微小RNA-125a-3p介导Notch1信号通路对骨质疏松性骨折大鼠愈合过程的影响  

Effects of mir-125a-3p mediated Notch1 signaling pathway on the healing process of osteoporotic fractures in rats

作　　者：张帅 张卓[2] 张韩瑜嘉 朱瑜琪[1] ZHANG Shuai;ZHANG Zhuo;ZHANGHAN Yujia;ZHU Yuqi(Department of Orthopedics,Eye Hospital,Chinese Academy of Chinese Medical Sciences,Beijing 100040,China;Department of Orthopedics,PLA General Hospital,Beijing 100853,China)

机构地区：[1]中国中医科学院眼科医院骨科,北京100040 [2]解放军总医院骨科,北京100853

出　　处：《安徽医药》2021年第3期546-552,I0004,共8页Anhui Medical and Pharmaceutical Journal

摘　　要：目的探讨微小核糖核酸(miR)-125a-3p靶向介导Notch1信号通路对Wistar大鼠骨质疏松性骨折愈合的影响。方法6月龄Wistar大鼠58只,假手术组8只(假手术组),其余50只鼠切除卵巢建立骨质疏松大鼠模型,测骨密度验证模型成立,再手术建立一侧股骨中点骨折模型,克氏针固定。去除模型不成功大鼠后分组转染,每组8只,包括:模型组、inhibitor NC组(转染抑制剂阴性对照组)、miR-125a-3p inhibitor组(转染miR-125a-3p抑制剂)、si-Notch1组(转染Notch si-RNA干扰)、miR-125a-3p inhibitor+si-Notch1组(miR-125a-3p抑制剂及Notch1 si-RNA联用组)。大鼠饲养8周后取断端组织,实时定量逆转录聚合酶链反应(qRT-PCR)检测miR-125a-3p及Notch1的表达,过生物信息学网站预测及双荧光素酶报告实验检测miR-125a-3p与Notch1的靶向作用,病理组织学及免疫组化观察、骨密度及生物力学测量共同分析各组骨折愈合情况。结果模型组较假手术组骨密度下降(P<0.05)。与假手术组相比,模型组中miR-125a-3p表达量(1.48±0.11)均上调,Notch1表达(0.75±0.02)下调(P<0.05)。miR-125a-3p mimic与野生型Notch1-WT共转染组中荧光素酶活性强度[萤火虫荧光素酶(0.58±0.02)]下降(P<0.05)。与inhibitor NC组相比,miR-125a-3p inhibitor组(0.62±0.29)及miR-125a-3p inhibitor+si-Notc1组(0.73±0.21)表达下调(P<0.05)。与inhibitor NC组相比,miR-125a-3p inhibitor组Notch1表达(0.93±0.35)提高,si-Notch1组(0.40±0.15)表达降低(P<0.05)。与si-Notch1组相比,miR-125a-3p inhibitor+si-Notc1组(0.79±0.33)Notch1表达提高(P<0.05)。转染8周后,假手术组骨小梁开始大量形成,厚度均匀,排列紧密、方向一致。模型组、inhibitor NC组骨细胞数大量减少,骨小梁较细,间隙较大且排列紊乱。miR-125a-3p inhibitor组骨小梁厚度增加,骨细胞数增多,骨质改善,与假手术组显微形态较为相似。si-Notch组骨细胞数大量缺失,骨小梁间隙极大,显微结构较模型组病理形态更明显Objective This study aims to investigate the roles of miR-125a-3p in the osteoporotic fracture recovery in Wistar rats via regulating the Notch1 signaling pathway.Methods Fifty-five 6-month old Wistar rats were included in this study.8 rats were subject⁃ed to sham-surgery group,while the remaining 50 rats were ovariectomized for osteoporotic rat model establishment.The bone mineral density was measured for model identification.The model rats were further used for fracture inducement at the midpoint of one-side fe⁃mur and fixed with K-wire.After removing the unsuccessful model rats,the rats were transfected in groups,each with 8 rats,including:Sham,Model,inhibitor NC,miR-125a-3p inhibitor,si-Notch1 and miR-125-3p+si-Notch-1 groups.The tissues from the broken ends were collected 8weeks later,and then the expression of miR-125a-3p and Notch was determined using RT-qPCR.Target relation be⁃tween miR-125a-3p and Notch1 was predicted via bio-information analysis and dual luciferase reporter gene assay.The bone recovery from fracture was detected using histopathology analysis,Immunohistochemistry,bone mineral density and biomechanics measure⁃ments.Results The BMD of model group was lower than that of sham operation group(P<0.05).Compared with sham group,the ex⁃pression of mir-125a-3p was up-regulated(1.48±0.11)and Notch1 was down regulated(0.75±0.02)in model group(P<0.05).The lu⁃ciferase activity intensity[firefly luciferase(0.58±0.02)]decreased in the co transfection group of mir-125a-3p mimic and wild-type Notch1 WT(P<0.05).Compared with the inhibitor NC group,the expression of mir-125a-3p inhibitor group(0.62±0.29)and mir-125a-3p inhibitor+Si notc1 group(0.73±0.21)were down regulated(P<0.05).Compared with the inhibitor NC group,the expression of Notch1 in mir-125a-3p inhibitor group(0.93±0.35)was increased,while that in Si Notch1 group(0.40±0.15)was decreased(P<0.05).Compared with si-notch1 group,the expression of Notch1 in mir-125a-3p inhibitor+si-notchc1 group was(0.79±0.33)(P<0.05).After 8 weeks

关 键 词：骨质疏松性骨折 骨折愈合 核糖核酸酶类 受体 Notch1 miR-125a-3p 骨密度 生物力学 大鼠 Wistar 

分 类 号：R580[医药卫生—内分泌] R683[医药卫生—内科学]