FoxO1对RAW264.7细胞向破骨细胞分化的作用研究

发布时间：2018-06-17 16:57

本文选题：RAW264.7细胞 + FoxO1　；参考：《兰州大学》2017年硕士论文

【摘要】：目的:观察叉头框转录因子(FoxO1)基因沉默对RAW264.7细胞向破骨细胞分化过程的影响及可能的机制。方法:1.利用RNA干扰技术,针对FoxO1基因靶点设计三条小干扰RNA(FoxO1-siRNA)沉默FoxO1在RAW264.7细胞中的表达,转染后检测转染效率,筛选出沉默效果最佳的FoxO1-si RNA;2.将细胞分为:对照组、S组(FoxO1-siRNA转染组)、H组(H_2O_2干预组)、N组(无义siRNA转染);各组均使用100ng/ml RANKL培养液培养,H_2O_2组使用100μmol/lH_2O_2处理细胞1h后更换为RANKL培养液。通过TRAP染色观察RAW264.7细胞向破骨细胞分化的程度;3.提取各组细胞RNA,经逆转录后使用RT-PCR法检测破骨细胞标志基因TRAP、BMMP-9、组织蛋白酶K(CathepsinK)在各组细胞中的表达情况;4.流式细胞仪检测各组细胞早期凋亡率;细胞内活性氧(ROS)水平;专用试剂盒检测MDA、SOD、GSH-PX水平。结果:1.FoxO1-siRNA沉默结果显示Si-FoxO1-2组在转染后24h时细胞FoxO1基因mRNA的表达量下降了约78%(P0.05),FoxO1蛋白的检测结果显示Si-FoxO1-2组FoxO1蛋白在转染后48h时表达量较其他各组显著降低,沉默效果最佳。2.RANKL诱导RAW264.7细胞向破骨细胞分化结果显示:TRAP染色可见高倍镜下各干预组均有多核巨细胞出现,胞浆呈酒红色,部分细胞内可见多个核。FoxO1基因沉默组及H_2O_2干预组的细胞的TRAP、BMMP-9、Cathepsin K的表达水平均较对照组升高(P0.05),且H_2O_2干预组升高幅度更大。3.FoxO1基因沉默组及H_2O_2干预组的细胞早期凋亡较对照组升高(P0.05),且FoxO1基因沉默组升高幅度较大;FoxO1基因沉默组、H_2O_2干预组均造成RAW264.7细胞内ROS水平及MDA含量升高,较对照组分别升高4倍和3.3倍(P0.05),SOD及GSH-PX活性较对照组分别下降约10%和74%(P0.05)。结论:FoxO1对破骨前体细胞RAW264.7细胞的破骨分化起抑制作用。其机制可能与其上调抗氧化酶活性、抑制细胞凋亡相关。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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