Runx2基因诱导人羊膜MSCs体外向韧带成纤维细胞定向分化及促进兔腱-骨愈合的研究  

Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits

作　　者：谢淘 仲鹤鹤 金瑛 刘修齐 陈方 向宽 吴术红 XIE Tao;ZHONG Hehe;JIN Ying;LIU Xiuqi;CHEN Fang;XIANG Kuan;WU Shuhong(Department of Orthopedics,Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou,563000,P.R.China)

机构地区：[1]遵义医科大学附属医院骨科,贵州遵义563000

出　　处：《中国修复重建外科杂志》2023年第12期1523-1532,共10页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：遵义市科技计划项目(2020-216);贵州省卫生健康委科学技术基金项目(gzwkj2021-257)。

摘　　要：目的探讨Runx2基因是否具有体外诱导人羊膜MSCs(human amniotic MSCs,hAMSCs)向韧带成纤维细胞分化以及在体内能否促进兔前交叉韧带重建后腱-骨愈合。方法取健康产妇自愿捐赠胎盘分离培养hAMSCs并传代后行流式细胞鉴定。构建携带Runx2基因的腺病毒载体(Ad-Runx2)以及空质粒载体腺病毒(Ad-NC),经病毒滴度测定后分别转染第3代hAMSCs(Ad-Runx2组、Ad-NC组),实时荧光定量PCR及Western blot检测Runx2基因及蛋白表达,验证Ad-Runx2基因转染hAMSCs有效性;然后于转染后培养3、7 d时,进一步采用实时荧光定量PCR检测韧带成纤维细胞相关基因[VEGF、Ⅰ型胶原、纤连蛋白(Fibronectin)和肌腱蛋白C(Tenascin-C)]表达;以单纯hAMSCs作为空白对照组。将单纯hAMSCs以及转染Ad-NC、Ad-Runx2的hAMSCs分别与基质胶(Matrigel)按照体积比1∶1和1∶2混合构建复合物,采用细胞计数试剂盒8(cell counting kit 8,CCK-8)检测细胞增殖,取增殖较好的对应复合物进行后续动物实验。将12只新西兰白兔随机分为假手术组(Sham组)、前交叉韧带重建组(ACLR组)、前交叉韧带重建+Ad-NC组(Ad-NC组)、前交叉韧带重建+Ad-Runx2组(Ad-Runx2组)4组,每组3只。制备前交叉韧带重建模型后,Ad-NC组、Ad-Runx2组于骨道内对应注射最佳hAMSCs-Matrigel复合物。术后1个月取材行大体、组织学(HE染色及天狼猩红染色)、免疫荧光染色观察,评价韧带组织中炎症细胞浸润以及Ⅰ/Ⅲ型胶原、Tenascin-C含量。结果流式细胞鉴定分离培养细胞符合MSCs表型特征。Runx2基因成功转染至hAMSCs;与Ad-NC组相比,Ad-Runx2组转染后VEGF及Ⅰ型胶原基因相对表达量于培养3、7 d时均增高(P<0.05),Fibronectin仅3 d时增高(P<0.05),而Tenascin-C于3 d时增高、7 d时降低(P<0.05)。CCK-8检测示两种比例混合培养后,细胞增殖组间以及组内各时间点间差异均无统计学意义(P>0.05),选择1∶1比例构建复合物进行后续实验。动物实验显�Objective To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells(hAMSCs)to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits.Methods hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged,and then identified by flow cytometric identification.Adenoviral vectors carrying Runx2 gene(Ad-Runx2)and empty vector adenovirus(Ad-NC)were constructed and viral titer assay;then,the 3rd generation hAMSCs were transfected with Ad-Runx2(Ad-Runx2 group)or Ad-NC(Ad-NC group).The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs;and at 3 and 7 days after transfection,real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes[vascular endothelial growth factor(VEGF),collagen typeⅠ,Fibronectin,and Tenascin-C].The hAMSCs were used as a blank control group.The hAMSCs,hAMSCs transfected with Ad-NC,and hAMSCs were mixed with Matrigel according to the ratio of 1:1 and 1:2 to construct the cell-scaffold compound.Cell proliferation was detected by cell counting kit 8(CCK-8)assay,and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments.Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group(Sham group),anterior cruciate ligament reconstruction group(ACLR group),anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group(Ad-NC group),and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group(Ad-Runx2 group),with 3 rabbits in each group.After preparing the ACL reconstruction model,the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly.The samples were taken for gross,histological(HE staining and si

关 键 词：Runx2基因 人羊膜MSCs 韧带成纤维细胞 腱-骨愈合 韧带组织工程 

分 类 号：R686.5[医药卫生—骨科学]