發(fā)布時間：2018-06-26 01:34

  本文選題：TWIST + Hep-2細胞��； 參考：《山東大學》2010年碩士論文

【摘要】： 喉癌是頭頸部常見惡性腫瘤之一,過去的20年中,多種治療方法的實施,并沒有使患者的生存率明顯升高。紫杉醇是目前臨床上常用化療藥物,聯(lián)合其他的化療藥物或作為放療增敏劑,廣泛的用于晚期腫瘤的治療,療效顯著,近年來,有報道紫杉醇用于晚期頭頸腫瘤化療,可延長患者的生存期。研究報道紫杉醇能誘導多種癌細胞發(fā)生凋亡,抑制腫瘤的生長。但紫杉醇在喉癌的臨床應用和基礎研究報道均較少。TWIST,屬于堿性螺旋-環(huán)-螺旋轉錄因子家族,在多種實體腫瘤中過表達,參與腫瘤的侵襲、轉移,并與腫瘤預后和患者生存有關。TWIST的過高表達,在腫瘤進展的過程中,不僅能夠促進腫瘤轉移,而且還與腫瘤細胞對化療藥物耐藥的產生有關。TWIST在腫瘤轉移和多藥耐藥方面存在調控作用,其具體機制還不是很清楚。本研究,針對轉錄因子TWIST在人喉癌細胞系Hep-2中的表達變化及其與紫杉醇誘導Hep-2細胞凋亡的關系進行初步研究,為探討喉癌化療敏感性及耐藥機制提供理論依據。 目的： 1.探討TWIST在喉癌Hep-2細胞中的表達； 2.初步探討TWIST在喉癌Hep-2細胞中的表達與紫杉醇誘導Hep-2細胞凋亡的關系。 方法： 以人喉癌Hep-2細胞為實驗材料,以1×10-9、5×10-9、10×-9、25×10-9mol/L的紫杉醇作用于Hep-2細胞,采用倒置相差顯微鏡觀察其形態(tài)變化,四甲基偶氮唑藍(MTT)比色法觀察不同濃度紫杉醇對Hep-2細胞的相對存活率的影響,吖啶橙細胞化學染色觀察紫杉醇對Hep-2細胞凋亡的影響,流式細胞術檢測Hep-2細胞的凋亡率,RT-PCR和Western-blotting分別檢測紫杉醇作用后的Hep-2細胞中TWIST mRNA和蛋白的表達變化情況。 結果： 1. TWIST在喉癌Hep-2細胞中存在表達 2.倒置相差顯微鏡下和吖啶橙細胞化學染色可見細胞凋亡的形態(tài)學改變,顯示加藥組細胞染色質固縮,細胞核聚集呈現塊狀、新月狀,出現凋亡小體。 3.MTT顯示紫杉醇對Hep-2細胞的生長抑制作用呈時間和濃度依賴性。 4.流式細胞術檢測10×10-9mol/L紫杉醇作用24 h、48 h及72 h Hep-2細胞的凋亡率分別為22.6%±5.30%、38.7%±7.90%、52.4%±14.27%,顯著高于對照組9.85%±5.83%,差異具有統(tǒng)計學意義(F=12.621,P＜0.05)。 5.在10×10-99mol/L紫杉醇作用的Hep-2細胞中,RT-PCR顯示TWIST mRNA在24 h、48 h及72 h的表達呈降低趨勢。與對照組相比,TWIST mRNA分別降低了16.70%±5.83%、46.85%±2.74%、76.87%±2.45%,差異具有統(tǒng)計學意義(F=10.407,P0.05)。 6.在10×10-9mol/L紫杉醇作用的Hep-2細胞中,Western-blotting檢測結果顯不,TWIST蛋白在24 h、48 h及72 h的表達亦呈降低趨勢。與對照組相比,TWIST蛋白分別降低了16.44%±4.95%、33.56%±2.00%、69.62%±5.69%,差異具有統(tǒng)計學意義.(F=18.013,P0.05)。 結論： 1. TWIST在喉癌細胞系Hep-2細胞表達 2. TWIST能夠參與紫杉醇誘導Hep-2細胞發(fā)生凋亡的過程,TWIST表達變化是紫杉醇誘導Hep-2細胞發(fā)生凋亡的機制之一。 3.TWIST作為與腫瘤耐藥相關一個新的指標,不僅有助于解釋腫瘤耐藥的機制,而且有可能成為以后腫瘤基因治療的新靶點。
[Abstract]:Laryngeal cancer is one of the most common malignant tumors in the head and neck. In the past 20 years, the implementation of various therapies has not significantly increased the survival rate of the patients. Paclitaxel is a commonly used chemotherapeutic drug, combined with other chemotherapeutic drugs or as a radiation sensitizer, widely used in the treatment of advanced tumor, and has been reported in recent years. Paclitaxel can prolong the survival period of patients with advanced head and neck cancer. It is reported that paclitaxel can induce apoptosis and inhibit the growth of cancer cells. But the clinical application and basic research of taxol in larynx cancer are less.TWIST, which belongs to the family of basic spiral cyclo rotations factor, and overwatch in various solid tumors. It is involved in tumor invasion, metastasis, and high expression of.TWIST associated with tumor prognosis and patient survival. In the process of tumor progression, it not only promotes tumor metastasis, but also has a regulatory effect on tumor metastasis and multidrug resistance with the production of chemotherapeutic drug resistance in tumor cells, and the specific mechanism is not yet. It is clear that the changes in the expression of transcription factor TWIST in human larynx cell line Hep-2 and the relationship with paclitaxel induced apoptosis in Hep-2 cells were studied in this study, which provided a theoretical basis for the study of chemosensitivity and resistance mechanism of larynx cancer.
