Research On The Effect Of Salmon Calcitonin Transformed Yeast To Osteoporosis And Arterial Calcification

Calcitonin is a 32-amino-acid-long peptide . Salmon calcitonin (sCT) is used to treat osteoporosis by injection or nasal administration, and the high price is the other limitation of sCT. To study the oral delivery of salmon calcitonin and to low the price of it, yeast yAGA2-sCT will be the best choice.To observe the therapeutic effect of yAGA2-sCT on osteoporosis in rat, at first we established a rat model of osteoporosis. Then we determined bone mineral density(BMD) and examined calcium(Ca),alkaline phosphatase(AKP),bone gla protein(BGP), insulin-like growth factor-1(IGF-1) in serum and Ca, hydroxyproline(HOP) in urine. A rat osteoporosis model induced by ovariectomy was established successfully, which was verified by X-ray, BMD and histological examination. When sCT and yAGA2-sCT were added, both the BMD and biochemical markers of bone metabolism improved significantly. There is no difference between the two groups of sCT and yAGA2-sCT. The result suggest that the bone resorption and bone loss of osteoporosis in rat can be prevented by sCT and yAGA2-sCT.To study the mechanism of yAGA2-sCT to osteoporosis, the osteoblasts of newborn SD rats were obtained through the enzymatic digestion- consecutive tissue method. sCT and yAGA2-sCT were added into the culture medium, and the proliferation, differentiation and mineralization of the osteoblasts were observed. The result showed us that the in vitro osteoblasts were successfully cultured , which was verified histologically. From the result we can conclud that both sCT and yAGA2-sCT can stimulate formation of bone through affecting the proliferation , differentiation and mineralization of osteoblasts in vitro . Osteoporosis is usually occur concomitantly with arterial calcification in elderly. Recent studies find that OPG/RANKL/RANK, newly defined members of TNF super-family of receptors, participate in regulation of osteoporosis and arterial calcification, and are the connection of these two disease process.To study the regulatory role of OPG/RANKL/RANK system in arterial calcification and the effect of calcitonin on OPG/RANKL/RANK system, at first we established a rat model of arterial calcification. Then we examined calcium and alkaline phosphatase (AKP) level in arterial tissue and determined the expression of OPG/RANKL in arterial tissue by immunohistochemistry. A rat arterial calcification model induced by Vitamin D3 was established successfully, which was verified by examining the arteries histologically. The calcium and alkaline phosphatase (AKP) level in arterial tissue of the rat model were higher than that of control group. The expression of OPG was prominently lower and RANKL was prominently higher in the arterial tissue of rat model than that of control. When sCT and yAGA2-sCT were added, the OPG expression was markedly increased and RANKL level was obviously decreased in arterial tissue of the rat than that of rat model without calcitonin. The result suggest that OPG/RANKL participates the regulation of arterial calcification, sCT and yAGA2-sCT effectively inhibit arterial calcification through OPG/RANKL/RANK system.To investigate the regulation effect of RANKL on in vitro calcification of rat aortic vascular smooth muscle cells (VSMC) and the role of calcitonin in this effect,we cultured rat aortic VSMC in vitro and established a calcification model with the cells. RANKL and CT were added into the culture media, then the calcium deposition, alkaline phosphatase (AKP) activity, and alterations of expression of osteocalcin were examined. Von Kossa staining VSMC demonstrate the in vitro calcification model was successfully established. The Calcium content, AKP activity and the expression of osteocalcin were markedly increased in calcification, RANKL , sCT and yAGA2-sCT group than that of control group. But they were obviously decreased in sCT and yAGA2-sCT group than that of RANKL group, which suggest that RANKL increase the calcium deposition, AKP activity, and expression of osteocalcin in VSMC, whereas sCT and yAGA2-sCT inhibit this effect. RANKL increase calcium deposition in VSMC, possibly through increasing AKP activity and expression of osteocalcium and thus promote the transformation of VSMC to osteoblast. sCT and yAGA2-sCT may inhibit VSMC calcification through OPG/RANKL/RANK system.