| BackgroungOsteoporosis is a systemic disease that leads to the decrease of bone strength and increased fragility and risk of fracture characterized by the reduce of the bone mineral density(BMD)and the disruption of bone microarchitecture:bone trabecula become thiner,breaked and reduced and OS integumentale become thiner with increased porosity. Osteoporosis is divided into primary osteoporosis and secondary osteoporosis. The Postmenopausal and senile osteoporosis are both belong to primary osteoporosis.Weightlessness-induced osteoporosis is a secondary osteoporosis, it is a special disuse osteoporosis caused by the microgravity environment. With the vigorous development of the space industry,it has become one of the prominent hampers. Exercise, drug and nutritional supplements taken by astronaut at present spaceflight, can’t effectively prevent bone loss occurrence. To assurance the health and effective performance of astronaut during future long-term spaceflight mission, it was required to clarify the mechanism of bone loss in space and develop effective countermeasure against bone loss based on the cellular and molecular intrinsic understanding of bone loss.Weightlessness is a kind of special mechanics environment that means the object does not represent manifest weight but mass. The special mechnics enviroment actually is Microgravity. Microgravity is a state in which gravity isreduced to almost negligible levels, such as during space flight. The space flight falls under small non-gravitational forces and results very a small acceleration. Usually,this non-gravitational acceleration is only one ten thousandth of the ground acceleration of gravity or even less. It is commonly defined as thel0-5～10-4g. In order to compare with the normal gravity,the micro-acceleration phenomenon is named microgravity. The less microgravity is. the more complete zero gravity is. In a word, zero gravity is only an ideal state. Just micro-gravity is practical circumstance.Weightlessness-induced osteoporosis is mainly caused by mechanical factors of osteoporosis. Osteoblasts are the most important mechanical feel osteoblast cells and effectorcells in bone tissue. The weightlessness osteoporosis occurrence is mainly the bone formation decreased which leads to the bone loss. Especially the proliferation、the differentiation function and the polymerization and depolymerization of cytoskeleton of osteoblasts were depressed,while the metabolic disorders of osteoblasts further inhibit the bone formation ability of the cells. Currently, research of changes of cytoskeleton feeling and conductingmechanical stimulation, signaling transduction, gene expression of osteoblast have made some progress. Studies have shown that the level of insulin-like growth factor I (IGF-1) are closely linked to the occurrence of osteoporosis. Insulin-like growth factor-1(IGF-1) is an important growth factor for the differentiation regulation of osteoblast, which can reduce degradationc of bone collagen, increase deposition of sclerotin, promote differentiation and maturation of osteoblast, and its level is related to bone mineral density.The level of IGF-1in blood serum of patients suffering from Osteoporosis decreaseand signaling cascade of IGF-1is inhibited in conditions of weightlessness. Integrin is important mechanical signal transduction transmembrane molecules. Mechanical signals can be converted to chemical signals to regulate physiological function of cells by the reaction between integrin and extracellular matrix proteins, cytoskeletal protein and focaladhe-sionkinase. Under conditionsof simulated weightlessness, the expression of integrin subunitsin osteoblasts is decreased. Insulin-like growth factor (IGFS) including IGF1and IGF2, two IGF receptors, six IGF binding protein(IGFBP1-6), IGFBPprotease. IGFS,the faction and coupling of osteoblast and osteoclast have intimate connection. Igf-1is an important part of IGFS. IGf-1is a necessary factor to the growth of long bone. It stimulates proliferation and differentiationof chondrocyte, plays an important role in the process of formation of corticaland cancellous bone, stimulates the proliferation and differentiation of osteoblasts, increases the synthesis of type Ⅰ collagen, activity of alkaline phosphatase and production of osteocalcin.It also restrains the degradation of collagen produced by osteoblasts and takes part in the composition of inorganic salt and1,25(OH)2D3composed by kidney. IGF-1is considered to be important regulatory agent of intestinal absorption and extracellular calcium and phosphorus. Recent studies suggest that the level of IGF-1in postmenopausal people declined by30%～40%compared with Young people. IGF-1is negatively correlated to age. Before puberty, IGF-1deficiency or in the form of resistance can slow the growth of long bones and reduct peak bone mass. Measure bone mineral density (BMD) of vertebral, tibia, and distal tibia with dual-energy x-ray absorptiometry in the rat model induced osteoporosis by ovariectomy and bury a special subcutaneous device that can release IGF-1penetratively in Subcutaneous of rats in for6weeks.the result show that BMD of the three bonescan points increased dose-dependently.The increase of BMD caused by IGF-1in this animal model is mainly through thethickening of the backbone. Osteoblasts not only possessthe IGF-1receptor,but also have the function Generating IGF-1. Therefore, IGF-1has an important relationship with bone