Strohbach Cassandra A, Rundle Charles H, Wergedal Jon E, Chen Shin-Tai, Linkhart Thomas A, Lau K-H William, Strong Donna D
Musculoskeletal Disease Center, Jerry L. Pettis Memorial VA Medical Center, 11201 Benton Street, Loma Linda, CA 92357, USA.
Calcif Tissue Int. 2008 Sep;83(3):202-11. doi: 10.1007/s00223-008-9163-0. Epub 2008 Aug 16.
LIM mineralization protein-1 (LMP-1) is a novel intracellular osteogenic factor associated with bone development that has been implicated in the bone morphogenetic protein (BMP) pathway. This preliminary study evaluated the possibility of LMP-1-based retroviral gene therapy to stimulate osteoblast differentiation in vitro and fracture repair in vivo. A Moloney leukemia virus (MLV)-based retroviral vector to express LMP-1 with a hemagglutinin (HA) tag was developed, and its effects were evaluated on MC3T3-E1 cell differentiation and in the rat femur fracture model. MC3T3-E1 osteoblasts transduced with the MLV-HA-LMP-1 vector demonstrated significantly increased osteoblast marker gene expression (P < 0.05) and mineral deposition compared to control transduced cells. Femoral midshaft fractures were produced in Fischer 344 rats by the three-point bending technique. The MLV-HA-LMP-1 or control vector was applied at the fracture site through percutaneous injections 1 day postfracture. Analysis of fracture healing of 10 MLV-HA-LMP-1-treated and 10 control MLV-beta-galactosidase (beta-gal)-treated animals was completed at 3 weeks by X-ray, peripheral quantitative computed tomography, and histology. MLV-HA-LMP-1-treated animals had 63% more bone mineral content at the fracture site (P < 0.01), 34% greater total hard callus area (P < 0.05), and 45% less cartilage in the fracture callus (P < 0.05) compared to MLV-beta-gal-treated animals. There was no effect of LMP-1 treatment on the density of the hard callus. Immunohistochemistry revealed expression of the LMP-1 transgene in the fracture callus at 21 days postfracture. Immunohistochemistry also revealed that LMP-1 transgene expression did not result in an increase in BMP-4 expression in the fracture callus. Compared to MLV-BMP-4 gene therapy studies, MLV-HA-LMP-1 gene therapy improved bony union of the fracture gap to a greater extent and did not cause heterotopic bone formation. This suggests that LMP-1 may be a better potential candidate for gene therapy for fracture repair than BMP-4. These exciting, albeit preliminary, findings indicate that LMP-1-based gene therapy may potentially be a simple and effective means to enhance fracture repair that warrants further investigation.
LIM矿化蛋白-1（LMP-1）是一种与骨发育相关的新型细胞内成骨因子，参与骨形态发生蛋白（BMP）信号通路。本初步研究评估了基于LMP-1的逆转录病毒基因疗法在体外刺激成骨细胞分化及体内促进骨折修复的可能性。构建了一种基于莫洛尼白血病病毒（MLV）的逆转录病毒载体，用于表达带有血凝素（HA）标签的LMP-1，并评估其对MC3T3-E1细胞分化及大鼠股骨骨折模型的影响。与对照转导细胞相比，用MLV-HA-LMP-1载体转导的MC3T3-E1成骨细胞显示成骨细胞标志物基因表达显著增加（P<0.05），矿物质沉积也增加。采用三点弯曲技术在Fischer 344大鼠中制造股骨中段骨折。骨折后1天，通过经皮注射将MLV-HA-LMP-1或对照载体应用于骨折部位。在3周时，通过X线、外周定量计算机断层扫描和组织学对10只接受MLV-HA-LMP-1治疗和10只接受对照MLV-β-半乳糖苷酶（β-gal）治疗的动物的骨折愈合情况进行分析。与接受MLV-β-gal治疗的动物相比，接受MLV-HA-LMP-1治疗的动物骨折部位的骨矿物质含量多63%（P<0.01），总硬骨痂面积大34%（P<0.05），骨折痂中的软骨少45%（P<0.05）。LMP-1治疗对硬骨痂密度无影响。免疫组织化学显示骨折后21天骨折痂中有LMP-1转基因表达。免疫组织化学还显示LMP-1转基因表达未导致骨折痂中BMP-4表达增加。与MLV-BMP-4基因治疗研究相比，MLV-HA-LMP-1基因治疗在更大程度上改善了骨折间隙的骨愈合，且未引起异位骨形成。这表明与BMP-4相比，LMP-1可能是骨折修复基因治疗更好的潜在候选者。这些令人兴奋的初步研究结果表明，基于LMP-1的基因治疗可能是一种简单有效的促进骨折修复的方法，值得进一步研究。
