| BackgroundThere are two components absolutely necessary for the basic process, namely RANKL (receptor activator of NF-KB ligand) and M-CSF (macrophage colony-stimulating factor). RANKL mostly expressed in osteoblastSN stromal cells and the tissue of thymus lymphoid node and spleen. It was proved that RANKL had the function of promoting the differentiation , maturation and activation of osteoclasts. M-CSF is a growth factor of monocyte-macrophage cell system and osteoclasts, which bring into effect through promoting the recruitment and differentiation of osteoclast. RANKL and M-CSF are known both expressed on the surface of the stromal cells of bone, as well as secreted into the local milieu, they both exist as two molecule forms: membrane-bone form and secrete form.Mechanical strain is one of the most important stimulating factor of regulating the synthesis and secretion of RANKL and M-CSF. Bone marrow stromal cell is a kind of excitable cell tomechanical stimulation. It had been proved by experiments in vitro that mechanical strain within certain range could affect the expression of RANKL and M-CSF by bone marrow stromal cells, and regulation of RANKL and RANK by mechanical strain is magnitude , frequency and time dependent. But present research reported focus on the effect of physiological dynamic strain on the expression of RANKL and M-CSF at gene level , the effect of over-physiological strain on the RANKL and M-CSF synthesis at protein level has yet to be elucidated.Research objective1. The four-point bending system was used to load the mouse bone marrow stromal cell line ST2 in this experiment, ST2 were subjected to physiological and pathological strain to investigate the effects of mechanical stretching on the change of cellular morphology and proliferative activity.2. ST2 were subjected to physiological and pathological strain in this experiment to investigate the effects of mechanical stretching on the expression of RANKL ( sRANKL and mRANKL) , M-CSF (sM-CSF and mM-CSF) .Material and method1.Mechanical strain device and mechanical stimulating The four-point bending system was used to load the cells in this experiment, which was invented by Huazhong Electron Science and Technology University. ST2 were inoculated onto the plastic platelet and were cultured for 24h.When the cell proportion reached 80%, the plastic platelet would be removed into the tailor-made loading plate of the loading system and the cyclical loading began. The bending displacement of plastic plateletrepresent the magnitude of tension force subjected to the cells, so the bigger the displacement, the more obvious the transmutation, the bigger tension force subjected to the cells.The loading frequency was 0.5Hz, the displacement were respectively lmm and 2mm namely 1785u and 3570u strain.2. MTT detect the effect of mechanical strain on the cell proliferation *of ST2The cells were divided into 3 groups by random after they grew into logarithmic phase on the plastic platelet, group 1 without any treatment, group 2 and group 3 were respectively subjected to tension force of lmm and 2mm displacement for 6h. After loading the cells were inoculated into 96 well plate with the density of 2X104, every group owning 21 wells. Then in the following 7 days, MTT technique was used to detect the absorbance at 570nm 3 wells per group day-to-day.3. Enzyme-linked immunosorbent assay (ELISA) for the content of soluble RANKU M-CSF in supernatant fluidThe cells were divided into 2 groups by random after they grew to 80% confluence on the plastic platelet, group 1 and group 2 were respectively subjected to tension force of lmm and 2mm displacement for 12h. In the course of loading, proper quantities of supernatant fluid was extracted at the time of Oh, 0.5h, lh, 21-u 3h, 61-u 9h> 12h to detect the content of soluble RANKU M-CSF according to the operation of ELISA kit.4. Immunodeposition Western blotting for mRANKU mM-CSF The cells were divided into 6 groups by random after they grewto 80% confluence, the loading time is Otn ltu 3h> 6h> 9h> 12h. Every group was respectively subjected to tension force of lmm and 2mm displacement for 3 times except for the control group. For theRANKL or M-CSF assay, plates were washed on ice with cold PBS and cells were lysed in 100 Hi of lysis buffer. Cells were lysed and transferred to Eppendorf tubes and centrifuged with 13000 r/min for lOmin.After measuring protein , transfer 200u g of the above total celluar protein, add 1p 1 primary antibody and incubate for lh at 4° C, then add 20n 1 Protein G PLUS-Agarose and incubate at 4° C on rotating device overnight. Wash pellet 4 times, discard supernatant in 40 P 1 electrophoresis sample buffer. Ten micrograms of protein was loaded onto a 12% polyacrylamide gel for chromatography before transfer to polyvinylidene difluoride (PVDF) membrane. After blocked with 5% milk for 2h, incubation with primary antibodies and secondary antibodies were performed before exposing membranes to an enhanced chemiluminescence (ECL) solution for 5 minutes before radiography, revealing RANKL or M-CSF bands. The films were scanned to detect the density ration of the specific protein band with analysis software.Resultl.MTT: The control group cells proliferated steady, the lmm strain group did not change much. The 2mm strain group proliferated slowly (P<0. 01) .2.ELISA.- Application of lmm strain did not cause evident change of secretion of sRANKL and sM-CSF, but 2mm strain caused secretion of sRANKL increasing after 3h of loading (K0.05) with sM-CSF increasing after 0. 5h (/X0.05) .3. Immunodeposition Western blotting: mRANKL decreased after ShlmmstrainC/XO. 05), but mM-CSF did not change under lmm strain, neither mRANKL nor mM-CSF changed under 2mm strain.Conclusionsl.The state of ST2 changed much after pathological force as the proliferation was suppressed.2. Physiological tension force restrained the expression of mRANKL, pathological force just promoted the secretion of sRANKL.3. Neither physiological nor pathological force had effect on mM-CSF, pathological force just promoted the secretion of sM-CSF. |
PDF Full Text Request