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

通过向培养基中加入小鼠钙调蛋白h1的反义寡核苷酸来增强重组人BMP-2（40 ng/ml）诱导的成肌细胞向成骨细胞的分化。用胰蛋白酶消化法从大腿肌肉中制备成肌细胞。设计并合成了小鼠钙调蛋白h1序列的反义寡核苷酸（18聚体），该序列包括起始密码子。80 μg/ml的反义寡核苷酸处理48小时后，Calponin h1 mRNA的表达明显降低，碱性磷酸酶的表达显示有32.4±3.9%的细胞分化为成骨细胞。我们还证明了骨折后48小时，通过用pluronic F127凝胶局部递送反义寡聚DNA，实验性骨折的加速愈合。骨折后3周的愈合率，通过放射学和组织学检查评估，在反义寡核苷酸存在下显著增加（50.9%，n=28/55）与对照组相比（正义寡聚DNA; 31.3%，n=16/51，突变的反义寡聚DNA; 28.6%，n=4/14，仅普朗尼克凝胶; 37.5%，n=6/16）。我们进一步构建了携带人钙调蛋白h1反义cDNA的腺病毒载体，用于抑制人间充质细胞中钙调蛋白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.