沉默微小RNA-378靶向调节骨形态发生蛋白2/母亲DPP同源物4信号通路对大鼠胫骨骨折愈合的影响实验研究  

Effect of of miR-378 silencing on tibial fracture healing by targeting BMP2/Smad4 signaling pathway in rats

作　　者：崔书伟 高小康 刘晓宁 CUI Shuwei;GAO Xiaokang;LIU Xiaoning(Third Department of Orthopedics,Handan Branch,Fengfeng General Hospital of North China Medical and Health Group,Handan 056200,China)

机构地区：[1]华北医疗健康集团峰峰总医院邯郸院区骨三科,河北邯郸056200

出　　处：《陕西医学杂志》2024年第6期748-753,共6页Shaanxi Medical Journal

基　　金：河北省医学科学研究课题(20230199)。

摘　　要：目的:探究沉默微小RNA-378(miR-378)靶向调节骨形态发生蛋白(BMP)2/母亲DPP同源物(Smad)4信号通路对大鼠胫骨骨折愈合的影响。方法:对38只大鼠建立胫骨骨折模型,另取18只正常大鼠作为假手术组。选取胫骨骨折模型造模成功大鼠中的8只作为造模组,假手术组中的8只作为对照组。实时荧光定量PCR(RT-qPCR)检测造模组胫骨骨折部位骨组织及对照组相同部位正常骨组织miR-378与BMP2、Smad4 mRNA表达。将30只胫骨骨折模型大鼠随机分为模型组、空载质粒组和miR-378沉默组,每组10只。空载质粒组、miR-378沉默组分别经尾静脉注射1μg空载质粒或miR-378小干扰RNA(siRNA)质粒;模型组和剩余10只假手术组大鼠经尾静脉注射0.9%氯化钠溶液1μg;每2天注射1次,连续干预30 d。利用X线观察各组大鼠胫骨骨折愈合情况并进行骨折愈合评分(Lane-Sandhu评分)。采用酶联免疫吸附法检测各组血清炎症因子和骨钙素(OC)、碱性磷酸酶(ALP)水平。采用HE染色观察各组骨组织形态学变化。采用RT-qPCR检测各组骨组织miR-378与BMP2、Smad4 mRNA表达。采用蛋白印迹检测各组骨组织BMP2、Smad4蛋白表达。结果:造模组骨组织miR-378表达水平高于对照组,BMP2、Smad4 mRNA表达水平低于对照组(均P<0.05)。假手术组大鼠骨组织结构清晰;模型组和空载质粒组骨折线清晰,无骨痂出现,骨组织结构严重破坏,仅有少量成骨细胞生成;miR-378沉默组骨折线模糊,出现骨痂,骨组织破坏程度得到缓解并有大量成骨细胞生成。与假手术组比较,模型组血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、OC、ALP及骨组织miR-378表达水平升高,BMP2、Smad4 mRNA和蛋白表达水平降低(均P<0.05)。与模型组和空载质粒组比较,miR-378沉默组Lane-Sandhu评分、OC、ALP水平,以及BMP2、Smad4 mRNA和蛋白表达水平升高,血清TNF-α、IL-1β及骨组织miR-378表达水平降低(均P<0.05)。结论:沉默miR-378�Objective:To investigate the effect of microRNA-378(miR-378)silencing on tibial fracture healing by targeting bone morphogenetic protein(BMP)2/maternal DPP homolog(Smad)4 signaling pathway in rats.Methods:The tibial fracture model was established in 38 rats,and 18 normal rats were selected as sham operation group.Eight rats in the tibial fracture model were selected as modeling group,and eight rats in the sham operation group were selected as control group.RT-qPCR was used to detect the expression of miR-378,BMP2 and Smad4 mRNA in bone tissue of tibial fracture site in the modeling group and normal bone tissue of the same site in the control group.Thirty tibial fracture model rats were randomly divided into model group,no-loaded plasmid group and miR-378 silencing group,with 10 rats in each group.The no-loaded plasmid group and the miR-378 silencing group were injected with 1μg no-loaded plasmid or miR-378 small interfering RNA(siRNA)plasmid through the tail vein,respectively;the model group and the remaining 10 rats in the sham operation group were injected with 1μg 0.9%sodium chloride solution through the tail vein;the rats were injected once every 2 days for 30 consecutive days.The tibial fracture healing of each group was observed by X-ray and the fracture healing score(Lane-Sandhu score)was performed;serum levels of inflammatory factors,osteocalcin(OC)and alkaline phosphatase(ALP)were detected by enzyme linked immunosorbent assay;the changes of bone morphology were observed by HE staining;the miR-378,BMP2 and Smad4 mRNA expression in bone tissues were detected by RT-qPCR;the protein expressions of BMP2 and Smad4 in bone tissues were detected by Western blot.Results:The expression level of miR-378 in bone tissue of modeling group was significantly higher than that of control group,and the mRNA expression levels of BMP2 and Smad4 were significantly lower than those of control group(all P<0.05).The bone structure of rats in sham operation group was clear;in the model group and the no-loaded plasmid group,the

关 键 词：胫骨骨折 微小RNA-378 骨形态发生蛋白2 母亲DPP同源物4 炎症因子 骨组织形态学 大鼠 

分 类 号：R683.42[医药卫生—骨科学]