Coxsackievirus and adenovirus receptor (CAR)-positive immature osteoblasts as targets of adenovirus-mediated gene transfer for fracture healing
Coxsackievirus and adenovirus receptor (CAR)-positive immature osteoblasts as targets of adenovirus-mediated gene transfer for fracture healing
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DOI：
10.1038/sj.gt.3302060
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发表时间：
2003-09-01
期刊：
影响因子：
5.1
通讯作者：
Endo, N
中科院分区：
文献类型：
--
作者：
Ito, T;Tokunaga, K;Endo, N
腺病毒载体有望成为基因治疗严重骨折的有力工具。腺病毒通过与细胞膜上的柯萨奇病毒和腺病毒受体（CAR）结合来侵入细胞。CAR在正常成年动物中表达较低，但在一些实验模型中，它在再生细胞上被诱导。建立小鼠肋骨骨折模型，分别于骨折后1、5、10、14、21 d采用RT-PCR方法检测组织学变化及CARmRNA表达。CAR mRNA在每个时间点仅在骨折肋骨中表达，而在正常肋骨中不表达。我们在骨折后第10天和第14天的骨折骨痂中的成纤维细胞样细胞中检测到CAR蛋白。原位杂交结果显示，这些成纤维细胞样细胞表达I型胶原和骨桥蛋白的mRNA，但不表达骨钙素，将这些细胞定义为未成熟的成骨细胞。然后，我们在第14天将小剂量（10（4）-10（8）PFU）的表达lacZ的腺病毒载体转移到未成熟的成骨细胞中。β-半乳糖苷酶仅在每种剂量的未成熟成骨细胞上检测到。未成熟的成骨细胞在骨折愈合的基质替代步骤中起重要作用。CAR介导的基因转移到未成熟的成骨细胞可以合理的腺病毒介导的治疗骨折愈合。
Adenovirus vectors are expected to be a powerful tool for gene therapy to treat severe fractures. Adenovirus invades cells through binding to the coxsackievirus and adenovirus receptor (CAR) on the cell membrane. CAR expression is low in normal adult animals, but it is induced on regenerating cells in some experimental models. We made a rib fracture model in mice and evaluated the histological changes and CAR mRNA expression by RT-PCR 1, 5,10,14, and 21 days after the fracture. CAR mRNA was expressed exclusively in the fractured ribs at each time point, but not in the normal ribs. We detected the CAR protein immunohistochemically in fibroblast-like cells in the fracture callus on days 10 and 14 after fracture. In situ hybridization showed that these fibroblast-like cells expressed mRNA of type I collagen and osteppontin, but not osteocalcin, defining the cells as immature osteoblasts. We then transferred small doses (10(4)-10(8) PFU) of lacZ-expressing adenovirus vector into immature osteoblasts on day 14. beta-galactosidase was detected only on the immature osteoblasts at every dose. Immature osteoblasts play an important role in the matrix replacement step in fracture healing. CAR-mediated gene transfer into immature osteoblasts can be reasonable for adenovirus-mediated treatment of fracture healing.