metabolism.Integrins are important mechanical signal transduction transmembrane olecules. Mechanical signals can be converted to chemical signals to regulate physiological function of cells by the reaction between integrin and extracellular matrix proteins, cytoskeletal proteinand focaladhe-sionkinase. Studyconfirmed that mechanical signal pathways of skeleton is inhibited in the weightless or simulated weightless environment.And one of the pathways is theextracellular matrix-integrin-cytoskeleton system.The decline expression of integrin subunits in weightless environment shows that weightlessness may regulate downstream molecules by inhibiting the expression of integrin.Studies have shown that IGF-1and integrin signaling pathway are connectwitheach other. Weightlessness may affect the role of the IGF-1pathway by integrins. This study was designed to investigate the effect of insulin-like growthfactor-1on expression of integrin subunits in osteoblasts under simulated microgravity in order to provide a theoretical and experimental basis for the weightlessness osteoporosis treatment.ObjectiveThis rearch dicussed the probable mechanisms of osteoporosis in eightlessnesson the Cell and molecular level. Discuss the effects of insulin-like growthfactor-1on expression of integrin subunits in osteoblasts under simulated microgravity,taking the mouse osteoblastic cell line (MC3T3-E1) as rearch object and imitating the effect of microgravity by gyrator. we expect to realize the interaction between IGF-1and integrin in the Weightlessness osteoporosis,in order to offer a new method for the treatment of osteoporosis.Subjects and Methods1. Cell lines and culture The mouse osteoblastic cell line MC3T3-E1was provided by the Department of Anatomy,Southern Medical University. We adopted the same batch of frozen cells to recovery. Cells culture with10%fetal bovine serum 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. Identification 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 interveneUsing the50ml volume of the High Aspect Ratio Vessel(HARV) of the rotary cell culture system(RCCS) to imitate the microgravity, we culture the osteoblast cell with Cephodex microcarrier.we adopted the third generation of cell after revived the same batch of frozen cells.Because the cells were in the logarithmic phase and the character was quite stable. Then change the culture medium to the low sugar DMEM without fetal bovine serum.24h later collect the cells and inoculate in5cyclotron culture cylinder prepositioned microcarrier by3x105cells/ml randomly. The5cylinders were divided into simulated microgravity group,simulated microgravity+10μmol/L IGF-1group, simulated microgravity+50μmol/L IGF-1group, simulated microgravity+100μmol/L IGF-1group, and normal gravity group. The simulated microgravity group and normal gravity group were filled with serum-free low glucose DMEM.Other groups were filled with10ng/mlIGF-1,50ng/mlIGF-1,100ng/mlIGF-1serum-free low glucose DMEM and drain the bubble.The normal gravity groupwere placed in37℃,5%CO2, the humidity saturation incubator to culture and the rest were placed on the gyrator. cylinders were in a low-speed intermittent rotary in the fomrt8h:stir1～2min in45min, then rotary cultureat30r/min. Observe whether there are air bubbles in the container everyday.If there are bubbles, exhaust bubbles under sterile conditions immediately. Cells were digested and collected after Cultivation of48h.4. Collect the total RNA for RT-PCRThe total RNA in cells was isolated using Trizol method for reverse transcription PCR. Reverse transcription PCR analysis was made to examine the gene expression of α2,a5and β1integrin subunits. Each value was normalized against that of GAPDH mRNA. Scan the photo of PCR products and calculatethe integral optical density ratio of integrina2/GAPDH、integrina5/GAPDH、integrinβ1/GAPDH,then calculate the relative expression level of integrina2、integrinαs、integrinβ1.5. Statistical methodsThe statistical data was statistical analyzed using SPSS19.0. The Same condition experiment repeat5times. Experimental measurement data was present as mean±standard deviation (x±S). Comparing mean of multiple groupswith analysis of variance by completely randomized design information, the descriptive statistical analysis of single-factor analysis of variance (One Way), thediversity of this pairwise mean comparisons ANOVA and LSD method, Levene’s test of homogeneity of variance. P<0.05was statistically significant.Result1.The growth of ostcoblasts:After MC3T3-E1cells were revived and cultured, cells were tri angle, 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.The expression of integrin subunits in each group:Compared with the normal gravity group, the mRNA expression of integrinα2、integrinα5、integrinβ1of the simulated microgravity group decreased obviously (P<0.05). Compared with the simulated microgravity group, the mRNA expression of integrinα2、integrinonα5、 integrinβ1of the simulated microgravity+IGF-1group increased obviously and dose dependent obviously.Conclusion1. MC3T3-E1osteoblast cell lines have the same biological characteristics with the primary cultured osteoblasts of rat.2. The mRNA expression of integrin subunits in osteoblasts under simulated microgravity decreased obviously.3. Insulin-like growth factor-1can increase the mRNA expression of integrin subunits in osteoblasts under simulated microgravity. |
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