牵张应力下沉默Girdin对人牙周膜成纤维细胞影响的实验研究

发布时间：2019-07-04 20:49

【摘要】：f目的]体外分离培养人牙周膜成纤维细胞(human periodontal ligament fibro-blasts, hPDLFs)。探讨体外牵张应力作用对hPDLFs和人成骨样MG63细胞中Girdin/Akt信号通路表达的影响。进一步研究正畸牙移动过程中牙周组织改建的机械生物力学机制。 [方法]1.采用改良组织块贴附法体外培养hPDLFs并进行细胞传代,通过细胞形态学及免疫组织化学方法进行鉴定,为后续应力实验提供细胞。2.以人牙周膜成纤维细胞和人成骨样MG63细胞为研究对象,采用Forcel四点弯曲细胞力学加载仪对两种细胞分别施加动态张应力(强度5000μ strain,频率0.5Hz),透射电镜观察细胞加力前后超微结构的改变。3.采用人工合成的siRNA序列干扰MG63细胞中Girdin蛋白,用Western Blot法检测干扰前后Girdin、磷酸化Girdin(P-Girdin)、Akt、磷酸化Akt (P-Akt)的表达情况；以及施加动态牵张应力后上述因子的表达情况和细胞超微结构的改变。 [结果]1.采用改良组织块贴附法成功培养出原代牙周膜细胞,并证实所培养的细胞是中胚层来源的hPDLFs。2.透射电镜观察结果：与对照组相比,实验组中牙周成纤维细胞呈梭形,突起多且长,有大小不等的空泡样结构,有的空泡含有胶原碎片,.细胞体积明显增大,均活跃的功能状态,胞质细胞器异常丰富,粗面内质网、线粒体发达,呈不同程度的扩张、肿胀；与对照组相比,实验组中MG63细胞体积明显增大,均活跃的功能状态,胞质细胞器异常丰富,粗面内质网、线粒体发达,呈不同程度的扩张、肿胀。3. Western Blot检测结果：与空白对照组相比,转染siRNA的MG63细胞Girdin、P-Girdin、Akt、P-Akt衣达明显下调；转染siRNA后再施加动态牵张力的MG63细胞Girdin、P-Girdin、Akt、P-Akt表达产生下调。 [结论]1.通过改良组织块贴附法能在体外成功培养出人牙周成纤维细胞。2.适宜的机械应力能够促进牙周组织的正常改建。3. Girdin/Akt信号通路参与调节成骨细胞的增殖与凋亡,Girdin的下调可诱导成骨细胞凋亡。
[Abstract]:F objective] isolation and culture of human periodontal ligament fibroblasts (human periodontal ligament fibro-blasts, hPDLFs). In vitro To investigate the effect of distraction stress on the expression of Girdin/Akt signaling pathway in hPDLFs and human osteoblast-like MG63 cells. The mechanical and biomechanical mechanism of periodontal tissue remodeling during orthodontic tooth movement was further studied. [method] 1. HPDLFs was cultured in vitro and subcultured by modified tissue block attachment method, and identified by cell morphology and immunohistochemistry, which provided cells for subsequent stress test. 2. Human periodontal ligament fibroblasts and human osteoblast-like MG63 cells were studied. Dynamic tensile stress (intensity 5000 渭 strain, frequency 0.5Hz) was applied to the two kinds of cells by Forcel four-point bending cell mechanical loading instrument. The ultrastructure of the cells before and after stress was observed by transmission electron microscope. The synthetic siRNA sequence was used to interfere with Girdin protein in MG63 cells. The expression of Girdin, phosphorylation Girdin (P-Girdin), Akt, phosphorylation Akt (P-Akt) before and after interference was detected by Western Blot assay, and the expression of the above factors and the changes of cell ultrastructure after dynamic stretch stress were applied. [result] 1. The primary periodontal ligament cells were successfully cultured by modified tissue block attachment method, and it was confirmed that the cultured cells were hPDLFs.2. from mesoderm. The results of transmission electron microscope showed that compared with the control group, the periodontal fibroblasts in the experimental group were fusiform, the protrusions were many and long, and there were vacuoles of varying sizes, and some vacuoles contained collagen fragments. Compared with the control group, the volume of MG63 cells in the experimental group was significantly larger, all of them were active, the cytoplasm organelle was abnormally rich, the rough endoplasmic reticulum, the mitochondria were developed, showing varying degrees of expansion and swelling, compared with the control group, the volume of MSCs cells in the experimental group was significantly increased, and the cytoplasm organelle was abnormally rich, the rough endoplasmic reticulum, the mitochondria were developed, and showed different degrees of expansion and swelling. The results of Western Blot detection: compared with the blank control group, the expression of Girdin,P-Girdin,Akt,P-Akt in MG63 cells transfected with siRNA was significantly down-regulated, and the expression of Girdin,P-Girdin,Akt,P-Akt in MG63 cells with dynamic tension was down-regulated after siRNA transfer. [conclusion] 1. Human periodontal fibroblasts can be successfully cultured in vitro by modified tissue block attachment method. 2. Suitable mechanical stress can promote the normal remodeling of periodontal tissue. 3. Girdin/Akt signaling pathway is involved in regulating the proliferation and apoptosis of osteoblasts. The down-regulation of Girdin can induce apoptosis of osteoblasts.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783.5

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