尼克酰胺通过下调SATB1诱导小鼠F9细胞的凋亡

发布时间：2018-05-05 03:17

本文选题：尼克酰胺 + SATB1　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：SATB1(Special AT rich sequence binding protein 1)是一种核基质结合蛋白,它的表达具有一定的组织特异性和时间特异性。近年来研究发现,SATB1在多种肿瘤细胞中过表达,通过改变多种基因的调控从而发挥着重要的作用,并且这种表达与肿瘤的迁移和增殖等行为相关。尼克酰胺是水溶性维生素B3的一种成分,它可以改变细胞的生存方式,并能诱导一些肿瘤细胞的的凋亡。本研究主要是通过荧光定量PCR、蛋白免疫印迹、细胞增殖、细胞周期、细胞凋亡等实验研究SATB1的表达量的变化对F9细胞增殖、周期和凋亡的影响,研究结果如下:1.实时荧光定量PCR检测结果表明,F9细胞中SATB1有大量表达。用不同浓度(0mmol/L,1.5 mmol/L,2 mmol/L,2.5 mmol/L)的尼克酰胺处理F9细胞48 h,通过荧光定量PCR和蛋白免疫印迹检测处理后的F9细胞中SATB1的表达,处理后SATB1的表达量明显下调,且随着尼克酰胺浓度的增加,SATB1的表达量逐渐下降。随后,对用0mmol/L,1.5 mmol/L,2 mmol/L,2.5 mmol/L浓度的尼克酰胺处理48 h后的细胞,换成新鲜的不含尼克酰胺的培养液,36 h后收集细胞同样进行荧光定量PCR和蛋白免疫印迹检测SATB1的表达,发现SATB1的表达出现了明显的回升,说明尼克酰胺的作用下调了SATB1的表达。2.利用流式细胞仪检测了细胞周期和细胞凋亡。发现用0 mmol/L,1.5 mmol/L,2mmol/L,2.5 mmol/L浓度的尼克酰胺处理后大部分细胞被阻碍在G1期,细胞凋亡率也随着尼克酰胺浓度的增加出现了明显的升高。通过CCK-8试剂盒检测了细胞的增殖情况,发现,三个实验组细胞增殖在12 h和24 h变化不显现,但在36 h和48 h,细胞增殖出现了明显的抑制,但实验组和对照组相比,细胞增殖在12 h就明显的被抑制,说明尼克酰胺能抑制F9细胞的增殖并诱导F9细胞的凋亡。3.设计和构建了3对靶向小鼠SATB1的siRNA,并通过实验筛选出干扰效率最高的si-984。结果表明,干扰后的F9细胞表现出了与尼克酰胺处理后相似的特征,大部分细胞被阻滞在G1期,细胞凋亡率也出现了明显的升高,细胞的增殖受到明显的抑制,说明尼克酰胺是通过下调SATB1诱导了F9细胞的凋亡。总之,本研究首次发现尼克酰胺作为诱导因子能通过下调SATB1抑制F9细胞的增殖和诱导F9细胞的凋亡。这一结果对尼克酰胺在肿瘤的治疗应用上提供了重要的理论基础,也为肿瘤的治疗提供了一新思路。
[Abstract]:SATB1(Special AT rich sequence binding protein 1 is a nuclear matrix binding protein. In recent years, it has been found that SATB1 is overexpressed in many kinds of tumor cells, which plays an important role by changing the regulation of many genes, and this expression is related to the behavior of tumor migration and proliferation. Nicotinamide, a component of water-soluble vitamin B _ 3, can change the way cells survive and induce apoptosis in some tumor cells. In this study, the effects of SATB1 expression on the proliferation, cycle and apoptosis of F9 cells were studied by fluorescence quantitative PCR, Western blot, cell proliferation, cell cycle and apoptosis. The results are as follows: 1. The results of real-time fluorescence quantitative PCR showed that there was a great deal of SATB1 expression in F9 cells. F9 cells were treated with nicotinamide (2.5 mmol / L) at different concentrations of 0 mmol / L ~ (1.5) mmol / L ~ (2. 5) mol / L ~ (2. 5) for 48 h. The expression of SATB1 in F9 cells was detected by fluorescence quantitative PCR and Western blot. After treatment, the expression of SATB1 was down-regulated. The expression of SATB1 decreased with the increase of nicotinamide concentration. Then the cells were treated with 0 mmol / L 1.5 mmol / L 2 mmol / L 2.5 mmol/L nicotinamide for 48 h, then the cells were changed to fresh culture medium without nicotinamide for 36 h. The SATB1 expression was also detected by fluorescence quantitative PCR and Western blot. It was found that the expression of SATB1 increased significantly, suggesting that nicotinamide down-regulated the expression of SATB1. 2. Cell cycle and apoptosis were detected by flow cytometry. It was found that most of the cells were blocked in G1 phase after treatment with 0 mmol / L 1.5 mmol / L 2. 5 mmol / L nicotinamide, and the apoptosis rate increased significantly with the increase of nicotinamide concentration. The cell proliferation was detected by CCK-8 kit. The results showed that the proliferation of the three experimental groups did not change at 12 h and 24 h, but at 36 h and 48 h, the cell proliferation was significantly inhibited, but compared with the control group. The proliferation of F9 cells was significantly inhibited at 12 h, indicating that nicotinamide could inhibit the proliferation of F9 cells and induce apoptosis of F9 cells. Three pairs of siRNAs targeting mouse SATB1 were designed and constructed, and si-984, which had the highest interference efficiency, was screened out by experiments. The results showed that the interfering F9 cells showed similar characteristics as those treated with nicotinamide. Most of the cells were blocked in G1 phase, the apoptosis rate was also increased, and the proliferation of F9 cells was inhibited obviously. It is suggested that nicotinamide induces apoptosis of F 9 cells by down-regulating SATB1. In conclusion, it is the first time that nicotinamide can inhibit the proliferation of F9 cells and induce apoptosis of F9 cells by down-regulating SATB1. This result provides an important theoretical basis for the application of nicotinamide in the treatment of cancer, and also provides a new idea for the treatment of cancer.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.79

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