网膜素-1对结肠癌干细胞增殖凋亡的影响及机制研究

发布时间：2018-08-09 19:26

【摘要】：目的研究网膜素-1(Omentin-1)对结肠癌干细胞增殖、凋亡的影响,观察网膜素-1对结肠癌干细胞PI3K/Akt信号通路的影响以及与PI3K/Akt信号通路抑制剂LY294002之间是否存在协同作用,探讨网膜素-1作用于结肠癌干细胞的可能机制。方法1.采用间接免疫磁珠方法,从结肠癌细胞株SW480中分选出结肠癌干细胞,在无血清培养基中培养结肠癌干细胞。2.结肠癌干细胞鉴定:克隆球形成实验检测结肠癌干细胞成球情况、分化实验检测结肠癌干细胞分化情况、流式细胞仪检测结肠癌干细胞表面标志物CD133。3.CCK-8实验:(1)结肠癌干细胞分为对照组(不加网膜素-1及LY294002)、Omentin1组(加入1μg/ml网膜素-1)、Omentin2组(加入2μg/ml网膜素-1)、Omentin LY组(加入1μg/ml网膜素-1及50μM LY294002)、LY组(加入50μM LY294002),干预24小时后,检测细胞OD值;(2)分别检测Omentin-11 ug/ml干预细胞0h、1h、6h、24h、48h后细胞OD值。4.结肠癌干细胞分为对照组(不加网膜素-1及LY294002)、Omentin1组(加入1μg/ml网膜素-1)、Omentin2组(加入2μg/ml网膜素-1)、Omentin LY组(加入1μg/ml网膜素-1及50μM LY294002)、LY组(加入50μM LY294002),干预24小时后,流式细胞术检测不同组别细胞凋亡情况。5.结肠癌干细胞分为对照组(不加网膜素-1)、Omentin1组(加入1μg/ml网膜素-1)、Omentin2组(加入2μg/ml网膜素-1),干预24小时后,提取出细胞蛋白,用Western blot方法检测不同浓度Omentin-1干预后Akt、p Akt蛋白表达。结果1.结肠癌干细胞在无血清培养基中培养至第1天可见小的干细胞球形成,第5天可见明显干细胞球形成,第10天干细胞球进一步增大且变圆,呈悬浮生长;分化实验显示在含血清培养基培养至第9天干细胞球全部分化成为梭状的结肠癌SW480细胞,呈贴壁生长;流式细胞仪分析显示CD133+结肠癌干细胞含量达80.3%。2.Omentin1组、Omentin2组、Omentin LY组、LY组,OD值均较对照组下降,差异具有统计学意义(P0.05);Omentin2组较Omentin1组的OD值更低,差异具有统计学意义(P0.05);Omentin LY组OD值低于Omentin1组和LY组,差异具有统计学意义(P0.05)。3.1μg/ml网膜素-1作用于结肠癌干细胞0h、1h、6h、24h、48h后,随干预时间增加,OD值呈逐渐下降趋势,差异具有统计学意义(P0.05)。4.Omentin1组、Omentin2组、Omentin LY组、LY组,细胞凋亡率均较对照组上升,差异具有统计学意义(P0.05);Omentin2组的凋亡率高于Omentin1组,差异具有统计学意义(P0.05);Omentin LY组凋亡率高于Omentin1组和LY组,差异具有统计学意义(P0.05)。5.Western blot结果显示Omentin-1干预后p Akt/Akt蛋白表达下降,其中Omentin2组与对照组相比,差异有统计学意义(P0.05)。结论1.网膜素-1抑制结肠癌干细胞增殖,其作用呈浓度-时间依赖性。2.网膜素-1促进结肠癌干细胞凋亡,呈浓度依赖性。3.网膜素-1对结肠癌干细胞的作用机制可能与抑制Akt通路有关。4.网膜素-1与LY294002在抑制结肠癌干细胞增殖、促进其凋亡的过程中具有协同作用。
[Abstract]:Objective to study the effects of omentin-1 (Omentin-1) on the proliferation and apoptosis of colon cancer stem cells, and to observe the effect of omentin-1 on the PI3K/Akt signaling pathway of colon cancer stem cells and whether there is a synergistic effect with PI3K/Akt signal pathway inhibitor LY294002. To explore the possible mechanism of omentin-1 acting on colon cancer stem cells. Method 1. Colon cancer stem cells were isolated from colon cancer cell line SW480 by indirect immunomagnetic beads and cultured in serum-free medium. Identification of Colon Cancer Stem cells: the Colon Cancer Stem Cell formation Test was used to detect the formation of Colon Cancer Stem cells, and the differentiation Test to detect the Colon Cancer Stem Cell differentiation. CD133.3.CCK-8 assay of surface markers of colon cancer stem cells: (1) Colon cancer stem cells were divided into control group (without omentin-1 and LY294002), Omentin1 group (adding 1 渭 g/ml omentin -1), Omentin2 group (adding 2 渭 g/ml omentin -1) and omentin LY group (adding 1 渭 g/ml omentin 1). The LY group (50 渭 M LY294002) was treated with 50 渭 M LY294002 for 24 hours. (2) the OD value of the cells treated with Omentin-11 ug/ml for 24 h or 48 h was measured respectively, and the OD value was 4. 