LMP-1 mRNA反义寡核苷酸抑制大鼠成骨细胞的分化

LMP-1 mRNA antisense oligonucleotide inhibited the differentiation of rat osteoblasts

为深入探讨LIM矿化蛋白 1(LMP 1)在成骨细胞分化中的作用 ,体外培养大鼠成骨细胞 ,在培养基中加入LMP 1反义寡核苷酸 (LMP 1antisense终浓度为 0 8mol L) ,以阻断大鼠成骨细胞中LMP 1mRNA的表达。对照组加nonsense(终浓度为 0 8mol L)。测定细胞的碱性磷酸酶 (ALP)活性、培养基中骨钙素 (OC)的含量 ,免疫组化分析Ⅰ型胶原蛋白的表达 ,Northernblotting检测Ⅰ型胶原mRNA的表达。结果显示 :LMP 1mRNA被LMP 1antisense阻断后 ,ALP活性降低、OC分泌减少、Ⅰ型胶原蛋白和mRNA的表达下降。上述结果表明 :LMP 1mRNA被LMP 1反义寡核苷酸阻断后 ,成骨细胞分化受到明显的抑制 ,提示LMP 1是成骨细胞分化必不可少的正调节因子。

To investigate the role of LIM mineralization protein-1(LMP-1) on osteoblastic differentiation, rat osteoblasts were cultured and LMP-1antisense( final concentration 0.8mol/L) was added into the medium to block the expression of LMP-1 mRNA. Nonsense( final concentration 0.8M) was added as a control. The activity of alkaline phosphatase (ALP) and synthesis of osteocalcin (OC) were measured. The expression of collagen type Ⅰprotein and collagen type ⅠmRNA by rat osteoblast was analyzed by immunohistochemical method and Northern blotting respectively. The results were as following: after LMP-1 mRNA was blocked by LMP-1 antisense, both the activity of ALP and synthesis of osteocalcin were reduced. The expression of collagen type Ⅰprotein and mRNA was down-regulated. It was suggested that the blocking of LMP-1 mRNA by LMP-1 antisense inhibited the differentiation of rat osteoblasts. Therefore, LMP-1 function as an essential positive regulator for osteoblastic differentiation.