摘要
目的 研究异补骨脂素(isopsoralen,ISO)对小鼠胫骨骨折愈合的影响及其作用机制。方法 取2月龄体质量为(20±2)g雄性C57BL/6小鼠50只,按随机数字表法分为模型(Model)组和异补骨脂素治疗(ISO)组,每组25只,ISO组灌服40 mg/kg的ISO,Model组灌服等体积生理盐水,每日1次。取服药第7、14、21、28天术侧胫骨,采用Micro-CT扫描骨折区域量化骨体积分数(bone volume/total volume,BV/TV),并通过HE染色观察骨折断端愈合及塑形情况。采用ELISA法检测服药14 d小鼠血清中骨碱性磷酸酶(bone alkaline phosphatase,BALP)和Ⅰ型前胶原氨基端原肽(procollagen Type I N-terminal peptide,PINP)骨形成指标的变化情况。采用Western blot检测服药7、14 d小鼠胫骨骨痂组织中CollagenⅠ、Runx2、BMP2、OSX和VEGF蛋白的表达含量。通过血管灌注观察服药28 d后骨折区域骨痂微血管,量化分析血管体积分数及血管直径。采用免疫组织化学染色观察服药14 d后胫骨中VEGF表达情况。结果 HE染色结果显示,ISO组的愈合进程快于Model组。Micro-CT量化结果显示,服药7 d后ISO组的BV/TV高于Model组,但差异无统计学意义;14 d后ISO组的BV/TV明显高于Model组(P<0.05);21 d后ISO组的BV/TV明显低于Model组(P<0.05);28 d后ISO组的BV/TV明显低于Model组(P<0.05)。血清骨形成生化指标显示ISO组BALP和PINP含量均显著高于Model组(P<0.05)。骨组织蛋白表达检测结果显示,ISO组的CollagenⅠ、Runx2、BMP2、OSX和VEGF表达量均明显高于Model组(P<0.05)。血管造影结果示,ISO组血管体积分数明显高于Model组(P<0.05),ISO组血管直径明显高于Model组(P<0.05)。免疫组织化学染色结果显示ISO组VEGF表达量明显高于Model组(P<0.05)。结论 异补骨脂素可以提高成骨细胞活性,提高相关骨形成蛋白的表达量,加快造血组织进入来促进骨折愈合。
Objective To determine the effect of isopsoralen(ISO) on the healing of tibia fracture in mice and explore its underlying mechanism.Methods Fifty male C57BL/6 mice(2 month old,20±2 g) were randomly divided into model group and ISO treatment group,with 25 animals in each group.From the 3rd day after modeling,the mice from the ISO group were given an intragastric gavage of 40 mg/kg ISO,once per day for 28 consecutive days,while those of the model group was given same volume of normal saline in same way.On the 7th,14th,21st,and 28th day after gavage,the tibia on the surgical side was taken,and the fracture area was quantified by bone volume/total volume(BV/TV) after micro-CT scanning.The healing and shaping of the fracture end were observed through HE staining.ELISA was used to detect the serum contents of bone alkaline phosphatase(BALP) and procollagen type I N-terminal peptide(PINP) on the 14th day of gavage.Western blotting was employed to determine the expression levels of Collagen Ⅰ,Runx2,BMP2,OSX,and VEGF in the tibial callus tissue in 7 and 14 d after gavage.Vascular perfusion was applied to observe the callus microvessels in 28 d to quantitatively analyze the vascular volume fraction and vessel diameter.Immunohistochemical staining was conducted to observe the expression of VEGF in the callus in 14 d after gavage.Results HE staining displayed that the ISO group had faster healing process than the model group.Micro-CT quantification results showed that the ISO group had higher BV/TV ratio in 7 d after gavage though no statistical difference,significantly higher ratio in 14 d(P<0.05),but obviously lower ratio in 21 and 28 d after gavage(both P<0.05) when compared with the model group.The serum contents of BALP and PINP were also remarkably higher in the ISO group than the model group(P<0.05).Western blotting results indicated that the expression levels of Collagen Ⅰ,Runx2,BMP2,OSX and VEGF in the ISO group were higher than those in the model group(P<0.05).The results of angiography revealed that the vascular volume fraction and vessel diameter were notably increased in the ISO group than the model group(both P<0.05).Immunohistochemical assay showed that the expression of VEGF was higher in the ISO group than the model group(P<0.05).Conclusion ISO can improve the activity of osteoblasts,increase the expression of osteogenesis-related proteins,and accelerate the angiogenesis to promote fracture healing.
作者
吴西
王中琪
高玉海
魏振龙
柏鑫
陈克明
WU Xi;WANG Zhongqi;GAO Yuhai;WEI Zhenlong;BAI Xin;CHEN Keming(Clinical College of Traditional Chinese Medicine,Gansu University of Traditional Chinese Medicine,Lanzhou,Gansu Province,730030,China;Basic Medical Laboratory,No.940 Hospital of PLA Joint Logistic Support Force,Lanzhou,Gansu Province,730050,China;Gansu Provincial Key Laboratory of Stem Cells and Gene Drug,Lanzhou,Gansu Province,730050,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2024年第3期240-248,共9页
Journal of Army Medical University
基金
国家自然科学基金面上项目(81770879)
军队后勤科研计划项目(CLB18J028)
甘肃省青年科技基金计划项目(22JR5RA020)。
关键词
异补骨脂素
骨折愈合
骨重建
血管造影
isopsoralen
bone fracture healing
bone remodeling
angiography
