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 type Ⅰ, 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 sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues.
Results: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups.
Conclusion: Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
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。动物实验显示，术后1个月时，大体观察各组移植肌腱连续性完整；HE染色示Ad-Runx2组组织修复情况优于ACLR组和Ad-NC组、炎症细胞更少；天狼猩红染色及免疫荧光染色均示Ad-Runx2组韧带组织的Ⅰ、Ⅲ型胶原纤维增多，趋于形成正常前交叉韧带结构，但Tenascin-C蛋白荧光强度与ACLR组和Ad-NC组相比减弱。.
结论: Runx2基因转染hAMSCs后可诱导其向韧带成纤维细胞定向分化，并促进兔前交叉韧带损伤重建的腱-骨愈合。.
Keywords: Runx2 gene; human amniotic mesenchymal stem cells; ligament fibroblasts; ligament tissue engineering; tenbon-bone healing.