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

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10-12mol/L分别干预Caco-2细胞5、10、20、40和80min;10-8mol/L1,25-(OH)2-D3干预Caco-2细胞2、4、8、16和24h,溶剂对照为0.1%乙醇;10-8mol/L1,25-(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20min,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/L1,25-(OH)2-D3干预Caco-2细胞2、4、8、16和24h,CaBP-D9k mRNA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,10-8mol/L1,25-(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/LPTH可能促进Caco-2细胞CaBP-D9k mRNA瞬时(1~20min)合成增加;10-8mol/L1,25-(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。

Effect of parathyroid hormone and 1,25-(OH)2-D3 on the expression of the mRNA of calbindin D9k in Caco-2 cells

Objective To study the effect of parathyroid hormone(PTH) on the expression of the mRNA of calbindin D9k(CaBP-D9k) in Caco-2 Cells and whether PTH influences the effect of 1,25-(OH)_2-D_3 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 -8 mol/L 1,25-(OH)_2-D_3 alone. The other groups of Caco-2 cells were treated for 2,4,8,16 and 24h at the dose of 10 -8 mol/L 1,25-(OH)_2-D_3 plus at the dose of 10 -8 , 10 -9 and 10 -12 mol/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 -12 mol/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-D_3 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 -8 mol/L 1,25-(OH)_2-D_3 plus PTH were more lower than those caused by 1,25-(OH)_2-D_3 alone.Conclusion 10 -8 and 10 -12 mol/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-D_3 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-D_3 in Caco-2 cells.