1 Department of Orthopedics, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China .
2 Department of Orthopedics, Navy General Hospital, Chinese People's Liberation Army, Beijing, China .
Hum Gene Ther. 2018 Aug;29(8):902-915. doi: 10.1089/hum.2018.013. Epub 2018 May 9.
A number of previous studies have indicated that the genetic variation at the collage type I alpha 1 (COLIA1) gene locus influences susceptibility to osteoporosis. However, seldom have studies reported the effect of gene delivery using an adenovirus vector carrying human recombinant COLIA1 cDNA on stimulating osteogenic activity of osteoblasts and enhancing fracture healing of ovariectomized rats. The current study was performed to demonstrate whether direct gene delivery using an adenovirus vector carrying human recombinant COLIA1 cDNA could stimulate osteogenic activity of osteoblast in vitro and enhance fracture healing of ovariectomized rats in vivo. In vitro, the tet-on system regulated COLIA1 gene adenovirus was constructed and transfected to osteoblasts. COLIA1 mRNA and collagen type I levels were assessed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay to determine whether adenovirus transfected successfully. Osteogenic activity of the osteoblasts was assessed by alkaline phosphatase activity, immunohistochemical staining, immunofluorescent staining, mineralized matrix formation, and extracellular calcium levels. In vivo, adenovirus-delivered COLIA1 gene was injected into the fracture site of the tibia in an ovariectomized rat model of osteoporosis, and bone callus condition was assessed to determine whether the COLIA1 gene could accelerate osteoporotic fracture healing. In vitro, the results showed that COLIA1 gene adenovirus transfection could increase osteoblast COLIA1 gene expression and collagen type I protein synthesis, increase alkaline phosphatase activity, and stimulate calcium nodules formation, which exhibited a direct osteogenic effect on the osteoblasts. In vivo, local injection of COLIA1 gene adenovirus increased collagen type I expression, restored bone mineral density, and accelerated fracture healing in ovariectomized rats, without increasing serum collagen type I and liver COLIA1 mRNA levels. This study suggests direct gene delivery using an adenovirus carrying human COLIA1 cDNA can stimulate the osteogenic activity of osteoblasts in vitro and enhance bone fracture healing in vivo. The tet-on system is an ideal gene regulatory system for effective and safe regulation of the therapeutic gene.
先前的一些研究表明，I 型胶原α 1 （COLIA1）基因座的遗传变异影响骨质疏松症的易感性。然而，很少有研究报道使用携带人重组 COLIA1 cDNA 的腺病毒载体进行基因传递对刺激成骨细胞的成骨活性和增强去卵巢大鼠骨折愈合的影响。本研究旨在证明使用携带人重组 COLIA1 cDNA 的腺病毒载体直接进行基因传递是否可以体外刺激成骨细胞的成骨活性，并增强去卵巢大鼠体内骨折的愈合。体外，构建并转染 COLIA1 基因腺病毒 tet-on 系统，通过逆转录聚合酶链反应和酶联免疫吸附试验评估 COLIA1 mRNA 和 I 型胶原水平，以确定腺病毒是否转染成功。通过碱性磷酸酶活性、免疫组织化学染色、免疫荧光染色、矿化基质形成和细胞外钙水平评估成骨细胞的成骨活性。体内，将腺病毒递送的 COLIA1 基因注射到骨质疏松症去卵巢大鼠模型的骨折部位，评估骨痂情况，以确定 COLIA1 基因是否可以加速骨质疏松性骨折愈合。体外结果表明，COLIA1 基因腺病毒转染可以增加成骨细胞 COLIA1 基因表达和 I 型胶原蛋白合成，增加碱性磷酸酶活性，刺激钙结节形成，对成骨细胞具有直接的成骨作用。体内，局部注射 COLIA1 基因腺病毒增加了 I 型胶原的表达，恢复了骨密度，加速了去卵巢大鼠的骨折愈合，而不增加血清 I 型胶原和肝 COLIA1 mRNA 水平。本研究表明，使用携带人 COLIA1 cDNA 的腺病毒进行直接基因传递可以体外刺激成骨细胞的成骨活性，并增强体内骨骨折的愈合。tet-on 系统是一种理想的基因调控系统，可有效、安全地调节治疗基因。
