發(fā)布時間：2018-06-17 16:57

  本文選題：RAW264.7細胞 + FoxO1　； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：目的:觀察叉頭框轉(zhuǎn)錄因子(FoxO1)基因沉默對RAW264.7細胞向破骨細胞分化過程的影響及可能的機制。方法:1.利用RNA干擾技術(shù),針對FoxO1基因靶點設(shè)計三條小干擾RNA(FoxO1-siRNA)沉默F(xiàn)oxO1在RAW264.7細胞中的表達,轉(zhuǎn)染后檢測轉(zhuǎn)染效率,篩選出沉默效果最佳的FoxO1-si RNA;2.將細胞分為:對照組、S組(FoxO1-siRNA轉(zhuǎn)染組)、H組(H_2O_2干預(yù)組)、N組(無義siRNA轉(zhuǎn)染);各組均使用100ng/ml RANKL培養(yǎng)液培養(yǎng),H_2O_2組使用100μmol/lH_2O_2處理細胞1h后更換為RANKL培養(yǎng)液。通過TRAP染色觀察RAW264.7細胞向破骨細胞分化的程度;3.提取各組細胞RNA,經(jīng)逆轉(zhuǎn)錄后使用RT-PCR法檢測破骨細胞標志基因TRAP、BMMP-9、組織蛋白酶K(CathepsinK)在各組細胞中的表達情況;4.流式細胞儀檢測各組細胞早期凋亡率;細胞內(nèi)活性氧(ROS)水平;專用試劑盒檢測MDA、SOD、GSH-PX水平。結(jié)果:1.FoxO1-siRNA沉默結(jié)果顯示Si-FoxO1-2組在轉(zhuǎn)染后24h時細胞FoxO1基因mRNA的表達量下降了約78%(P0.05),FoxO1蛋白的檢測結(jié)果顯示Si-FoxO1-2組FoxO1蛋白在轉(zhuǎn)染后48h時表達量較其他各組顯著降低,沉默效果最佳。2.RANKL誘導(dǎo)RAW264.7細胞向破骨細胞分化結(jié)果顯示:TRAP染色可見高倍鏡下各干預(yù)組均有多核巨細胞出現(xiàn),胞漿呈酒紅色,部分細胞內(nèi)可見多個核。FoxO1基因沉默組及H_2O_2干預(yù)組的細胞的TRAP、BMMP-9、Cathepsin K的表達水平均較對照組升高(P0.05),且H_2O_2干預(yù)組升高幅度更大。3.FoxO1基因沉默組及H_2O_2干預(yù)組的細胞早期凋亡較對照組升高(P0.05),且FoxO1基因沉默組升高幅度較大;FoxO1基因沉默組、H_2O_2干預(yù)組均造成RAW264.7細胞內(nèi)ROS水平及MDA含量升高,較對照組分別升高4倍和3.3倍(P0.05),SOD及GSH-PX活性較對照組分別下降約10%和74%(P0.05)。結(jié)論:FoxO1對破骨前體細胞RAW264.7細胞的破骨分化起抑制作用。其機制可能與其上調(diào)抗氧化酶活性、抑制細胞凋亡相關(guān)。
[Abstract]:Objective: To observe the effect of FoxO1 gene silencing on the differentiation of RAW264.7 cells to osteoclast differentiation and its possible mechanism. Methods: 1. using RNA interference technique, the expression of three small interference RNA (FoxO1-siRNA) silent FoxO1 in RAW264.7 cells was designed to target FoxO1 gene target, and transfection efficiency was detected after transfection, and precipitation was screened. FoxO1-si RNA with the best silent effect; 2. cells were divided into two groups: control group, S group (FoxO1-siRNA transfection group), group H (H_2O_2 intervention group), N group (non sense siRNA transfection); all groups were cultured with 100ng/ml RANKL culture, H_2O_2 group was replaced by 100 micron mol/lH_2O_2 cells. The degree of cell differentiation; 3. RNA was extracted from each group, and the expression of osteoclast marker gene TRAP, BMMP-9, cathepsin K (CathepsinK) was detected by RT-PCR, and the rate of early apoptosis, intracellular reactive oxygen (ROS) level, and MDA, SOD, GSH-PX levels were detected by the 4. flow cytometer. Results: the results of 1.FoxO1-siRNA silencing showed that the expression of FoxO1 gene mRNA in Si-FoxO1-2 group decreased by about 78% (P0.05) at 24h after transfection. The detection results of FoxO1 protein showed that the expression of FoxO1 protein in Si-FoxO1-2 group was significantly lower than that of other groups after transfection, and the best silence effect was to induce RAW264.7 cells to osteoclasts. The results of differentiation showed that TRAP staining showed that there were multinucleated giant cells in all the intervention groups and the cytoplasm was wine red. In some cells, the TRAP, BMMP-9, Cathepsin K expression levels of multiple nuclear.FoxO1 gene silencing group and H_2O_2 intervention group were higher than those of the control group (P0.05), and the increase of H_2O_2 intervention group was greater.3.FoxO1. The early apoptosis of the cells in the gene silencing group and the H_2O_2 intervention group was higher than that in the control group (P0.05), and the FoxO1 gene silencing group increased significantly. The ROS level and the MDA content in the RAW264.7 cells increased by 4 and 3.3 times (P0.05) in the FoxO1 gene silencing group, and the SOD and GSH-PX activities were lower than those of the control group respectively. About 10% and 74% (P0.05). Conclusion: FoxO1 inhibits the osteoclast differentiation of RAW264.7 cells in osteoclast cells. The mechanism may be related to the up-regulation of antioxidant enzyme activity and inhibition of apoptosis.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R580

【參考文獻】

相關(guān)期刊論文 前2條

1 孟慶陽;鄭嶸;朱陽;王丹丹;吳立鵬;孫宏晨;;破骨細胞分化因子及其信號轉(zhuǎn)導(dǎo)通路[J];國際口腔醫(yī)學(xué)雜志;2015年02期

2 毛文晴;田甜;;原發(fā)性骨質(zhì)疏松的病因及發(fā)病機制[J];中國骨質(zhì)疏松雜志;2011年10期

相關(guān)碩士學(xué)位論文 前1條

1 劉進進;FoxO1/β-catenin信號通路在成骨細胞氧化應(yīng)激中的作用及機制研究[D];蘭州大學(xué);2014年

，

本文編號：2031741