本文選題：RAW264.7細(xì)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(duì)RAW264.7細(xì)胞向破骨細(xì)胞分化過(guò)程的影響及可能的機(jī)制。方法:1.利用RNA干擾技術(shù),針對(duì)FoxO1基因靶點(diǎn)設(shè)計(jì)三條小干擾RNA(FoxO1-siRNA)沉默F(xiàn)oxO1在RAW264.7細(xì)胞中的表達(dá),轉(zhuǎn)染后檢測(cè)轉(zhuǎn)染效率,篩選出沉默效果最佳的FoxO1-si RNA;2.將細(xì)胞分為:對(duì)照組、S組(FoxO1-siRNA轉(zhuǎn)染組)、H組(H_2O_2干預(yù)組)、N組(無(wú)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(xì)胞1h后更換為RANKL培養(yǎng)液。通過(guò)TRAP染色觀察RAW264.7細(xì)胞向破骨細(xì)胞分化的程度;3.提取各組細(xì)胞RNA,經(jīng)逆轉(zhuǎn)錄后使用RT-PCR法檢測(cè)破骨細(xì)胞標(biāo)志基因TRAP、BMMP-9、組織蛋白酶K(CathepsinK)在各組細(xì)胞中的表達(dá)情況;4.流式細(xì)胞儀檢測(cè)各組細(xì)胞早期凋亡率;細(xì)胞內(nèi)活性氧(ROS)水平;專(zhuān)用試劑盒檢測(cè)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(shí)細(xì)胞FoxO1基因mRNA的表達(dá)量下降了約78%(P0.05),FoxO1蛋白的檢測(cè)結(jié)果顯示Si-FoxO1-2組FoxO1蛋白在轉(zhuǎn)染后48h時(shí)表達(dá)量較其他各組顯著降低,沉默效果最佳。2.RANKL誘導(dǎo)RAW264.7細(xì)胞向破骨細(xì)胞分化結(jié)果顯示:TRAP染色可見(jiàn)高倍鏡下各干預(yù)組均有多核巨細(xì)胞出現(xiàn),胞漿呈酒紅色,部分細(xì)胞內(nèi)可見(jiàn)多個(gè)核。FoxO1基因沉默組及H_2O_2干預(yù)組的細(xì)胞的TRAP、BMMP-9、Cathepsin K的表達(dá)水平均較對(duì)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(xì)胞早期凋亡較對(duì)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(xì)胞內(nèi)ROS水平及MDA含量升高,較對(duì)照組分別升高4倍和3.3倍(P0.05),SOD及GSH-PX活性較對(duì)照組分別下降約10%和74%(P0.05)。結(jié)論:FoxO1對(duì)破骨前體細(xì)胞RAW264.7細(xì)胞的破骨分化起抑制作用。其機(jī)制可能與其上調(diào)抗氧化酶活性、抑制細(xì)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(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R580

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本文編號(hào)：2031741