EGR1对TSCs分化影响及其在腱骨愈合中作用的研究

发布时间：2018-06-01 08:14

本文选题：EGR1 + 肌腱干细胞　；参考：《第三军医大学》2017年博士论文

【摘要】：腱-骨愈合(tendon-bone healing)是运动医学与骨科面临的主要科学问题之一。肩袖止点断裂后局部应用生物学制剂(如细胞成分,细胞外基质或各种生长因子)可有效增加手术修复的成功率,改善修复术后的长期疗效。然而,生物力学结果表明,生物学制剂并不能真正提高肩袖修补的生物力学。如何更好地解决腱-骨愈合问题,成为运动医学面临的热点、难点。以往的研究表明,生长因子如骨形态发生蛋白2、7(BMP-2、BMP-7),转化生长因子β1和β3(TGF-β1、TGF-β3),碱性成纤维细胞生长因子(FGF)等有效提高动物模型中腱-骨愈合的治愈率。由于过分强调骨侧“被动”愈合,修复后生物力学强度并未见明显增加,应用此类因子提高手术成功率的结论存在质疑。最近的研究表明,早期生长反应因子(early growth response-1,Egr1)参与了肌腱分化。EGR1不仅促进胚胎时期四肢肌腱细胞的分化,并且能诱导肌腱形成标志物Scleraxis(SCX)的表达和肌腱胶原的形成。体内研究进一步证实,过表达Egr1的间充值干细胞(mesenchymal stem cells,MSCs)对损伤跟腱有明显的修复作用。前期课题组大量研究发现,肌腱干细胞(tendon stem cells,TSCs)是修复肌腱损伤最有效的前体细胞之一。如何刺激TSCs向肌腱方向分化,进而促进腱侧“主动”愈合成为本课题主要重点。本部分拟观察EGR1诱导TSC细胞向肌腱分化的作用。本研究拟从EGR1对TSCs分化作用的影响,观察影响TSCs成肌腱分化的信号通路,最后将EGR1导入TSCs,观察EGR1-TSCs对腱-骨愈合影响的表现。第一部分EGR1对TSCs多向分化影响的初步观察本部分拟观察EGR1对TSCs的多向分化能力的观察,对比成肌腱分化与病理性分化的差异。一、实验方法双酶切法将EGR1 c DNA编码序列从pCMV-EGR1切除,回收后并进一步克隆至pcDNA3.1+载体上,获得pCDNA-EGR1。细胞转染pCDNA-EGR1或载体(对照)单独使用Lipofectamine 2000转染试剂。G418(800μg/ml)筛选稳定转染细胞。TSCs与pCDNA-EGR1转染TSCs(EGR1-TSCS)进行向肌腱方向培养培养,细胞接种于含有DMEM的6孔板中。采用免疫荧光染色和荧光定量PCR检测,对分化相关的基因SCX、TNMD、TNC、Col I表达的检测。二、实验结果免疫荧光染色显示:过表达EGR1的TSCs可以表达较高水平的肌腱标记物SCX、TNMD与TNC,表明EGR1基因影响这些细胞腱性分化相关基因的转录水平。细胞特殊染色显示,过表达EGR1的TSCs诱导分化抑制成脂肪分化、成骨分化、成软骨分化。此外,PPARγ、Runx2、Sox9基因转录水平下降,表明EGR1下调非腱性分化(成脂、成骨、成软骨等)相关基因的转录。三、小结1.将EGR1转染至TSCs后,获得过表达EGR1的稳定TSCs细胞株。2.从蛋白表达、基因转录水平检测显示,EGR1促进TSCs向成肌腱方向分化。3.EGR1特异性促进TSCs向成肌腱分化,同时抑制TSCs向脂肪细胞、成骨细胞、软骨细胞方向分化。第二部分EGR1对TSCs向成肌腱方向分化的通路研究本部分在第一部分研究基础上进一步研究EGR1影响TSCs成肌腱分化的信号通路。一、实验方法在成肌腱分化研究过程中,分析BMP12/Smad1/5/8通路蛋白的产量,两组在不同时间点收集细胞。用BCA蛋白浓度测定试剂盒测定总蛋白浓度与等量的蛋白质,通过SDS-聚丙烯酰胺凝胶电泳。然后将蛋白转移到聚偏氟乙烯膜,依次用第一和第二抗体阻断。使用LICOR Odyssey成像仪观察结果。二、实验结果在EGR1-TSCs组,BMP12基因m RNA和蛋白水平和p-Smad1/5/8蛋白水平相似,逐渐增加并呈时间依赖性过表达,并促进向成肌腱分化,但在TSCs组中并不明显。肌腱分化中Smad1蛋白水平总量没有增加。EGR1-TSCS组肌腱相关基因的表达,SCX、TNMD、TNC、Col I呈时间依赖性逐渐增加。三、小结1.BMP12能诱导TSC向成肌腱分化,并得出最优剂量及最优持续时间。2.BMP12与BMPRI型受体结合后激活Smad表达,EGR1通过BMP12/Smad1/5/8信号通路促进TSCs向成肌腱分化。3.EGR1-TSCs向成肌腱分化过程中,EGR1对BMP12、Smad1/5/8调节呈时间依赖性。第三部分过表达EGR1的TSCs对兔肩袖慢性损伤的影响本部分建立兔肩袖慢性损伤模型,将EGR1-TSCs植入腱-骨界面,观察对腱-骨愈合的影响。一、实验方法将24只成年新西兰大白兔的双侧冈上肌腱切断,右侧为实验组,左侧假手术组。6周后行肩袖陈旧性损伤修补术。TSCs与EGR1-TSCS混合纤维蛋白胶中植入肩袖界面间。将实验用兔分为三组:1组(R),修复手术;2组(TSCs),修补+植入含TSCs的纤维蛋白胶;3组(EGR1-TSCS),修复+植入含EGR1-TSCS的纤维蛋白胶。修复手术8周后处死所有兔。组织学分析和免疫组化检测评估之间的差异。二、实验结果术后8周,TSCs组呈平行的纤维组织,可见沿应力方向少量的Sharpey纤维;在EGR1-TSC组,胶原纤维排列更为有序,止点处Sharpey纤维较TSC组更明显。三、小结1.通过建立稳定的兔肩袖慢性损伤模型,为腱-骨愈合提供了良好的动物模型。2.在兔肩袖慢性损伤模型中,腱-骨界面应用含有EGR1-TSCs的纤维凝胶可有效促进腱-骨愈合。
[Abstract]:Tendon bone healing (tendon-bone healing) is one of the major scientific problems in sports medicine and Department of orthopedics. The local application of biological agents (such as cell composition, extracellular matrix or various growth factors) after the rotator cuff break can effectively increase the success rate of surgical repair and improve the long-term effect after the repair. However, the biomechanical results show that Biological agents do not really improve the biomechanics of rotator cuff repair. How to better solve the problem of tendon bone healing has become a hot and difficult problem in sports medicine. Previous studies have shown that growth factors such as bone morphogenetic protein 2,7 (BMP-2, BMP-7), transforming growth factor beta 1 and beta 3 (TGF- beta 1, TGF- beta 3), basic fibroblast growth factors FGF and so on effectively improve the healing rate of tendon bone healing in animal models. Due to the excessive emphasis