| BackgroundWeightlessness is the object shows only for the quality but not for the weight in the microgravity (10-6-10-9g). In the weightlessness flying, Changes in other physiological systems will achieve a new balance and can quickly recover after the return to the ground, while the regulation of bone metabolism would be disequilibrium and the lost of bone mass might keep developing after return to the earth and result in Osteoporosis. Imbalance in the regulation of bone metabolism is the main problem of astronaut health. It has been proved that bone loss of weightlessness osteoporosis was mainly due to decreased bone formation rather than increased bone resorption. Currently used in space have failed to play a variety of counter measures to the desired results. In order to explore more effective prevention, treatment method of weightlessness bone loss, the research of the change in bone metabolism and bone cell metabolism under weightlessness has become the focus and hot topics of aerospace medical research field domestic and abroad.The present study show that Levels of insulin like growth factor-1 (IGF-1) is closely-related to The incidence of osteoporosis. It has been reported that Blood levels of GH,IGF-1 in diabetic patients with osteoporosis and osteoporosis patients decreased. The results of foreign clinical epidemiological survey showed that levels of PTH and markers of bone resorption elevated while IGF-1,IGFBP3 and bone formation markers levels,bone density decreased bone mass in obesity, diabetes osteoporosis, postmenopausal osteoporosis patients. Low serum IGF-1 always associates with increased risk of hip fracture. Animal, bone cells in vitro experiments results showed that IGF-1 can stimulate synthesis of bone collagen and bone matrix, promote the survival of osteoblasts, inhibition of osteoblasts apoptosis by self-secrete.In mouse bone tissue, IGF-1 has important implications in the stable state of collagen I mRNA and gene expression of procollagen bone. IGF-1 also inhibited the transcription of interstitial collagenase to reduce collagen degradation, that maintained bone mass. IGF-1 is important factor which maintained bone mass and promoted bone cells growth. This topic is the study of the change of cell proliferation, secretion of function and IGF-1 gene expression of rat osteoblasts in vitro under simulated weightlessness and intervention of calcitonin. To provide experimental evidence for going further explore about the change of osteoblast in gene or protein levels under simulated weightlessness.ObjectiveTo observate the change of cell proliferation, secretion of function and IGF-1 gene expression of rat osteoblasts in vitro under simulated weightlessness and intervention of calcitonin.Method1.Isolation, culture, amplification of Osteoblasts:Primary culture of osteoblasts made of from the rat cranial parietal bones. Adherent culture based on isolation and amplification of osteoblast, detected the cell proliferation rate by MTT assay and draw growth curve.4-6 generation of cells were taken into study.2. Identification of osteoblast function:alkaline phosphatase (ALP) Activity in medium and observed mineralized nodules induced mineralized nod3. Construction of alginate microcapsules of osteoblasts.4. Grouping:Experiment was divided into:normal weight group, simulated weightlessness, simulated weightlessness+calcitonin group (10 IU/L,40 IU/L,80 IU/L). Normal gravity group osteoblasts were cultured in normal gravity for 72 hours, simulated weightlessness and simulated weightlessness+calcitonin group osteoblasts were cultured in the rotating bioreactor simulated weightlessness for 72 hours. Cells cultured after 72 hours, all experimental groups alginate microencapsulation were dissolved by sodium citrate at 37℃, osteoblasts were detected after collected by centrifugation.5. Osteoblasts in each group were detected proliferation by MTT assay, alkaline phosphatase activity in the medium, apoptotic bodies by hoechst staining, mRNA expression of IGF-1, ALP, osteocalcin gene by two-step RT-PCR.6. Statistical data was statistical analyzed using SPSS13.0. Experimental measurement data was present as mean±standard deviation (x±s). Comparing mean of two groups was using two independent samples t test, multiple groups were compared with one-way classification (One-way ANOVA), pairwise comparied between groups by LSD t test, detected homogeneity of variance by Levene test. P <0.050 was statistically significant.Result1. The growth of osteoblasts:Osteoblasts were extracted in the way of "two-step digestion-tissue culture". Under the microscope, osteoblasts displayed irregular shapes, such as triangle, rhombus, polygon, with good transparency. Rich cytoplasm-osteoblasts linked processes with near osteoblasts processes. Osteoblasts had gathered into a group phenomenon (Colony). Osteoblasts had clear nucleus with 1 to 2 nucleoli, displaying round or oval. On the third day of tissue culture, a lot of osteoblasts grew along surrounding tissue; on the fifth day, the number of cells was increased significantly. Taking tissue as the center, Osteoblasts grew radioctively. The purification of osteoblasts was performed through different speed of adherence. On the seventh day, osteoblasts covered with single layer, while it needed 10 days for tissue culture. With culture time extending, osteoblasts would be multi-layered growth.2. Determination of cell viability:MTT proliferation growth curve, taken in the logarithmic phase of the experiment,4-6 cells.3. Identification of osteoblasts(1) Gomori calcium-cobalt staining showed that endochylema of osteoblasts contained black particles located in the activity of the department, to prove the expression of ALP activity.(2) Ability to detect mineralization in vitro:induced osteoblasts secreting mineralized nodules by 10 mmol/Lβ-glycerophosphate+50 mg/L of vitamin C mineralization induced medium. After 3-4 weeks' growth, cells were re-layered growth. In the center of cells, density calcification can occur to form a visible white dots scattered in the tilting.4. The proliferation of osteoblasts:Compared with normal gravity group, the cell proliferation of osteoblasts in simulated weightlessness group was significantly lower(P<0.01). Compared with simulated weightlessness group, simulated weightlessness+calcitonin groups were significantly increased cell proliferation rate (P<0.05), no significant dose-dependent effect relationship was observed.5. Alkaline phosphatase activity in the medium:Compared with the normal gravity group, medium alkaline phosphatase activity in simulated weightlessness group decreased significantly (P<0.01). Compared with simulated weightlessness group, medium ALP activity in simulated weightlessness+e calcitonin groups were significantly higher (P<0.05), no significant dose-dependent effect relationship was observed.6. Apoptosis of osteoblasts:hoechest staining:Cultured under normal gravity, osteoblasts evenly distributed chromatin, filled with uniform low-intensity fluorescent; Cultured under simulated weightlessness, osteoblasts (including simulated weightlessness, and CT plus drug group) were observed more apoptotic cells, cells were compact fluorescent condensation-like or particle-like, what was the tips of osteoblasts cultured in simulated weightlessness appeared condensed nuclei, chromatin condensation, DNA fragmentation and apoptosis. There was not observed significant differences between simulated weightlessness and simulated weightlessness+CT plus drug groups. 7. Expression of IGF-1, osteocalcin, alkaline phosphatase mRNA:RT-PCR results showed that:Compared with the normal gravity group, IGF-1, osteocalcin, alkaline phosphatase mRNA expression decreased simulated in weightlessness and simulated weightlessness+calcitonin groups. Compared with simulated weightlessness group, IGF-1, IGF-1, osteocalcin, alkaline phosphatase mRNA expression simulated weightlessness+calcitonin groups increased.Conclusion1."Two-step digestion-tissue culture" is a scientific and effective way of primary culture rat osteoblasts.2. In vitro, simulated microgravity environment reduces the activity, bone matrix formation, maturation and mineralization of osteoblasts; the rate of osteoblasts apoptosis increased significantly.3,Calcitonin partially Increased IGF-1, ALP, osteocalcin mRNA expression of genes and reversed the Inhibition on maturation and mineralization of osteoblasts under simulated weightlessness. |
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