can differentiate into bone cells, fat cells, cartilage cells, muscle cells and nerve cells[3-5].The experiment was designed of the bone marrow mesenchymal stem cells composite in microcarriers simulate with microgravity rotating culture system (RCCS) to the osteoblast differentiation induced by activation and inhibition of PPARy pathway, detection of the effect of bone marrow mesenchymal stem cells into bone in the process of the differentiation. Expect our results for the signal transduction mechanism of osteoporosis further accumulation of scientific basis and provide a potential new direction of treatment.ObjectiveTo observe the effect of the peroxisome proliferator-activated receptor y cellular pathways on rat bone marrow mesenchymal stem cells into osteoblast differentiation in vitro simulated microgravity and pioglitazone, GW9662peroxisome proliferator activated receptor y gene expression.Method1. The BMSCs separation, culture, amplification:drawn from the5-week-old SD rat femur and tibia bone marrow for BMSCs as primary culture, to break out of the bone marrow cell suspension into the prepared15ml centrifuge tube at room temperature, with1000r/min centrifugal5min, supernatant, fat and impurities was removed, cells were resuspended and washed with DMEM/F12; Resuspend cells in DMEM/F12containing10%fetal bovine serum, and seeded in25cm2flasks, adding complete culture medium (10%FCS, the penicillin G100U/ml, streptomycin100U/ml DMM/F12medium), culturing in37℃,5%CO2incubators. According to adherent culture cells were isolated and passaged, taking the4-5generation of bone marrow-derived mesenchymal stem cells as an object of study.2. Identification of BMSCs nature:with flow cytometry to detect cell surface CD29, CD34, CD44molecule expression and draw-flow diagram.3. Preparation bone induction medium:dexamethasone sodium phosphate injection1.03mg0.206ml, ascorbic acid1.00mg and (3-glycerophosphate3.06mg with5ml complete medium dissolution, magnetic stirring to completely dissolve sterile0.22μm membrane filtration sterilization combined with complete medium to20ml, with that became osteogenic induction liquid:containing50mg/L ascorbic acid0.1μmol/L dexamethasone,0.5mmol/Lβ-glycerophosphate,0.1volume fraction FBS DMEM-LG medium.4.Grouping:Experiment was divided into:normal gravity group (NG), analog micro-gravity group (MG), simulated microgravity+10μmol/L pioglitazone ketone group (MG+PIO), analog micro-gravity+10μmol/L GW9662group (MG+GW), simulated microgravity+10μmol/L the pioglitazone+10μmol/L GW9662group (MG+the PIO+GW). Normal gravity group adherent culture in normal gravity were cultured for14d, simulated microgravity group and simulated microgravity+pioglitazone and (or) GW9662group were in simulated microgravity cultured for14d in the Rotary gravity reactor. Cell culture after14d, the experimental groups with0.02%EDTA solution+2ml of0.25%trypsin were digested at37℃for1-2min, collected by centrifugation OB for detecting.5. Cells in each group cultured for14d were detected bone nodules by alizarin red staining, alkaline phosphatase activity within the cells, fat cells by oil red-O staining and calculations into fat percentage, the mRNA expression of PPARy gene by two-step RT-PCR (semi-quantitative reverse recorded-polymerase chain reaction).6. The statistical data was statistical analyzed using SPSS13.0. Experimental measurement data was present as mean±standard deviation (x±S). Comparing mean of multiple groups with analysis of variance by completely randomized design information, the descriptive statistical analysis of single-factor analysis of variance (One Way), the diversity of this pairwise mean comparisons ANOVA and LSD method, Levene’s test of homogeneity of variance. P<0.05was statistically significant. Result1. Growth of BMSCs:after primary rat BMSCs were inoculated, the cell morphology of different sizes, the cylindrical type. Medium was changed after24hours, the cells adherent growth3d, colony of fibroblast-like cells, cells projecting different lengths and uneven thickness protruding, abundant cytoplasm, nucleolus clear can be observed, every two days under sterile conditions, the full amount of the medium was changed to eliminate the impurities. After7to9d cells were colony growth, as the same direction arranged in a spiral shape. After10to12d cells were tightly packed, gradually integrated into the film. In cell passage purification training, every3to4d families were passaged. After continued passage4-5generation, cell growth strongly, with uniform shape, fusiform growth, abundant cytoplasm and nucleus obviously, and each cell with one or two nucleolis.2. Identification of BMSCs nature:expression of cell surface CD molecules were detected by flow cytometry, results showed that CD29(99.52%), CD34(0.58%),, CD44(41.88%), identification showed a mesenchymal stem cell