Effects Of Ipriflavone And1,25-（OH）₂D₃on Cell Proliferation And The Expression Of Calcium Channel Trpv6of Osteoblaet In Chicken Emtryonic Tibia

The epithelial Ca2+channel TRPV5/TRPV6performance to have sensitive selectivity to the calcium ion, thus it has the very important effect on the calcium ion metabolizes and the bone metabolism. Rescent studies have shown that estrogen,1,25dihydroxyvitamin D3, PTH, diet calcium and drugs can regulate the expression of TRPV5/TRPV6. The previous study showed TRPV5mainly existed in kidney, the TRPV6mainly existed in small intestines. TRPV5/TRPV6has been tested in the osteoblast of bone tissue. The foreign research has already proven to make exchange the calcium ion to keep the extracellular level of calcium. Researchers have observed the expression of TRPV5and TRPV6in osteoblasts which come from the differentiation of BMSCs. The study on the expression of TRPV5and TRPV6in the cultured neonatal rat calvarial osteoblasts and the role of the expression of TRPV5and TRPV6in mechanism of post-menopausal osteoporosis has yet been reported. But the study on the expression of TRPV6in the cultured osteoblasts from tibia of embryonic chicken and the role of the expression of TRPV6in mechanism of post-menopausal osteoporosis has not been reported.The osteoblasts from tibia of embryonic chicken were the best culture model in the research of the ability of osteoblastic bone formation. We isolated osteoblasts from embryonic chicken’s tibia with enzyme digestion and tissular pieces migratory growth methods. The cells were assayed with methyl thiazolyl tetrazolium (MTT) and identified with invert microscope. Alkaline phosphorase (ALP) stain, stain of calcified nodules, and Giemsa stain were also performed to explore biological characteristics of these cultures. The results showed that the embryonic chicken’s tibia osteoblasts had the classic morphological feature, ALP activity, the function as in vivo. They were consistent with the growth characteristics of cells, and had the ability of calcification in vitro. The experiment is to study the possible mechanism of Ipriflavone and1,25-(OH)2D3treatment to osteoblast in chicken embryos tibia in vitro. In the present study, the osteoblasts were cultured and subcultured to observe the proliferation, ALP activity, the expression of TRPV6, PMCAlb, ALP and the calcium ion content after exposed to Ipriflavone and1,25-(OH)2D3. Companied with the increase of Ipriflavone concentration and1,25-(OH)2D3concentration, the proliferation of osteoblast increased significantly(P<0.05), but when the concentration each reached10-4mol·L-1and10-8mol·L-1, the proliferation of osteoblast decreased. After Ipriflavone and1,25-(OH)2D3treatment to osteoblast, ALP secretion increased significantly(P<0.05). The level of TRPV6mRNA expression increased significantly at the concentration of10-8mol·L-1(P<0.01), and it increased significantly at the concentration of10-10mol·L-1and10-6mol·L-1(P<0.05), and the level of TRPV6mRNA expression decreased significantly at the concentration of10-10mol·L-1and10－9mol·L-1(P<0.01). The level of PMCAlb mRNA expression decreased, it decreased significantly at the concentration of10－6mol·L-1(P＜0.01) and decreased significantly at the concentration of10-10mol·L-1and10－8mol·L-1(P<0.05). But the level of PMCAlb mRNA expression had no regular trend, it increased significantly at the concentration of10-11mol·L-1(P<0.01) and decreased significantly at the concentration of10-12mol·L-1and10-10mol·L-1(P<0.01). The calcium ion content of osteoblast increased significantly at the concentration of10-8mol·L-1(P<0.01), and it increased significantly at the concentration of10-10mol·L-1and10-6mol·L-1(P<0.05), and1,25-(OH)2D3could affect the calcium ion content of osteoblast(p<0.05). The results showed that Ipriflavone and1,25-(OH)2D3could affect the proliferation, ALP activity, was correlation with the level of TRPV6mRNA, PMCAlb mRNA, ALP mRNA expression, and also affect the calcium ion content.