Objective:
1. to investigate the expression of TWIST in the Hep-2 cells of laryngeal carcinoma.
2. to investigate the relationship between the expression of TWIST and the apoptosis of Hep-2 cells induced by paclitaxel in Hep-2 cells.
Method:
Hep-2 cells of human larynx were used as experimental materials. The morphological changes of Hep-2 cells were observed by paclitaxel with 1 x 10-9,5 x -9,25 x 10-9mol/L. The effect of paclitaxel on the relative survival rate of Hep-2 cells was observed by four methyl azazolus (MTT) colorimetry, and the acridine orange cytochemical staining was observed. The effect of paclitaxel on the apoptosis of Hep-2 cells, the apoptosis rate of Hep-2 cells by flow cytometry, and the expression of TWIST mRNA and protein in Hep-2 cells after paclitaxel action were detected by RT-PCR and Western-blotting respectively.
Result:
Expression of 1. TWIST in Hep-2 cells of laryngeal carcinoma
2. inverted phase contrast microscope and acridine orange cytochemical staining showed the morphological changes of cell apoptosis, which showed that the cell chromatin was fixed in the dosing group, and the nucleus aggregation presented massive, crescent shape and apoptotic body.
3.MTT showed that paclitaxel inhibited the growth of Hep-2 cells in a time and concentration dependent manner.
4. flow cytometry was used to detect the apoptosis rate of 10 x 10-9mol/L paclitaxel, 48 h and 72 h Hep-2 cells, respectively, 22.6% + 5.30%, 38.7% + 7.90%, 52.4% + 14.27%, significantly higher than those of the control group 9.85% + 5.83%, and the difference was statistically significant (F=12.621, P <).
5. in the 10 x 10-99mol/L paclitaxel Hep-2 cells, RT-PCR showed that the expression of TWIST mRNA in 24 h, 48 h and 72 h decreased. Compared with the control group, TWIST mRNA decreased by 16.70% + 5.83%, 46.85% + 2.74% and 76.87% +, respectively, and the difference was statistically significant (F=10.407, P0.05).
6. in the 10 x 10-9mol/L paclitaxel Hep-2 cells, the results of Western-blotting detection were not significant, and the expression of TWIST protein in 24 h, 48 h and 72 h decreased. Compared with the control group, TWIST protein decreased by 16.44% + 4.95%, 33.56% + 2% and 69.62% +, respectively, and the difference was statistically significant. (F=18.013, P0.05).
Conclusion:
Expression of 1. TWIST in Hep-2 cell line of laryngeal carcinoma cell line
2. TWIST can participate in the process of paclitaxel induced apoptosis in Hep-2 cells. The change of TWIST expression is one of the mechanisms of paclitaxel induced apoptosis in Hep-2 cells.
As a new index of tumor resistance, 3.TWIST not only helps to explain the mechanism of tumor resistance, but also may be a new target for tumor gene therapy in the future.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R739.65

【參考文獻】

相關期刊論文 前1條

1 劉婷;耿敬姝;馮美燕;;Twist蛋白在胃癌中的表達及其意義[J];實用腫瘤學雜志;2008年01期

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本文編號：2068461