發(fā)布時間：2018-05-05 03:17

  本文選題：尼克酰胺 + SATB1　； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：SATB1(Special AT rich sequence binding protein 1)是一種核基質(zhì)結(jié)合蛋白,它的表達(dá)具有一定的組織特異性和時間特異性。近年來研究發(fā)現(xiàn),SATB1在多種腫瘤細(xì)胞中過表達(dá),通過改變多種基因的調(diào)控從而發(fā)揮著重要的作用,并且這種表達(dá)與腫瘤的遷移和增殖等行為相關(guān)。尼克酰胺是水溶性維生素B3的一種成分,它可以改變細(xì)胞的生存方式,并能誘導(dǎo)一些腫瘤細(xì)胞的的凋亡。本研究主要是通過熒光定量PCR、蛋白免疫印跡、細(xì)胞增殖、細(xì)胞周期、細(xì)胞凋亡等實驗研究SATB1的表達(dá)量的變化對F9細(xì)胞增殖、周期和凋亡的影響,研究結(jié)果如下:1.實時熒光定量PCR檢測結(jié)果表明,F9細(xì)胞中SATB1有大量表達(dá)。用不同濃度(0mmol/L,1.5 mmol/L,2 mmol/L,2.5 mmol/L)的尼克酰胺處理F9細(xì)胞48 h,通過熒光定量PCR和蛋白免疫印跡檢測處理后的F9細(xì)胞中SATB1的表達(dá),處理后SATB1的表達(dá)量明顯下調(diào),且隨著尼克酰胺濃度的增加,SATB1的表達(dá)量逐漸下降。隨后,對用0mmol/L,1.5 mmol/L,2 mmol/L,2.5 mmol/L濃度的尼克酰胺處理48 h后的細(xì)胞,換成新鮮的不含尼克酰胺的培養(yǎng)液,36 h后收集細(xì)胞同樣進(jìn)行熒光定量PCR和蛋白免疫印跡檢測SATB1的表達(dá),發(fā)現(xiàn)SATB1的表達(dá)出現(xiàn)了明顯的回升,說明尼克酰胺的作用下調(diào)了SATB1的表達(dá)。2.利用流式細(xì)胞儀檢測了細(xì)胞周期和細(xì)胞凋亡。發(fā)現(xiàn)用0 mmol/L,1.5 mmol/L,2mmol/L,2.5 mmol/L濃度的尼克酰胺處理后大部分細(xì)胞被阻礙在G1期,細(xì)胞凋亡率也隨著尼克酰胺濃度的增加出現(xiàn)了明顯的升高。通過CCK-8試劑盒檢測了細(xì)胞的增殖情況,發(fā)現(xiàn),三個實驗組細(xì)胞增殖在12 h和24 h變化不顯現(xiàn),但在36 h和48 h,細(xì)胞增殖出現(xiàn)了明顯的抑制,但實驗組和對照組相比,細(xì)胞增殖在12 h就明顯的被抑制,說明尼克酰胺能抑制F9細(xì)胞的增殖并誘導(dǎo)F9細(xì)胞的凋亡。3.設(shè)計和構(gòu)建了3對靶向小鼠SATB1的siRNA,并通過實驗篩選出干擾效率最高的si-984。結(jié)果表明,干擾后的F9細(xì)胞表現(xiàn)出了與尼克酰胺處理后相似的特征,大部分細(xì)胞被阻滯在G1期,細(xì)胞凋亡率也出現(xiàn)了明顯的升高,細(xì)胞的增殖受到明顯的抑制,說明尼克酰胺是通過下調(diào)SATB1誘導(dǎo)了F9細(xì)胞的凋亡�？傊�,本研究首次發(fā)現(xiàn)尼克酰胺作為誘導(dǎo)因子能通過下調(diào)SATB1抑制F9細(xì)胞的增殖和誘導(dǎo)F9細(xì)胞的凋亡。這一結(jié)果對尼克酰胺在腫瘤的治療應(yīng)用上提供了重要的理論基礎(chǔ),也為腫瘤的治療提供了一新思路。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.79

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本文編號：1845943