Development of a novel bone formation therapy by inhibition of the calponin gene expression

重组人BMP-2 （40 ng/ml）诱导成肌细胞向成骨细胞分化的过程中，加入小鼠钙钙蛋白h1反义寡odna。用胰蛋白酶消化法制备成肌细胞。设计合成了含起始密码子的小鼠钙钙蛋白h1序列的磷酸硫代修饰反义寡核苷酸（18 mers）。80 μg/ml的反义寡核苷酸（oligoDNA）处理48小时后，calponin h1 mRNA的表达减少，碱性磷酸酶的表达表明，32.4±3.9%的细胞分化为成骨系细胞。我们还证明了在骨折后48小时用pluronic F127凝胶局部递送反义寡核苷酸可以加速实验性骨折的愈合。骨折后3周的愈合率，通过x线和组织学检查，反义寡核苷酸存在组（50.9%,n=28/55）明显高于对照组（反义寡核苷酸；31.3%,n=16/51，突变的反义寡核苷酸；28.6%,n=4/14，纯pluronic gel； 37.5%, n=6/16）。我们进一步构建了含有人钙钙蛋白h1反义cDNA的腺病毒载体，用于抑制人间充质细胞钙钙蛋白mRNA的表达。

Myoblast differentiation into osteoblast induced by recombinant human BMP-2 (40 ng/ml) was enhanced by addition of anti-sense oligoDNA of mouse calponin h1 into the culture medium. Myoblast was prepared from the thigh muscle by trypsin digestion. Phosphothioate-modified anti-sense oligoDNA (18 mers) to the mouse calponin h1 sequence including the initiation codon was designed and synthesized. Treatment with the anti-sense oligoDNA but not with control oligoDNA at the concentrations of 80 μg/ml for 48 hr reduced the calponin h1 mRNA expression and 32.4±3.9% of the cells were differentiated into the osteogenic-lineage cells as demonstrated by the expression of alkaline phosphatase. We also demonstrated the accelerated healing of experimental bone fracture by local delivery of the anti-sense oligoDNA with the pluronic F127 gel at 48 h after bone fracture. The rate of union three weeks after fracture, as assessed by radiographic and histological examination, was significantly increased in the presence of the anti-sense oligoDNA (50.9%, n=28/55) as compared with control groups (sense oligoDNA ; 31.3%, n=16/51, mutated anti-sense oligoDNA ; 28.6%, n=4/14, pluronic gel only ; 37.5%, n=6/16). We further constructed adenovirus vector harboring anti-sense cDNA of human calponin h1 for inhibition of calponin mRNA expression in human mesenchymal cells.