蒜素抑制人甲狀腺乳頭狀癌TPC-1細胞的作用及機制
發(fā)布時間：2018-12-18 10:09
【摘要】：目的探討蒜素(Allicin)抑制人甲狀腺乳頭狀癌TPC-1細胞生長的作用及機制。方法選取處于對數(shù)生長期的人甲狀腺乳頭狀癌TPC-1細胞用于實驗,采用不同濃度的Allicin干預細胞(對照組0μg/ml;實驗組10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml);四甲基偶氮唑藍法(MTT法)觀察同一時間不同濃度的Allicin和同濃度的Allicin干預不同的時間對TPC-1細胞生長情況的影響,計算IC50;應用倒置顯微鏡和透射電鏡分別觀察Allicin對細胞形態(tài)和內(nèi)部超微結構的影響;采用劃痕實驗觀察Allicin對TPC-1細胞遷移能力的影響;流式細胞術(FCM)檢測應用Allicin后TPC-1細胞周期中各時相細胞比例的變化;免疫細胞化學與Western blot實驗檢測經(jīng)Allicin干預后TPC-1細胞中Bcl-2和Bax蛋白的表達情況;實時熒光定量PCR實驗檢測Allicin對TPC-1細胞內(nèi)Bcl-2 m RNA和Bax m RNA表達水平的影響結果MTT實驗結果顯示不同濃度Allicin均抑制TPC-1細胞的增殖,與對照組相比差異有統(tǒng)計學意義(P0.05),且Allicin對細胞增殖的抑制率呈顯出濃度和時間的依賴性;倒置顯微鏡和透射電鏡觀察可見,實驗組培養(yǎng)48h后細胞皺縮體積變小,輪廓不清,透光度降低,細胞表面微絨毛減少甚至消失,核質(zhì)比例減小,染色質(zhì)分布不規(guī)則且呈邊集、固縮樣改變,而對照組細胞生長狀態(tài)較好,細胞器無明顯變化;劃痕實驗顯示,培養(yǎng)48h后的對照組劃痕距離明顯變小,實驗組中隨著藥物濃度的提高,遷移率逐漸降低,與對照組相比P0.05,Allicin呈濃度依賴性抑制TPC-1細胞的遷移能力(P0.05);不同濃度Allicin干預后細胞周期各時相的細胞比例發(fā)生變化,當Allicin濃度為10μg·m L-1時各時相細胞的比例與對照組相比P0.05,但隨著Allicin濃度的提高G2/M期細胞所占比例明顯增多而G0/G1期細胞所占比例降低,同對照組相比較P0.05差異具有統(tǒng)計學意義;免疫細胞化學與Western blot結果顯示,對照組與實驗組均有Bcl-2蛋白和Bax蛋白的表達,但隨著Allicin濃度的提高Bcl-2的表達量減少,相反Bax的表達增加,與對照組相比P0.05,且具有濃度依賴性;PCR實驗發(fā)現(xiàn),對照組與實驗組均有Bcl-2 m RNA和Bax m RNA的表達,但隨著Allicin濃度的增高Bcl-2 m RNA的表達量減少,相反Bax m RNA的表達增加,與對照組相比P0.05,且具有濃度依賴性。結論1 Allicin能夠抑制TPC-1細胞的增殖,抑制效果具有濃度和時間依賴性;2Allicin能夠調(diào)控TPC-1細胞的細胞周期,主要表現(xiàn)為細胞周期阻滯于G2/M期;3Allicin能夠抑制TPC-1細胞的遷移與侵襲能力;4 Allicin可能通過上調(diào)Bax、下調(diào)Bcl-2誘導TPC-1細胞凋亡。
[Abstract]:Objective to investigate the inhibitory effect and mechanism of allicin (Allicin) on the growth of human papillary thyroid carcinoma (TPC-1) cells. Methods Human papillary thyroid carcinoma (TPC-1) cells in logarithmic growth phase were treated with different concentrations of Allicin (control group 0 渭 g / ml; experimental group 10 渭 g / ml 20 渭 g / ml 30 渭 g / ml 40 渭 g / ml). The effects of different concentrations of Allicin at the same time and the same concentration of Allicin on the growth of TPC-1 cells were observed by MTT method. IC50; was calculated. The effects of Allicin on the morphology and ultrastructure of TPC-1 cells were observed by inverted microscope and transmission electron microscope, and the effects of Allicin on the migration ability of TPC-1 cells were observed by scratch test. The expression of Bcl-2 and Bax protein in TPC-1 cells after Allicin intervention was detected by immunocytochemistry and Western blot assay. The effect of Allicin on the expression of Bcl-2 m RNA and Bax m RNA in TPC-1 cells was detected by real-time fluorescence quantitative PCR assay. The results of MTT assay showed that Allicin at different concentrations inhibited the proliferation of TPC-1 cells. Compared with the control group, the difference was statistically significant (P0.05), and the inhibitory rate of Allicin on cell proliferation showed a concentration-and time-dependent manner. Observation by inverted microscope and transmission electron microscope showed that after 48 hours of culture, the cells in the experimental group became smaller in volume, unclear in outline, decreased in transmittance, decreased or even disappeared of microvilli on the cell surface, reduced the ratio of nucleus to cytoplasm, and the chromatin distributed irregularly and showed edge-set. The changes of pyknosis were similar to those of the control group, but the growth state of the cells in the control group was better, and the organelle had no obvious change. The scratch test showed that the distance of scratch in the control group decreased significantly after 48 hours of culture, and the mobility decreased with the increase of the drug concentration in the experimental group, compared with the control group (P0.05). Allicin inhibited the migration of TPC-1 cells in a concentration-dependent manner (P0.05). After different concentrations of Allicin, the proportion of cells in each phase of cell cycle changed. When the concentration of Allicin was 10 渭 g mL ~ (-1), the proportion of cells in each phase was higher than that in control group (P 0.05). However, with the increase of Allicin concentration, the proportion of G2 / M phase cells increased significantly, while the proportion of G0/G1 phase cells decreased, compared with the control group (P0.05), the difference was statistically significant. The results of immunocytochemistry and Western blot showed that the expression of Bcl-2 protein and Bax protein were found in both the control group and the experimental group, but the expression of Bcl-2 decreased with the increase of Allicin concentration, whereas the expression of Bax increased, compared with the control group (P0.05). And it is concentration-dependent. PCR assay showed that the expression of Bcl-2 m RNA and Bax m RNA was found in both the control group and the experimental group, but with the increase of Allicin concentration, the expression of Bcl-2 m RNA decreased, whereas the expression of Bax m RNA increased, compared with the control group (P 0.05). And it is concentration-dependent. Conclusion 1 Allicin can inhibit the proliferation of TPC-1 cells in a concentration-and time-dependent manner, and 2Allicin can regulate the cell cycle of TPC-1 cells, mainly in G _ 2 / M phase. 3Allicin could inhibit the migration and invasion of TPC-1 cells, and Allicin might up-regulate the apoptosis of TPC-1 cells by down-regulating Bcl-2.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R736.1