on the "passive" healing of the bone side, the biomechanical strength after repair has not been significantly increased, and the conclusion of the application of such factors to improve the success rate of the operation is questioned. Recent studies have shown that the early growth response factor (early growth response-1, Egr1) participated in the study. Tendon differentiation.EGR1 not only promotes the differentiation of tendon cells in the embryonic period, but also induces the expression of Scleraxis (SCX) and the formation of tendon collagen in the tendon formation markers. In vivo studies have further confirmed that the revalued stem cells (mesenchymal stem cells, MSCs) over expressed Egr1 have obvious repair effect on the injured Achilles tendon. A large number of studies have found that tendon stem cells (TSCs) is one of the most effective precursor cells for the repair of tendon injury. How to stimulate the direction differentiation of TSCs to the tendons and promote the "active" healing of the tendon side has become the main focus of this topic. This part is to observe the effect of EGR1 induced TSC cells to the differentiation of the tendon to the tendon. This study is to be taken from EGR1 pairs. The effect of TSCs differentiation, observing the signal pathway affecting the differentiation of TSCs tendon, and finally introducing EGR1 into TSCs to observe the effect of EGR1-TSCs on the healing of tendon bone. The first part of the preliminary observation on the effect of EGR1 on the multidirectional differentiation of TSCs, this part is to observe the observation of the multidirectional differentiation energy of EGR1 to TSCs, and to contrast the differentiation and pathological differentiation of the tendon. First, the EGR1 C DNA coding sequence was removed from pCMV-EGR1 by double enzyme digestion method, and then recovered and cloned to pcDNA3.1+ vector, and pCDNA-EGR1. cells were transfected to pCDNA-EGR1 or carrier (control) to use Lipofectamine 2000 transfection reagent.G418 (800 mu g/ ml) to screen the stable transfection cell.TSCs and transfection GR1-TSCS) was cultured in the direction of tendon, and the cells were inoculated in the 6 orifice plate containing DMEM. Immunofluorescence staining and fluorescence quantitative PCR detection were used to detect the expression of SCX, TNMD, TNC, Col I related genes. Two, the experimental results of immunofluorescence staining showed that the TSCs of overexpressed EGR1 could express the higher level of tendon markers SCX, TNM D and TNC show that EGR1 genes affect the transcriptional level of the genes related to the differentiation of these cells. Cell specific staining shows that the TSCs induced differentiation of overexpressed EGR1 can inhibit adipose differentiation, osteogenesis and chondrodifferentiation. In addition, the transcriptional level of PPAR gamma, Runx2, Sox9 gene decreases, indicating that EGR1 downplays non Achilles differentiation (lipid, osteogenesis, cartilage, etc.) The transcription of related genes. Three, after transfection of EGR1 to TSCs, the stable TSCs cell line.2. expressing EGR1 was obtained from the protein expression, and the gene transcriptional level detection showed that EGR1 promoted TSCs to differentiate.3.EGR1 specificity from TSCs into tendons to promote the differentiation of TSCs into tendon, and inhibit TSCs to adipocytes, osteoblasts