characteristics, CD34mesenchymal stem cell surface marker antigen negative expression, CD29and CD44mesenchymal stem cell surface marker antigen positive expression. The above results are consistent with Li Xiaofeng[6], the results verifiable rat bone marrow mesenchymal stem cells. While BMSCs induced osteoblast differentiation solution, identification of osteogenic differentiation.3. Identification of OB:after the osteogenic induction liquid continuous differentiation of culture for14d, alizarin red staining under an inverted microscope can be visibled intracellular red or brown mineralized nodules. Simulated microgravity group within the field of vision mineralized nodules than the group of normal gravity decreases, simulated microgravity+pioglitazone group within the field of vision mineralized nodules compared with microgravity group significantly reduced, cell arrangement disorder, the simulated microgravity+GW9662group within the field of vision is visible mineralization nodules compared to simulated microgravity group slightly increased, mineralized nodule within the field of vision simulated microgravity+pioglitazone+GW9662group compare to the microgravity group increased, compared to simulated microgravity+GW9662group decreased.4. Detect of alkaline phosphatase (ALP) activity:compared with normal gravity and simulated microgravity cultured cells alkaline phosphatase activity difference was statistically significant (P<0.01), alkaline phosphatase activity of simulated microgravity experimental groups cultured reduction. Simulated microgravity group compared to simulated microgravity+pioglitazone group alkaline phosphatase activity is lower, the difference was statistically significant (P=0.001). The simulated microgravity+GW9662group of higher alkaline phosphatase activity, the difference was statistically significant (P=0.002). Simulated microgravity+pioglitazone group compared to simulated microgravity+GW9662group and simulated microgravity+pioglitazone+GW9662group of cells alkaline phosphatase activity is higher, the difference was statistically significant (P<0.01).5. Identification of fat cells into fat percentage calculated:compared with normal gravity group, the number of fat cells and adipogenic rate difference with simulated microgravity cultured cells was statistically significant (P<0.01), the experimental group of simulated microgravity culture higher adipogenic rate. Compared with the simulated microgravity group, simulated microgravity+pioglitazone group and simulated microgravity+GW9662group into fat rate difference significant (P<0.05), simulated microgravity+pioglitazone group fat rate increases, simulated microgravity+GW9662group adipogenic rate decrease. Compared to simulated microgravity+pioglitazone group, simulated microgravity+ GW9662group and simulated microgravity+pioglitazone+GW9662group into fat rate differences were statistical significance (P<0.05), were lower than the simulated microgravity+pioglitazone group.6. PPARγ mRNA of the OB expression:RT-PCR results show that compared with normal gravity, PPARγ mRNA expression of simulated microgravity group and simulated microgravity+pioglitazone group were increased, the difference was statistically significant (P<0.05). Compared with the simulation of microgravity+pioglitazone group, PPARγ the mRNA expression of simulated microgravity group, simulated microgravity+pioglitazone group and simulated microgravity+pioglitazone+GW9662group was reduced, the difference was statistically significant (P<0.05).Conclusion1."Whole bone marrow adherent culture" to cultivate primary cultured rat bone marrow mesenchymal stem cells is a scientific and effective, simple and reliable bone marrow mesenchymal stem cell isolation, culture, purification and amplification method.2. In vitro, simulation microgravity training bone marrow mesenchymal stem cells to the osteoblast differentiation, reduce the ossification differentiation rate and increase stem cell to fat cell differentiation chance to break the balance between bone cells and fat cells, which make a bone matrix formation, mature and mine of the role of lower raised, which increase the expression of PPARy mRNA gene.3. Pioglitazone can further reduce the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and promote the differentiation of stem cells into fat cells, aggravating the shape of the bone matrix maturation and mineralization obstacles, which raised PPARy mRNA expression. 4. GW9662can partially reverse under simulated microgravity state on bone marrow mesenchymal stem cells inhibition of osteoblasts differentiate, inhibition of fat cells, which down PPARγ mRNA gene expression.5. PPARγ cell signaling pathways leading to activation may be caused by simulated microgravity induced osteoporosis pathogenesis... |
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