本文編號：2385695
[Abstract]:Objective to investigate the inhibitory effect and mechanism of allicin (Allicin) on the growth of human papillary thyroid carcinoma (TPC-1) cells. Methods Human papillary thyroid carcinoma (TPC-1) cells in logarithmic growth phase were treated with different concentrations of Allicin (control group 0 渭 g / ml; experimental group 10 渭 g / ml 20 渭 g / ml 30 渭 g / ml 40 渭 g / ml). The effects of different concentrations of Allicin at the same time and the same concentration of Allicin on the growth of TPC-1 cells were observed by MTT method. IC50; was calculated. The effects of Allicin on the morphology and ultrastructure of TPC-1 cells were observed by inverted microscope and transmission electron microscope, and the effects of Allicin on the migration ability of TPC-1 cells were observed by scratch test. The expression of Bcl-2 and Bax protein in TPC-1 cells after Allicin intervention was detected by immunocytochemistry and Western blot assay. The effect of Allicin on the expression of Bcl-2 m RNA and Bax m RNA in TPC-1 cells was detected by real-time fluorescence quantitative PCR assay. The results of MTT assay showed that Allicin at different concentrations inhibited the proliferation of TPC-1 cells. Compared with the control group, the difference was statistically significant (P0.05), and the inhibitory rate of Allicin on cell proliferation showed a concentration-and time-dependent manner. Observation by inverted microscope and transmission electron microscope showed that after 48 hours of culture, the cells in the experimental group became smaller in volume, unclear in outline, decreased in transmittance, decreased or even disappeared of microvilli on the cell surface, reduced the ratio of nucleus to cytoplasm, and the chromatin distributed irregularly and showed edge-set. The changes of pyknosis were similar to those of the control group, but the growth state of the cells in the control group was better, and the organelle had no obvious change. The scratch test showed that the distance of scratch in the control group decreased significantly after 48 hours of culture, and the mobility decreased with the increase of the drug concentration in the experimental group, compared with the control group (P0.05). Allicin inhibited the migration of TPC-1 cells in a concentration-dependent manner (P0.05). After different concentrations of Allicin, the proportion of cells in each phase of cell cycle changed. When the concentration of Allicin was 10 渭 g mL ~ (-1), the proportion of cells in each phase was higher than that in control group (P 0.05). However, with the increase of Allicin concentration, the proportion of G2 / M phase cells increased significantly, while the proportion of G0/G1 phase cells decreased, compared with the control group (P0.05), the difference was statistically significant. The results of immunocytochemistry and Western blot showed that the expression of Bcl-2 protein and Bax protein were found in both the control group and the experimental group, but the expression of Bcl-2 decreased with the increase of Allicin concentration, whereas the expression of Bax increased, compared with the control group (P0.05). And it is concentration-dependent. PCR assay showed that the expression of Bcl-2 m RNA and Bax m RNA was found in both the control group and the experimental group, but with the increase of Allicin concentration, the expression of Bcl-2 m RNA decreased, whereas the expression of Bax m RNA increased, compared with the control group (P 0.05). And it is concentration-dependent. Conclusion 1 Allicin can inhibit the proliferation of TPC-1 cells in a concentration-and time-dependent manner, and 2Allicin can regulate the cell cycle of TPC-1 cells, mainly in G _ 2 / M phase. 3Allicin could inhibit the migration and invasion of TPC-1 cells, and Allicin might up-regulate the apoptosis of TPC-1 cells by down-regulating Bcl-2.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R736.1
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