miRNA219-5P靶向调控Smad4表达影响TGF-β_1诱导肌腱成纤维细胞纤维化的研究

EFFECTIVENESS EVALUATION OF PERCUTANEOUS MONOAXIAL SCREW COMBINED WITH INJURED VERTEBRAE POLYAXIAL PEDICAL SCREW FIXATION FOR TREATMENT OF THORACOLUMBAR FRACTURES

目的 探讨mi RNA219-5P（mi R219-5P）调控Smad4在TGF-β1诱导人肌腱成纤维细胞（tendon derived fibroblasts,TDFs）纤维化过程中的作用。方法 取患者自愿捐赠的肌腱组织分离培养TDFs,取第3代细胞进行实验。化学合成mi R219-5P类似物（mimics）、阻遏物（inhibitor）及阴性对照序列,分别转染TDFs 48 h后,采用实时定量PCR及Western blot法分别检测mi R219-5P基因及Smad4蛋白表达情况;以正常细胞作为空白对照。信息学软件预测mi R219-5P与Smad4基因3’UTR区结合位点,构建Smad4预测基因3’UTR-PGL3重组质粒,构建野生型及突变型报告基因表达载体,并通过荧光素酶报告基因实验进行靶位验证。取正常及转染后TDFs,采用TGF-β1诱导细胞发生纤维化,于培养24、48、72 h采用细胞计数试剂盒8法检测细胞增殖活性变化,72 h时采用Western blot法检测纤维化相关蛋白指标。结果 mi R219-5P mimics转染TDFs可明显上调mi R219-5P表达、下调Smad4蛋白表达,而mi R219-5P inhibitor抑制细胞内mi R219-5P表达、增加Smad4蛋白表达,与空白对照组比较差异均有统计学意义（P〈0.05）。荧光素酶实验显示,与p GL3-WT-Smad4转染组比较,p GL3-WT-Smad4＋mimics转染组细胞内荧光素酶活性降低、p GL3-WT-Smad4＋inhibitor转染组活性增强（P〈0.05）;而p GL3-MT-Smad4＋mimics转染组、p GL3-MT-Smad4＋inhibitor转染组组间比较无明显变化（P〉0.05）。与空白对照组比较,TGF-β1诱导培养72 h后细胞增殖活性明显增强（P〈0.01）,同时上调细胞纤维化相关蛋白指标的表达（P〈0.01）;与直接诱导组比较,mi R219-5P过表达且诱导组TDFs细胞增殖活性和纤维化蛋白指标升高趋势得到抑制,而mi R219-5P表达抑制且诱导组细胞增殖活性和纤维化蛋白指标则进一步增强,差异有统计学意义（P〈0.01）。结论 mi R219-5P可通过抑制其靶基因Smad4表达,显著抑制TGF-β1诱导TDFs纤维化。

[ Abstract ] Objective To explore the effectiveness percutaneous monoaxial screw combined with polyaxial pedical screw for treating thoracolumbar fracture by comparing with simple polyaxial pedic｜e screw fixation. Methods Between January 2012 and June 2014, 56 cases of thoracolumbar fractures were treated by percutaneous pedicle screw fixation, the clinical data were retrospectively analyzed. Of 56 cases, 30 were treated with percutaneous monoaxial screw combined with percutaneous polyaxial pedical screw fixation （group A）, 26 patients with only percutaneous polyaxial pedicle screw fixation （group B）. There was no significant difference in gender, age, body mass index, injury causes, time from injury to admission, involved segments, fracture type, and preoperative American Spinal Injury Association （ASIA） stage, visual analogue scale （VAS）, the anterior height of the injured vertebrae, Cobb angle, and sagittai index between 2 groups （P〉0.05）. The operation time, intraoperative blood loss, and complications were recorded and compared between the 2 groups. The VAS score was used to evaluate the improvement of the pain. The sagittal kyphosis Cobb angle, the anterior height of the injured vertebrae, sagittal index, and the average correction （difference between 3 days after oeration and preoperation） and loss degrees （difference between last follow-up and 3 days after operation） were measured on the X-ray films at preoperation, 3 days after operation, and last follow-up. Results Incision healing at stage I was obtained, no related complications occurred. The operation time and intraoperative blood loss showed no significant difference between 2 groups （P〉0.05）. The patients were followed up 20-42 months （mean, 32 months） in group A and 21-44 months （mean, 30 months） in group B. VAS score of group A was significantly lower than that of group B at 3 days after operation （t= -2.277, P=0.027）, but no significant difference was found at last follow-up （t= -0.289, P=0.774）. X-ray examination showed good position of internal fixation, with no broken nails or exit of nail. There were significant differences in the anterior height of the injured vertebrae, Cobb angle, and sagittal index between at preoperation and at 3 days and last follow-up, and between at 3 days and last follow-up in 2 groups （P〈0.05）. The anterior height of the iniured vertebrae, Cobb angle, and sagittal index of group A were significantly better than those of group B at 3 days and last follow-up （P〈0.05）, and correction degree were significantly higher than those of group B （P〈0.05）, but loss degree was not significant between 2 groups （P〉0.05）. Conclusion Percutaneous monoaxial screw combined with polyaxial pedicle screw fixation is better than simply polyaxial pedicle screw in effects of treating thoracolumbar fracture under the premise of strictly holding indications.