網(wǎng)膜素-1對結(jié)腸癌干細(xì)胞增殖凋亡的影響及機(jī)制研究
發(fā)布時間：2018-08-09 19:26
【摘要】：目的研究網(wǎng)膜素-1(Omentin-1)對結(jié)腸癌干細(xì)胞增殖、凋亡的影響,觀察網(wǎng)膜素-1對結(jié)腸癌干細(xì)胞PI3K/Akt信號通路的影響以及與PI3K/Akt信號通路抑制劑LY294002之間是否存在協(xié)同作用,探討網(wǎng)膜素-1作用于結(jié)腸癌干細(xì)胞的可能機(jī)制。方法1.采用間接免疫磁珠方法,從結(jié)腸癌細(xì)胞株SW480中分選出結(jié)腸癌干細(xì)胞,在無血清培養(yǎng)基中培養(yǎng)結(jié)腸癌干細(xì)胞。2.結(jié)腸癌干細(xì)胞鑒定:克隆球形成實驗檢測結(jié)腸癌干細(xì)胞成球情況、分化實驗檢測結(jié)腸癌干細(xì)胞分化情況、流式細(xì)胞儀檢測結(jié)腸癌干細(xì)胞表面標(biāo)志物CD133。3.CCK-8實驗:(1)結(jié)腸癌干細(xì)胞分為對照組(不加網(wǎng)膜素-1及LY294002)、Omentin1組(加入1μg/ml網(wǎng)膜素-1)、Omentin2組(加入2μg/ml網(wǎng)膜素-1)、Omentin LY組(加入1μg/ml網(wǎng)膜素-1及50μM LY294002)、LY組(加入50μM LY294002),干預(yù)24小時后,檢測細(xì)胞OD值;(2)分別檢測Omentin-11 ug/ml干預(yù)細(xì)胞0h、1h、6h、24h、48h后細(xì)胞OD值。4.結(jié)腸癌干細(xì)胞分為對照組(不加網(wǎng)膜素-1及LY294002)、Omentin1組(加入1μg/ml網(wǎng)膜素-1)、Omentin2組(加入2μg/ml網(wǎng)膜素-1)、Omentin LY組(加入1μg/ml網(wǎng)膜素-1及50μM LY294002)、LY組(加入50μM LY294002),干預(yù)24小時后,流式細(xì)胞術(shù)檢測不同組別細(xì)胞凋亡情況。5.結(jié)腸癌干細(xì)胞分為對照組(不加網(wǎng)膜素-1)、Omentin1組(加入1μg/ml網(wǎng)膜素-1)、Omentin2組(加入2μg/ml網(wǎng)膜素-1),干預(yù)24小時后,提取出細(xì)胞蛋白,用Western blot方法檢測不同濃度Omentin-1干預(yù)后Akt、p Akt蛋白表達(dá)。結(jié)果1.結(jié)腸癌干細(xì)胞在無血清培養(yǎng)基中培養(yǎng)至第1天可見小的干細(xì)胞球形成,第5天可見明顯干細(xì)胞球形成,第10天干細(xì)胞球進(jìn)一步增大且變圓,呈懸浮生長;分化實驗顯示在含血清培養(yǎng)基培養(yǎng)至第9天干細(xì)胞球全部分化成為梭狀的結(jié)腸癌SW480細(xì)胞,呈貼壁生長;流式細(xì)胞儀分析顯示CD133+結(jié)腸癌干細(xì)胞含量達(dá)80.3%。2.Omentin1組、Omentin2組、Omentin LY組、LY組,OD值均較對照組下降,差異具有統(tǒng)計學(xué)意義(P0.05);Omentin2組較Omentin1組的OD值更低,差異具有統(tǒng)計學(xué)意義(P0.05);Omentin LY組OD值低于Omentin1組和LY組,差異具有統(tǒng)計學(xué)意義(P0.05)。3.1μg/ml網(wǎng)膜素-1作用于結(jié)腸癌干細(xì)胞0h、1h、6h、24h、48h后,隨干預(yù)時間增加,OD值呈逐漸下降趨勢,差異具有統(tǒng)計學(xué)意義(P0.05)。4.Omentin1組、Omentin2組、Omentin LY組、LY組,細(xì)胞凋亡率均較對照組上升,差異具有統(tǒng)計學(xué)意義(P0.05);Omentin2組的凋亡率高于Omentin1組,差異具有統(tǒng)計學(xué)意義(P0.05);Omentin LY組凋亡率高于Omentin1組和LY組,差異具有統(tǒng)計學(xué)意義(P0.05)。5.Western blot結(jié)果顯示Omentin-1干預(yù)后p Akt/Akt蛋白表達(dá)下降,其中Omentin2組與對照組相比,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論1.網(wǎng)膜素-1抑制結(jié)腸癌干細(xì)胞增殖,其作用呈濃度-時間依賴性。2.網(wǎng)膜素-1促進(jìn)結(jié)腸癌干細(xì)胞凋亡,呈濃度依賴性。3.網(wǎng)膜素-1對結(jié)腸癌干細(xì)胞的作用機(jī)制可能與抑制Akt通路有關(guān)。4.網(wǎng)膜素-1與LY294002在抑制結(jié)腸癌干細(xì)胞增殖、促進(jìn)其凋亡的過程中具有協(xié)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.35
本文編號：2175083
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.35
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