蒜素抑制人甲状腺乳头状癌TPC-1细胞的作用及机制
发布时间：2018-12-18 10:09
【摘要】：目的探讨蒜素(Allicin)抑制人甲状腺乳头状癌TPC-1细胞生长的作用及机制。方法选取处于对数生长期的人甲状腺乳头状癌TPC-1细胞用于实验,采用不同浓度的Allicin干预细胞(对照组0μg/ml;实验组10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml);四甲基偶氮唑蓝法(MTT法)观察同一时间不同浓度的Allicin和同浓度的Allicin干预不同的时间对TPC-1细胞生长情况的影响,计算IC50;应用倒置显微镜和透射电镜分别观察Allicin对细胞形态和内部超微结构的影响;采用划痕实验观察Allicin对TPC-1细胞迁移能力的影响;流式细胞术(FCM)检测应用Allicin后TPC-1细胞周期中各时相细胞比例的变化;免疫细胞化学与Western blot实验检测经Allicin干预后TPC-1细胞中Bcl-2和Bax蛋白的表达情况;实时荧光定量PCR实验检测Allicin对TPC-1细胞内Bcl-2 m RNA和Bax m RNA表达水平的影响结果MTT实验结果显示不同浓度Allicin均抑制TPC-1细胞的增殖,与对照组相比差异有统计学意义(P0.05),且Allicin对细胞增殖的抑制率呈显出浓度和时间的依赖性;倒置显微镜和透射电镜观察可见,实验组培养48h后细胞皱缩体积变小,轮廓不清,透光度降低,细胞表面微绒毛减少甚至消失,核质比例减小,染色质分布不规则且呈边集、固缩样改变,而对照组细胞生长状态较好,细胞器无明显变化;划痕实验显示,培养48h后的对照组划痕距离明显变小,实验组中随着药物浓度的提高,迁移率逐渐降低,与对照组相比P0.05,Allicin呈浓度依赖性抑制TPC-1细胞的迁移能力(P0.05);不同浓度Allicin干预后细胞周期各时相的细胞比例发生变化,当Allicin浓度为10μg·m L-1时各时相细胞的比例与对照组相比P0.05,但随着Allicin浓度的提高G2/M期细胞所占比例明显增多而G0/G1期细胞所占比例降低,同对照组相比较P0.05差异具有统计学意义;免疫细胞化学与Western blot结果显示,对照组与实验组均有Bcl-2蛋白和Bax蛋白的表达,但随着Allicin浓度的提高Bcl-2的表达量减少,相反Bax的表达增加,与对照组相比P0.05,且具有浓度依赖性;PCR实验发现,对照组与实验组均有Bcl-2 m RNA和Bax m RNA的表达,但随着Allicin浓度的增高Bcl-2 m RNA的表达量减少,相反Bax m RNA的表达增加,与对照组相比P0.05,且具有浓度依赖性。结论1 Allicin能够抑制TPC-1细胞的增殖,抑制效果具有浓度和时间依赖性;2Allicin能够调控TPC-1细胞的细胞周期,主要表现为细胞周期阻滞于G2/M期;3Allicin能够抑制TPC-1细胞的迁移与侵袭能力;4 Allicin可能通过上调Bax、下调Bcl-2诱导TPC-1细胞凋亡。
[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.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R736.1
本文编号：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.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R736.1
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