甲状旁腺素、维生素D_3对Caco-2细胞钙结合蛋白D9k mRNA表达的影响

Effect of parathyroid hormone and 1,25-(OH)_2-D_3 on the expression of the mRNA of calbindin D9k in Caco-2 cells

目的 探讨甲状旁腺素（PTH）对小肠细胞钙结合蛋白（CaBP）D9k mRNA表达的影响以及探讨PTH是否影响1，25-（OH）2-D3，对Caco-2细胞钙结合蛋白D9k mRNA表达的促进作用。方法 以人结肠癌上皮细胞株caco-2细胞作为小肠细胞体外模型，PTH分三个剂量：10^-8、10^9和10^-12。mol／L分别干预Caco-2细胞5、10、20、40和80min；10^-8mol/L 1，25-（OH）2，-D3，干预Caco-2细胞2、4、8、16和24h，溶剂时照为0．1％乙醇；10^-8mol／L 1，25-（OH）2，-D3，联合以上三剂量PTH干预Caco-2细胞2、4、8、16和24h，溶剂对照亦为0．1％乙醇。运用RT-PCR方法 进行CaBP-D9k mRNA测定，以GAPDH作为内对照。结果 10^-8mol／LPTH作用20rain，10^12mol／L作用10min，CaBP-D9k mRNA表达量高于空白组，其余时间点低于空白组；10^-8mol／L 1，25-（OH）2．D，干预Caco-2细胞2、4、8、16和24h，CaBP．D9kmRNA的表达均高于溶剂对照（0、1％乙醇）；与10^-8mol／L1，25．（OH）2-D3单独作用比较，10^-8mo/L 1，25-（OH）2-D3，联合以上三剂量PTH作用，CaBP-D9k mRNA相对表达量均低于单独作用的表达量。结论 10^-8mol／L、10^-12mol/L PTH可能促进Caco-2细胞CaBP-D9k mRNA瞬时（1～20min）合成增加；10^-8mol／L 1，25-（OH）2，-D3，可明显诱导Caco-2细胞CaBP-D9k mRNA的表达；PTH可能抵消或者抑制1，25-（OH）2，-D3，促进Caco-2细胞CaBP-D9k mRNA表达的作用。

To study the effect of parathyroid hormone（PTH） on the expression of the mRNA of calbindin D9k（CaBP-Dgk） in Caco-2 Cells and whether PTH influences the effect of 1,25-（OH）2-D3 on the expression of calbindin mRNA in Caco-2 Cells. Methods Caco-2 cells were used as the model of human small intestinal enterocytes. Caco-2 cells were treated 5, 10,20,40 and 80min at the dose of 10^-8, 10^-9 and 10^-12 mol/L PTH, another groups of Caco-2 cells were treated for 2,4,8,16 and 24h at the dose of 10^-8mol/L 1,25-（OH）2-D3 alone. The other groups of Caco-2 cells were treated for 2,4,8,16 and 24h at the dose of 10^-8mol/L 1,25-（OH）2-D3 plus at the dose of 10^-8, 10^-9 and 10^-12mol/L of PTH respectively. 0.1% ethanol solutions were used as the vehicle for the later two groups. The expression of calbindin D9k mRNA was determined by RT-PCR （the level of GAPDH was taken as internal control）.Results The expressions of calbindin D9k mRNA were more higher than those of blank controls when Caco-2 cells were treated at the dose of 10^-8 mol/L PTH for 20min and at the dose of 10^-12mol/L PTH for 10min, and were more lower than those of blank controls at other treating points Caco-2 cells were administrated at the dose of 10^-8 mol/L 1,25-（OH）2-D3 for 2,4,8,16 and 24h, the expressions of CaBP-D9k mRNA were higher than those of vehicle. The increases of CaBP-D9k mRNA levels caused by 10^-8mol/L 1,25-（OH）2-D3 plus PTH were more lower than those caused by 1,25-（OH）2-D3 alone. Conclusion 10^-8 and 10^-12mol/L PTH probably caused increase on the expression of CaBP-D9k mRNA in short period（ 1-20min）. 10^-8 mol/L 1,25-（OH）2-D3 increases CaBP-D9k mRNA levels in Caco-2 cells. PTH may inhibit or counteract the increase in CaBP-D9k mRNA caused by 1,25-（OH）2-D3 in Caco-2 cells.