| To establish a high-performance induction culture system for RAW264.7 cells to differentiate into osteoclasts(OC)in vitro by improving the cell culture program,solved the problem of OC induction process in vitro.On the other hand,this study also analyzed the effect of Cystatin of silver carp purified in our laboratory on mouse RAW264.7 cells differentiate into OC cells in vitro with celll?biochemical?morphological level,and its effect on Mature OC,so as to investigate the bioactivity of Cystatin of silver carp in bone resorption of bone remodeling.This study will establish necessary experiment foundation and provide dependable theory for utilizing the offcuts of silver carp efficiently and reasonably,and turning waste into wealth.1.The establishment of a high-performance induction culture system for RAW264.7cells to differentiate into OC in vitroThis study established a high-performance induction culture system for Raw264.7 cells to differentiate into OC in vitro induced by combination of M-CSF?RANKL and 1?,25-(OH)2D3 At the same time,to guarantee the cells remained to grow in single layers all the time in most areas of the well during the whole induction we have brief digest with low concentrations of trypsin.The cells remained to grow in single layers all the time in most areas of the well during the whole induction by the improved culture program.The observation results of inverted microscope and HE staining showed that the growth area of the polykaryotic OC reached to 70%of the well on day 12.FITC-phalloidin staining showed that in the maturation of the OC,the cluster-shaped podosomes in the pseudopodia gradually transformed into rings,which finally fused to form a large belt surrounding the periphery of the cytoplasm.Calcitionin receptor(CTR)was markedly enhanced compared with the precursor cells by expression of immunofluroescence staining,and a large number of red granules appeared in the cytoplasm of OC with TRAP staining on day 12.These results that the obtained comfirmed OC were maturated and owned phagotrophic function.2.Effect of Cystatin of silver carp on bone resorptionFirst of all,this study explored the effects of Cystatin of silver carp for RAW264.7 cells to differentiate into OC.Alexa Fluor546-phalloidin fluorescent staining shows,podosome belt fusion from podosome clusters surrounding the cytoplasm in OC in the control group,high fluorescence intensity;10ng/mL Cystatin of silver carp group only surrounding many small podosome rings in OC cytoplasm;100ng/mL Cystatin of silver carp group only surrounding pointlike podosome clusters in OC cytoplasm;while 500ng/mLconcentration group has no podosome cluster,Tubulin ring changes in OC observed by immunofluorescence staining,tubulin ring surrounding the cytoplasm in OC in control group;With the concentration of Cystatin of silver carp increasing,tubulin ring thickness becomes smaller,the diameter decreases,the fluorescence intensity decreased,The 500ng/mL group has no tubulin ring formation.Expression of Cathepsin K in OC observed by immunofluorescence staining shows,cultured for 12 days a large number of CTSK expressied in OC in control group,cell membrane and perinuclear cytoplasm showed strong green fluorescence;While the other groups,the expression of CTSK in OC have low levels,the intensity of green fluorescence reduced,only faint green fluorescence surrounding a small amount of cytoplasm around the nucleus in 500 ng/mL group.Indirect ELISA test results show that,with the culture time increasing(3d?7d and 11d),the concentration of CTSK in culture supernatant of the control group increased gradually,while in 10-500 ng/mL group,the concentration of CTSK in culture supernatant decreased,the decline is positively correlated with Cystatin of silver carp concentration,the CTSK concentration in 500 ng/mL group is zero.and the decreasing amplitude of group 500 ng/mL was the most obvious.Concentration of Ca2+ changes in supernatant observed by micro plate method shows,with the culture time increasing(3d?7d and 1d),the concentration of Ca2+ in culture supernatant of the control group increased gradually,while in 10-500 ng/mL group,the concentration of Ca2+ in culture supernatant decreased,the decline is positively correlated with Cystatin of silver carp concentration,and the decreasing amplitude of group 500 ng/mL was the most obvious,to 11d the concentration of Ca2+ in culture supernatant decreased zero.This study also examined the regulation of Cystatin of silver carp on mature OC function,Alexa Fluor546-phalloidin fluorescent staining shows,podosome belt surrounding the cytoplasm in OC in the control group,high fluorescence intensity;10ng/mL Cystatin of silver carp group the podosome belt surrounding the cytoplasm in OC depolymerized into a large number of punctate aggregation podosome clusters;100ng/mL Cystatin of group only surrounding pointlike podosome clusters in OC cytoplasm;while 500ng/mLconcentration group podosome disappeared.Tubulin ring changes in mature OC observed by immunofluorescence staining,shows that,stable tubulin ring surrounding the cytoplasm in mature OC in control group;10ng/mL of Cystatin of silver carp group tubulin ring thickness becomes smaller,the fluorescence intensity decreased,while the 100 ng/mL and 500ng/mL group tubulin ring disappeared.Expression of Cathepsin K in OC observed by immunofluorescence staining shows,cultured for 12 days a large number of CTSK expressied in OC in control group,cell membrane and perinuclear cytoplasm showed strong green fluorescence;While the other groups,the expression of CTSK in OC have low levels,the intensity of green fluorescence reduced,only faint green fluorescence surrounding a small amount of cytoplasm around the nucleus in 500 ng/mL group.Indirect ELISA test results show that,with the culture time increasing,the concentration of CTSK in culture supernatant of the control group increased gradually,while in 10-500 ng/mL group,the concentration of CTSK in culture supernatant decreased,the decline is positively correlated with Cystatin of silver carp concentration,the CTSK concentration in 500 ng/mL group is zero.Concentration of Ca2+ changes in supernatant observed by micro plate method shows,with the culture time increasing,the concentration of Ca2+ in culture supernatant of the control group increased gradually,while in 10-500 ng/mL group,the concentration of Ca2+ in culture supernatant decreased,the decline is positively correlated with Cystatin of silver carp concentration,the Ca2+ concentration in 500 ng/mL group disappeared.The effection of Cystatin of silver carp on mature OCs apoptosis observed by Annexin V-FITC apoptosis detection kit,in the control group did not appear apoptosis OC,10ng/mL of Cystatin of silver carp lead to 30%OC apoptosis,100ng/mL of Cystatin of silver carp lead to 65%OC enter the stage of advanced apoptosis,while OC in 500 ng/mL group are all in the stage of advanced apoptosis.It came to the conclusion from the result above,this study established a high-performance induction culture system for RAW264.7 cells to differentiate into OC in vitro by improving the cell culture program,successful solved the problem of OC induction process in vitro.In this study,the Cystatin of silver carp not only inhibits the differentiation into OC also inhibited the function of mature OC,down regulate of CTSK expression,reduce the activity of OC of bone resorption,this may be due to the Cystatin of silver carp lead to mature OC apoptosis and necrosis. |
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