牽張應力下沉默Girdin對人牙周膜成纖維細胞影響的實驗研究
發(fā)布時間：2019-07-04 20:49
【摘要】：f目的]體外分離培養(yǎng)人牙周膜成纖維細胞(human periodontal ligament fibro-blasts, hPDLFs)。探討體外牽張應力作用對hPDLFs和人成骨樣MG63細胞中Girdin/Akt信號通路表達的影響。進一步研究正畸牙移動過程中牙周組織改建的機械生物力學機制。 [方法]1.采用改良組織塊貼附法體外培養(yǎng)hPDLFs并進行細胞傳代,通過細胞形態(tài)學及免疫組織化學方法進行鑒定,為后續(xù)應力實驗提供細胞。2.以人牙周膜成纖維細胞和人成骨樣MG63細胞為研究對象,采用Forcel四點彎曲細胞力學加載儀對兩種細胞分別施加動態(tài)張應力(強度5000μ strain,頻率0.5Hz),透射電鏡觀察細胞加力前后超微結(jié)構(gòu)的改變。3.采用人工合成的siRNA序列干擾MG63細胞中Girdin蛋白,用Western Blot法檢測干擾前后Girdin、磷酸化Girdin(P-Girdin)、Akt、磷酸化Akt (P-Akt)的表達情況；以及施加動態(tài)牽張應力后上述因子的表達情況和細胞超微結(jié)構(gòu)的改變。 [結(jié)果]1.采用改良組織塊貼附法成功培養(yǎng)出原代牙周膜細胞,并證實所培養(yǎng)的細胞是中胚層來源的hPDLFs。2.透射電鏡觀察結(jié)果：與對照組相比,實驗組中牙周成纖維細胞呈梭形,突起多且長,有大小不等的空泡樣結(jié)構(gòu),有的空泡含有膠原碎片,.細胞體積明顯增大,均活躍的功能狀態(tài),胞質(zhì)細胞器異常豐富,粗面內(nèi)質(zhì)網(wǎng)、線粒體發(fā)達,呈不同程度的擴張、腫脹；與對照組相比,實驗組中MG63細胞體積明顯增大,均活躍的功能狀態(tài),胞質(zhì)細胞器異常豐富,粗面內(nèi)質(zhì)網(wǎng)、線粒體發(fā)達,呈不同程度的擴張、腫脹。3. Western Blot檢測結(jié)果：與空白對照組相比,轉(zhuǎn)染siRNA的MG63細胞Girdin、P-Girdin、Akt、P-Akt衣達明顯下調(diào)；轉(zhuǎn)染siRNA后再施加動態(tài)牽張力的MG63細胞Girdin、P-Girdin、Akt、P-Akt表達產(chǎn)生下調(diào)。 [結(jié)論]1.通過改良組織塊貼附法能在體外成功培養(yǎng)出人牙周成纖維細胞。2.適宜的機械應力能夠促進牙周組織的正常改建。3. Girdin/Akt信號通路參與調(diào)節(jié)成骨細胞的增殖與凋亡,Girdin的下調(diào)可誘導成骨細胞凋亡。
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R783.5
本文編號：2510223
[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.
【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R783.5
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