Effect Of Calcium And Phosphorus On STC-1 MRNA And Protein Expression In Cow RTECs

Calcium and phosphorus metabolism diseases is one of the major diseases of dairy cow. At present, such diseases poses serious threat to dairy farms, seriously affecting the economic benefits of dairy farms. In our country and abroad, the studies of Calcium and phosphorus metabolism diseases focused on parathyroid hormone (PTH), vitamin D, calcitonin, insulin-like growth factor (IGF-1), osteocalcin (OC) and so on in recent years.Stanniocalcin(STC), is a glycoprotein hormone first discovered in bony fish, and then in the human, mouse and other mammals. It has been confirmed that STC-1 involved in regulating calcium and phosphorus homeostasis. Therefore, our experimental design focused on researching the relationship between STC-1 and calcium/phosphorus metabolism, to provide more theories of calcium and phosphorus metabolism diseases.And the experimental studies from the following three aspects.First, the culture of cows'RTECS.Do RTECS primary culture and subculture using neonatal dairy cows'renal cortex and identify RTECS by immunohistochemical methods.The cell identification results show that the cells was the cow renal tubular epithelial cells, and through the purification and subculture, we are able to get pure,stable renal tubular epithelial cells.Second, Effect of Calcium and phosphorus on STC-1 mRNA expression in cow RTECsAccording to the cell proliferation curve, added calcium and phosphorus to logarithmic growth phase cells, calcium, phosphorus factors were respective divided into control group, 100μmol/L,500μmol/L, 1000μmol/L and 5000μmol/L five different concentrations group. Extract total RNA respectively, in 6h,12h,24h,48h groups, using fluorescence quantitative PCR to detect the effect of calcium and phosphorus on STC-1 mRNA.Fluorescent quantitation PCR showed that in cow renal tubular epithelial cells,with Short-term effects of low concentrations of phosphate ions, the STC-1 mRNA expression levels were promoted. But when high concentrations phosphate treatmented, the STC-1 mRNA expression levels were significantly inhibited; with low concentration phosphorus ions for a long time treatmented, the expression of STC-1 mRNA level were inhibited.For calcium, with the high concentration of 5000μmol/L treatment, the STC-1 mRNA expression levels were inhibited; With 48h treatment,all four groups of concentration calcium inhibited the STC-1 mRNA expression. With 12h and 24h treatment, 100μmol/L,500μmol/L and 1000μmol/L concentration of calcium ions can promote the STC-1 mRNA expression levels.Third, Effect of Calcium and phosphorus on STC-1 protein expression in cow RTECsDivided into different concentrations groups like above.Extract total protein respectively, in 6h,12h,24h,48h groups, using Western bloting to detect the effect of calcium and phosphorus on STC-1 protein expression.Through Western-blot method the STC-1 protein expression results show that,With short-term treatment,low levels concentration Phosphate can promote STC-1 protein production; With higher levels concentration Phosphate treatment, the STC-1 protein expression was inhibited;48h treatment With concentration Phosphate, the STC-1 protein expression levels were inhibited in both low concentrations and high concentrations.For calcium, Different concentrations of calcium ions in 6h,12h,24h and 48h have the similar results, with the calcium ion concentration rising, STC-1 protein expression was declining,With high concentration calcium treatment,the STC-1 protein expression was significantly reduced compared with the control group.