重组人甲状旁腺素在骨折愈合过程中促进Osterix蛋白表达的实验研究

Intermittent low-dose administration of recombinant human parathyroid hormone （1-34） promotes the expression ofOsterix during early stage of fracture healing

目的探讨间断性低剂量应用重组人甲状旁腺素（1—34）[recombinanthumanparathyroidhormonef1—34），rhPTH（1-34）]在骨折愈合早期对Osterix蛋白表达的促进作用。方法应用2月龄雄性SD大鼠48只，建立单侧闭合性股骨骨折内固定模型。随机分为甲状旁腺素组（24只）和安慰剂组（24只），术后分别于皮下注射rhPTH（1—34）10mg/kg/d和等量生理盐水。于术后第2、7、14和21天取股骨标本，提取骨折部位骨组织RNA和蛋白质，行RT．PCR和蛋白印迹检测，评估Osterix的mRNA表达和蛋白质水平。对第7、14、21天的股骨标本行X线检查，观察骨愈合情况。结果x线片示术后第7天两组骨折愈合情况无明显差异，第14、21天甲状旁腺素组较安慰剂组有更好的骨折愈合。术后第2、7天，甲状旁腺素组骨折端Osterix的mRNA表达分别为1．87±0．5、1．90±0．5，安慰剂组分别为1．90±0．6、1．93±0．6，差异均无统计学意义；甲状旁腺素组蛋白质水平（Photoshop灰度值）分别为731．08±70、900．30±131，安慰剂组分别为729．08±68、740．34±100，差异均无统计学意义。术后第14、21天，甲状旁腺组骨折端Osterix蛋白的mRNA表达分别为5．02_±0．5、10．03±0．8，安慰剂组分别为2．30_±0．4、4．01±0．7，差异有统计学意义（P〈0．05）；甲状旁腺素组蛋白质水平分别为3164．03±131、3509．02±126，高于安慰剂组的1053．04±121、2721．03±123，差异均有统计学意义（P〈0．05）。结论骨折愈合早期应用10mg／kg／d剂量的rhPTH（1-34）可上调转录因子Osterix蛋白的基因表达，促进骨折愈合。

Objective To study the effects of intermittent low-dose administration of recombinant human parathyroid hormone （1-34） [rhPTH（1-34）] in the expression of Osterix （Osx） during early stage of fracture healing. Methods Forty-eight 2- month old male Sprague-Dawley rats were underwent close unilateral femoral fracture and intramedullary nail fixation. The sub- jeets were divided into 2 equal groups randomly： treatment group undergoing subcutaneous injection of rhPTH（1-34） 10 mg/kg/d immediately after the operation and control group undergoing subcutaneous injection of normal saline of the same dose. Six rats in each group were sacrifice at 2, 7, 14, and 21 days after operation. X-ray photography study was conducted at 7, 14 and 21 days. Tissue RNA and protein were extracted from the bone tissues of bilateral femurs and the expression levels of Osx mRNA and protein were evaluated via real time quantitative PCR and Western-blotting. Results Fracture healing was significant at 14 days after operation, and the progress of fracture healing was better in the rhPTH（1-34） group than in the control group at 14 and 21 days. The relative expression of Osx mRNA and protein in the fractured femurs of the rhPTH（1-34） group （5.02±0.5 and 10.03±0.8 for Osx mRNA, 3164.03＋131 and 3509.02±126 for protein） at 14 and 21 days after the operation were significantly higher than those of the control group （2.30±0.4 and 4.01 ±0.7 for Osx mRNA, 1053.04± 121 and 2721.03± 123 for protein）. However, there was no significant difference at 2 and 7 days after operation between the rhPTH（1-34） and control group. Conclusion Intermittent lowdose administration of rhPTH（1-34） up-regulates the expression levels of osteogenesis-specific Osx mRNA and protein in rats. It will accelerate the early phase of fracture healing process.