4 after the intervention of Omentin-11 ug/ml for 24 h or 48 h. Colon cancer stem cells were divided into two groups: control group (without omentin 1 and LY294002) and Omentin1 group (with 1 渭 g/ml omentin -1) and Omentin2 group (with 2 渭 g/ml omentin 1) Oimentin LY group (1 渭 g/ml omentin -1 and 50 渭 M LY294002) and LY group (50 渭 M LY294002). Flow cytometry was used to detect apoptosis in different groups of cells. Colon cancer stem cells were divided into control group (without omentin 1) and Omentin1 group (adding 1 渭 g/ml omentin -1) and Omentin2 group (2 渭 g/ml omentin 1). After 24 hours of intervention, the cell protein was extracted and the expression of Akt protein was detected by Western blot method. Result 1. Colon cancer stem cells were cultured in serum-free medium to form small stem cell balls on the first day, obvious stem cell balls on the fifth day, and further enlarged and rounded stem cells balls on the 10th day. The differentiation test showed that the stem cells were differentiated into fusiform colon cancer SW480 cells in serum-containing medium until the 9th day, and the cells were adherent to the wall. Flow cytometry analysis showed that the OD value of CD133 colon cancer stem cells in Omentin 2 group was lower than that in control group (P0.05), and the OD value of Omentin2 group was lower than that of Omentin1 group. The difference was statistically significant (P0.05) the OD value of Omentin LY group was lower than that of Omentin1 group and LY group, and the difference was statistically significant (P0.05) .3.1 渭 g/ml omentin--1 was applied to colon cancer stem cells for 48 h, and the OD value decreased gradually with the increase of intervention time. The difference was statistically significant (P0.05). 4. The apoptosis rate of Omentin1 group was higher than that of control group (P0.05). The apoptosis rate of Omentin2 group was significantly higher than that of Omentin1 group (P0.05). The apoptosis rate of Omentin1 group was higher than that of Omentin1 group and LY group, and the apoptosis rate of Omentin2 group was higher than that of Omentin1 group and LY group, and the apoptosis rate of Omentin1 group was higher than that of Omentin1 group and LY group. The difference was statistically significant (P0.05). 5. Western blot results showed that the expression of p Akt/Akt protein decreased after Omentin-1 intervention, and the difference between the Omentin2 group and the control group was statistically significant (P0.05). Conclusion 1. Omentin-1 inhibits the proliferation of colon cancer stem cells in a concentration-time dependent manner. Omentin-1 promotes apoptosis of colon cancer stem cells in a concentration dependent manner. The mechanism of omentin-1 on colon cancer stem cells may be related to the inhibition of Akt pathway. 4. Omentin-1 and LY294002 have synergistic effects in inhibiting the proliferation and promoting the apoptosis of colon cancer stem cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35

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