Study On The Differentiation And Function Of Osteoblast And Osteoclast Of Carassius Auratus Sialyglycoproteins

Normal adult bone metabolism is a process of dynamic equilibrium with thebone formation in osteoblast and bone resorption in osteoclast. When this balancedisorders, bone resorption is greater than bone formation, leading to osteoporosis.Fish eggs has lots of bioactive substances, such as glycoproteins, EPA, DHA,vitamins and mineral substances, with a variety of biological activities:anti-cardiovascular disease, improvement of learning ability, promotion of children’sgrowth etc. Nowadays, the research of the fish eggs mainly focuses on thedevelopment and utilization of functional lipid. And the research of roe glycoproteinmainly focuses on its separation and purification, biochemical property, but there arefew studies on the bioactivities. In this study, the sialyglycoproteins were separatelyextracted from Carassius auratus, the promotion effect of bone formation and theinhibitory effect of bone resorption were determined, as well as the mechanism wassystematically undertaken at the cellular level. The main results in this thesis are asfollows:The content of sialic acid of different roes were detected by using DAD/FLDHPLC, and MTT assay was used to determined the proliferation ability of differentroes. The results suggest that the content of sialic acid of different roes has a positivecorrelation with the proliferation ability of MC3T3-E1cells. Sleeve-fish dose notcontain sialic acid, as well as has no effect on the MC3T3-E1cell proliferation.Carassius auratus sialyglycoproteins (Ca-SGP) has the highest content of sialic acid,and Ca-SGP could significantly promote MC3T3-E1cell proliferation mostsensitively by MTT assay, with a time and dose-dependent effect.The proliferating, differentiating and mineralized-stimulation effects andmechanism of Ca-SGP were studied on MC3T3-E1cells. And Ca-SGP was divided into two parts with the hyperfiltration membrane of10KDa. The MTT assay indicatedthat Ca-SGP (>10KDa) and Ca-SGP (<10KDa) showed significant promotion effectson the proliferation of MC3T3-E1cells, the proliferation rate was153.57%and137.28%, respectively. The ELISA assay showed that Ca-SGP could markedlyincrease the secretion of ALP、OCN and COL I, and then promote the differentiationof MC3T3-E1cells. The results of alizarin red staining exhibited that Ca-SGP(>10KDa) and Ca-SGP (<10KDa) could improve the mineralized bone nodular by196.41%and158.37%(P<0.01). The RT-PCR and Western blotting assaydemonstrated that Ca-SGP remarkably increased the expression level of BMP-2andOPG, decreased the expression of RANKL. OPG and RANKL plays an important rolein the differentiation of osteoclast, RANKL can promote the process and OPG playsan opposite effects. It suggested that Ca-SGP exhibited a bone formation inducingactivity in MC3T3-E1cell through rising of the expression of BMP-2and the ratio ofOPG/RANKL. Besides, the effects of Ca-SGP (>10KDa) were better than Ca-SGP(>10KDa).In this study, the osteoclastogenesis-inhibition effects and the mechanism ofCa-SGP were investigated on osteoclast model by using M-CSF and RANKL. MTTand LDH assay indicated that Ca-SGP had no effect on the proliferation andcytotoxicity of BMMs and osteoclast. The TRAP staining and activity assay showedthat Ca-SGP significantly inhibited the differentiation from BMM to osteoclast cellwith a time-and dose-dependent effect, which were most effective at the early periodof differentiation. The proinflammatory factors, such as IL-1β、IL-6and TNF-αpossessed the effect of promoting differentiation of osteoclast. And the enzyme-linkedimmunosorbent assay (ELISA) indicated that Ca-SGP could remarkably decreased thesynthesis of IL-1β、IL-6and TNF-α. It suggested that Ca-SGP exhibited inhibitioneffect of osteoclast differentiation and the content of proinflammatory factors withoutaffecting the cell proliferation.The mechanism of osteoclast differentiation are as follows: RANKL, which issecreted by osteoblast, combined with RANK which is on the surface of osteoclast.Then TRAF6bound with RANK, promoting the activity of NF-κB and MAPK signal pathway. Thereby increasing the expression level of c-fos and NFATc1. And thusaccelerating the activity of TRAP、MMP-9、CTR and cathepsin K. QRT-PCR andWestern blotting results showed that Ca-SGP could decrease the expression ofTRAP、MMP-9、CTR and Cathepsin K. The expression level of TRAF6and thephosphorylation of NF-κB、I-κB、ERK、JNK and p38were also significantly inhibitedby Ca-SGP, as well as the nuclear transcription factor: c-fos and NFATc1. The resultssuggested that Ca-SGP descended osteoclast differentiation by down-regulating theNF-κB/MAPK/NFATc1signal pathway.In conclusion, the present study provides strong evidence that Ca-SGP possessesmarked pro-bone formation and anti-bone resorption activity at the cellular level.Furthermore, we have done lots of work to illustration of the mechanism of its effect.Researches in this thesis would provide credible theoretical and experimentalfoundation for the development of anti-osteoporosis drug and the high value ofutilization of fish eggs.