过表达miR-378a修饰骨髓间充质干细胞复合胶原蛋白海绵支架对大鼠股骨缺损的修复作用  

Repair effect of bone marrow mesenchymal stem cells modified by overexpressed miR-378a combined with collagen sponge scaffold for femoral defect in rats

作　　者：孙皓 蒲胤瑄 刘佳林 吾凡别克・巴合提 都曼别克・阿曼台 韩祥祯 何惠宇[1,2] Sun Hao;Pu Yin-Xuan;Liu Jia-Lin;Wufanbieke Baheti;Dumanbieke Amantai;Han Xiang-Zhen;He Hui-Yu(Department of Dental Restoration and Implantation,the First Affiliated Hospital of Xinjiang Medical University(Affiliated Stomatological Hospital),Urumqi,Xinjiang 830054,China;Institute of Stomatology of Xinjiang Uygur Autonomous Region,Urumqi,Xinjiang 830054,China;Department of Oral Implantology,Affiliated Stomatological Hospital of Southwest Medical University,Luzhou,Sichuan 646000,China;Department of Stomatology,the People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi,Xinjiang 830001,China)

机构地区：[1]新疆医科大学第一附属医院(附属口腔医院)口腔修复种植科,新疆乌鲁木齐830054 [2]新疆维吾尔自治区口腔医学研究所,新疆乌鲁木齐830054 [3]西南医科大学附属口腔医院口腔种植科,四川泸州646000 [4]新疆维吾尔自治区人民医院口腔科,新疆乌鲁木齐830001

出　　处：《解放军医学杂志》2023年第2期175-182,共8页Medical Journal of Chinese People's Liberation Army

基　　金：新疆医科大学研究生创新创业项目(CXCY2022001);新疆维吾尔自治区自然科学基金计划青年项目(2021D01C337)。

摘　　要：目的 探究过表达miR-378a修饰骨髓间充质干细胞(BMMSCs)复合胶原蛋白海绵(CS)支架修复大鼠股骨缺损的效果。方法 分离培养BMMSCs并采用流式细胞术鉴定细胞表型。使用过表达miR-378a的慢病毒及阴性空载慢病毒转染BMMSCs,将转染细胞分为3组:过表达miR-378a转染BMMSCs组(LV-miR-378a组)、阴性对照慢病毒转染BMMSCs组(LV-miR-NC组)、未转染BMMSCs组(对照组),并采用流式细胞术检测转染效率。制备转染细胞与CS支架的复合物,使用扫描电镜观察细胞与支架的相容性。建立SD大鼠股骨缺损回植模型,将15只SD大鼠随机分为3组(n=5):LV-miR-378a+CS组(植入过表达miR-378a慢病毒的BMMSCs复合CS支架)、LV-miR-NC+CS组(植入阴性空载慢病毒转染的BMMSCs复合CS支架)、CS组(只植入CS支架)。术后第8周,使用CO_(2)吸入法处死SD大鼠,取手术侧股骨样本进行大体观察、微型CT扫描重建,定量分析骨密度(BMD)及骨体积分数(BV/TV),并采用HE、Masson及骨桥蛋白(OPN)免疫组化染色观察骨修复情况。结果 流式细胞术检测BMMSCs细胞表型显示,细胞表面抗原CD44阳性表达率95.5%,CD29阳性表达率94.7%,而CD45阳性表达率0.8%,CD34阳性表达率0.7%。流式细胞术检测各组转染效率显示,与对照组比较,LV-miR-378a组和LV-miR-NC组转染效率明显增高(P<0.05),而LV-miR-378a组与LV-miR-NC组比较差异无统计学意义(P>0.05)。扫描电镜观察显示,CS支架材料具有良好的三维空洞结构,两组细胞与CS支架共培养7 d后,LV-miR-378a组较LV-miR-NC组细胞在支架材料表面铺展区域更大,细胞呈团簇状生长,伸出的伪足更多。大体观察结果显示,术后8周,LV-miR-378a+CS组骨缺损区修复较为完全,LV-miR-NC+CS组及CS组骨缺损中心均可观察到未完全矿化的新生骨质,且CS组仍可见缺损区大致轮廓。微型CT三维重建及定量分析结果显示,术后8周,LV-miR-378a+CS组骨缺损区修复效果优于其余两组,新骨沉积量较多,Objective To explore the effect of miR-378a overexpression in modifying bone marrow mesenchymal stem cells(BMMSCs) combined with collagen sponge(CS) scaffold in repairing femoral defects of rats. Methods The BMMSCs were isolated and cultured, and the cell phenotype was identified by flow cytometry. The BMMSCs were transfected with an overexpressed miR-378a lentivirus and a negative unloaded lentivirus, and the transfected cells were divided into three groups: a BMMSCs transfected with overexpressed miR-378a group(LV-miR-378a group), BMMSCs transfected with negative control lentivirus group(LV-miR-NC group) and an untransfected BMMSCs group(control group). The transfection efficiency was detected by flow cytometry. The composite of transfected cells with CS scaffold was prepared, and the compatibility of the cells with the scaffold was observed by scanning electron microscopy. A femoral defect replantation model was established in SD rats, and 15 SD rats were randomly divided into three groups(n=5): LV-miR-378a+CS group(with the lentivirus complex CS scaffold overexpressing miR-378a implanted), LV-miR-NC+CS group(with the BMMSCs complex CS scaffold transfected with negative non-load lentivirus),and CS group(with the CS scaffold implanted only). At the 8th week after operation, the SD rats were sacrificed by CO2 inhalation.The femur on the operation side was taken for gross observation and micro-CT scanning reconstruction. Bone mineral density(BMD) and bone volume/total volume(BV/TV) were quantified, and HE, Masson and osteopontin(OPN) immunohistochemical staining were used to observe bone repair. Results The phenotyping results of BMMSCs by flow cytometry showed that the positive expression rate of cell surface antigen CD44 and CD29 were 95.5% and 94.7%, respectively, while the positive expression rate of CD45 and CD34 were 0.8% and 0.7%, respectively. Transfection efficiency detected by flow cytometry: compared with the control group, the transfection efficiency of LV-miR-378a group and LV-miR-NC group increased

关 键 词：微小RNA miR-378 骨髓间充质干细胞 成骨分化 

分 类 号：R318[医药卫生—生物医学工程] R459.9[医药卫生—基础医学]