【摘要】：目的:本實(shí)驗(yàn)采用MTT實(shí)驗(yàn),Western Blot實(shí)驗(yàn),平板克隆實(shí)驗(yàn)及流式細(xì)胞實(shí)驗(yàn)檢測(cè)微管蛋白抑制劑ZP-20f對(duì)體外培養(yǎng)的口腔鱗癌細(xì)胞的增殖、凋亡及細(xì)胞周期的影響,并進(jìn)行相關(guān)數(shù)據(jù)分析。探討微管蛋白抑制劑Zp-20f在口腔鱗狀細(xì)胞癌中的作用機(jī)制。方法:1、口腔鱗癌SCC-9、CAL-27細(xì)胞的細(xì)胞培養(yǎng)。2、MTT法檢測(cè)Zp-20f對(duì)口腔鱗癌細(xì)胞SCC-9、CAL-27細(xì)胞的毒性作用,并計(jì)算最實(shí)試驗(yàn)濃度IC50。3、流式細(xì)胞術(shù)檢測(cè)口腔鱗癌細(xì)胞SCC-9、CAL-27細(xì)胞的周期及凋亡率。4、應(yīng)用平板克隆形成實(shí)驗(yàn)分析Zp-20f對(duì)口腔鱗狀細(xì)胞癌細(xì)胞SCC-9、CAL-27的毒性效應(yīng)。5、Wetern Blot檢測(cè)SCC-9、CAL-27細(xì)胞中相關(guān)蛋白的表達(dá)變化。結(jié)果:1、ZP-20f可顯著抑制口腔鱗癌細(xì)胞增殖ZP-20f在一定濃度范圍內(nèi)能顯著抑制口腔鱗癌的增殖作用,且其抑制活性具有濃度依賴(lài)作用。平板克隆實(shí)驗(yàn)可以看出ZP-20f能濃度依賴(lài)的抑制腫瘤細(xì)胞克隆的形成。2、ZP-20f可在G2/M期阻滯細(xì)胞周期ZP-20f在一定濃度范圍內(nèi)能顯著誘導(dǎo)口腔鱗癌細(xì)胞G2/M期阻滯,并具隨著化合物濃度的增加,G2/M期細(xì)胞比例隨之增加。3、ZP-20f可在G2/M期阻滯細(xì)胞周期ZP-20f在一定濃度范圍內(nèi)能顯著誘導(dǎo)口腔鱗癌細(xì)胞G2/M期阻滯,并具隨著化合物濃度的增加,G2/M期細(xì)胞比例隨之增加。4、細(xì)胞周期相關(guān)蛋白量發(fā)生變化化合物在一定濃度范圍內(nèi)能引起細(xì)胞內(nèi)周期和凋亡相關(guān)蛋白含量的變化,使細(xì)胞中周期與凋亡相關(guān)蛋白Cycling B1、p-H3和cleaved-PARP的量顯著增加,且具有濃度依賴(lài)性(圖5)。結(jié)論:1、ZP-20f對(duì)口腔鱗癌細(xì)胞SCC-9和Cal-27的體外增殖抑制活性較強(qiáng),能明顯將細(xì)胞周期阻滯在G2/M期,并能誘導(dǎo)細(xì)胞凋亡;2、ZP-20f在體外有較好的抗腫瘤作用。其作用機(jī)制可能與p-H3或cleaved-PARP的上調(diào)及對(duì)微管蛋白的抑制作用相關(guān),是一個(gè)一個(gè)非常有前景的抗腫瘤藥物。
[Abstract]:Objective: to investigate the effects of tubulin inhibitor (ZP-20f) on the proliferation, apoptosis and cell cycle of oral squamous carcinoma cells cultured in vitro by MTT, Western Blot assay, plate clone assay and flow cytometry. And carries on the correlation data analysis. To investigate the mechanism of tubulin inhibitor Zp-20f in oral squamous cell carcinoma. Methods: 1. The cell culture of oral squamous cell carcinoma (SCC-9,CAL-27) cells. 2MTT assay was used to detect the toxic effect of Zp-20f on oral squamous cell carcinoma SCC-9,CAL-27 cells, and the concentration of IC50.3, was calculated. Flow cytometry was used to detect the cell cycle and apoptosis rate of oral squamous cell SCC-9,CAL-27 cells. 4. The toxic effect of Zp-20f on SCC-9,CAL-27 of oral squamous cell carcinoma cells was analyzed by plate clone formation assay. The expression of related proteins in SCC-9,CAL-27 cells was detected by Wetern Blot. Results: 1ZP-20f could significantly inhibit the proliferation of oral squamous cell carcinoma (OSCC) cell line ZP-20f in a certain concentration range, and its inhibitory activity was concentration-dependent. Plate cloning assay showed that ZP-20f could inhibit the formation of tumor cell clone in a concentration-dependent manner. 2ZP-20f could significantly induce G _ 2 / M phase arrest of oral squamous carcinoma cells in a certain concentration range by blocking cell cycle ZP-20f in G _ 2 / M phase. With the increase of the concentration of compounds, the proportion of G2 / M phase cells increased. 3ZP-20f could significantly induce the G _ 2 / M phase arrest of oral squamous carcinoma cells in a certain concentration range by blocking cell cycle ZP-20f in G _ 2 / M phase. With the increase of the concentration of compounds, the proportion of cells in G _ 2 / M phase increased. 4. The change of cell cycle associated protein content in a certain concentration range could cause changes in cell cycle and apoptosis-related protein content. Cell cycle and apoptosis-related protein Cycling B1, p-H3 and cleaved-PARP were significantly increased in a concentration-dependent manner (Fig. 5). Conclusion: (1) ZP-20f can inhibit the proliferation of oral squamous cell SCC-9 and Cal-27 in vitro, block the cell cycle at G _ 2 / M phase and induce apoptosis, and ZP-20f has a good anti-tumor effect in vitro. The mechanism may be related to the upregulation of p-H3 or cleaved-PARP and the inhibition of tubulin. It is a promising antitumor drug.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R739.8

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