and chondrocytes. Second part of the pathway of EGR1 to the direction differentiation of TSCs into the tendons of the tendon. This part further studies the signal pathway that EGR1 affects the differentiation of TSCs tendon in the first part of the study. One, the experimental method analyses the production of BMP12/Smad1/5/8 pathway protein in the process of tendon differentiation, and the two groups collect cells at different time points. CA protein concentration assay kit was used to determine the total protein concentration and equal amount of protein by SDS- polyacrylamide gel electrophoresis. Then the protein was transferred to polyvinylidene fluoride membrane, and the first and second antibodies were interrupted in turn. The results were observed by the LICOR Odyssey imager. Two, the experimental results were in the EGR1-TSCs group, the BMP12 gene m RNA and protein levels and p-. Smad1/5/8 protein levels were similar, gradually increased and showed time dependent overexpression, and promoted the differentiation of the tendon into the tendon, but it was not obvious in the TSCs group. The total amount of Smad1 protein in the tendon differentiation did not increase the expression of the gene related genes in the.EGR1-TSCS group. SCX, TNMD, TNC, Col I showed a gradual increase in time dependence. Three, nodules 1.BMP12 could induce TSC. The optimal dose and optimal duration of.2.BMP12 are combined with BMPRI receptor to activate Smad expression. EGR1 through BMP12/Smad1/5/8 signaling pathway promotes TSCs into tendon differentiation.3.EGR1-TSCs into tendon differentiation process, EGR1 is time dependent on BMP12, Smad1/5/8 regulation. The third part overexpresses EGR1 TSCs pairs. The chronic injury of the rotator cuff in rabbits was used to establish a chronic injury model of the rabbit's rotator cuff. EGR1-TSCs was implanted into the tendon bone interface and the effect of the tendon bone healing was observed. One, the experimental method was used to cut the bilateral supraspinatus tendon of 24 adult New Zealand white rabbits, the right was the experimental group, and the left rotator cuff old injury repair operation was performed after the left hand prosthesis group.6 weeks after the repair of.TSCs and the repair of the rotator cuff. EGR1-TSCS mixed fibrin glue was implanted into the rotator cuff interface. The experimental rabbits were divided into three groups: 1 groups (R), repair operation; 2 groups (TSCs), repair + implantation of fibrin glue containing TSCs; 3 groups (EGR1-TSCS), repair and implantation of fibrin glue containing EGR1-TSCS. All rabbits were killed after 8 weeks of repair. Histology analysis and immunohistochemical detection assessment Two, 8 weeks after the operation, group TSCs showed a parallel fibrous tissue, and a small amount of Sharpey fibers along the stress direction; in group EGR1-TSC, the collagen fibers were arranged more orderly, and the Sharpey fibers were more obvious at the stop point. Three, nodule 1., by establishing a stable chronic injury model of the rabbit rotator cuff, provided a good movement for the tendon bone healing. Object model.2. in rabbit chronic rotator cuff injury model, fibrous gel containing EGR1-TSCs can be used to promote tendon bone healing in tendon bone interface.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R687

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