【摘要】：目的探討蒜素(Allicin)抑制人甲狀腺乳頭狀癌TPC-1細(xì)胞生長(zhǎng)的作用及機(jī)制。方法選取處于對(duì)數(shù)生長(zhǎng)期的人甲狀腺乳頭狀癌TPC-1細(xì)胞用于實(shí)驗(yàn),采用不同濃度的Allicin干預(yù)細(xì)胞(對(duì)照組0μg/ml;實(shí)驗(yàn)組10μg/ml、20μg/ml、30μg/ml、40μg/ml、50μg/ml);四甲基偶氮唑藍(lán)法(MTT法)觀察同一時(shí)間不同濃度的Allicin和同濃度的Allicin干預(yù)不同的時(shí)間對(duì)TPC-1細(xì)胞生長(zhǎng)情況的影響,計(jì)算IC50;應(yīng)用倒置顯微鏡和透射電鏡分別觀察Allicin對(duì)細(xì)胞形態(tài)和內(nèi)部超微結(jié)構(gòu)的影響;采用劃痕實(shí)驗(yàn)觀察Allicin對(duì)TPC-1細(xì)胞遷移能力的影響;流式細(xì)胞術(shù)(FCM)檢測(cè)應(yīng)用Allicin后TPC-1細(xì)胞周期中各時(shí)相細(xì)胞比例的變化;免疫細(xì)胞化學(xué)與Western blot實(shí)驗(yàn)檢測(cè)經(jīng)Allicin干預(yù)后TPC-1細(xì)胞中Bcl-2和Bax蛋白的表達(dá)情況;實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)檢測(cè)Allicin對(duì)TPC-1細(xì)胞內(nèi)Bcl-2 m RNA和Bax m RNA表達(dá)水平的影響結(jié)果MTT實(shí)驗(yàn)結(jié)果顯示不同濃度Allicin均抑制TPC-1細(xì)胞的增殖,與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05),且Allicin對(duì)細(xì)胞增殖的抑制率呈顯出濃度和時(shí)間的依賴性;倒置顯微鏡和透射電鏡觀察可見,實(shí)驗(yàn)組培養(yǎng)48h后細(xì)胞皺縮體積變小,輪廓不清,透光度降低,細(xì)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(duì)照組細(xì)胞生長(zhǎng)狀態(tài)較好,細(xì)胞器無明顯變化;劃痕實(shí)驗(yàn)顯示,培養(yǎng)48h后的對(duì)照組劃痕距離明顯變小,實(shí)驗(yàn)組中隨著藥物濃度的提高,遷移率逐漸降低,與對(duì)照組相比P0.05,Allicin呈濃度依賴性抑制TPC-1細(xì)胞的遷移能力(P0.05);不同濃度Allicin干預(yù)后細(xì)胞周期各時(shí)相的細(xì)胞比例發(fā)生變化,當(dāng)Allicin濃度為10μg·m L-1時(shí)各時(shí)相細(xì)胞的比例與對(duì)照組相比P0.05,但隨著Allicin濃度的提高G2/M期細(xì)胞所占比例明顯增多而G0/G1期細(xì)胞所占比例降低,同對(duì)照組相比較P0.05差異具有統(tǒng)計(jì)學(xué)意義;免疫細(xì)胞化學(xué)與Western blot結(jié)果顯示,對(duì)照組與實(shí)驗(yàn)組均有Bcl-2蛋白和Bax蛋白的表達(dá),但隨著Allicin濃度的提高Bcl-2的表達(dá)量減少,相反Bax的表達(dá)增加,與對(duì)照組相比P0.05,且具有濃度依賴性;PCR實(shí)驗(yàn)發(fā)現(xiàn),對(duì)照組與實(shí)驗(yàn)組均有Bcl-2 m RNA和Bax m RNA的表達(dá),但隨著Allicin濃度的增高Bcl-2 m RNA的表達(dá)量減少,相反Bax m RNA的表達(dá)增加,與對(duì)照組相比P0.05,且具有濃度依賴性。結(jié)論1 Allicin能夠抑制TPC-1細(xì)胞的增殖,抑制效果具有濃度和時(shí)間依賴性;2Allicin能夠調(diào)控TPC-1細(xì)胞的細(xì)胞周期,主要表現(xiàn)為細(xì)胞周期阻滯于G2/M期;3Allicin能夠抑制TPC-1細(xì)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(dǎo)TPC-1細(xì)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(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R736.1

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