TWIST在紫杉醇诱导喉癌Hep-2细胞凋亡中的作用研究

发布时间：2018-06-26 01:34

本文选题：TWIST + Hep-2细胞　；参考：《山东大学》2010年硕士论文

【摘要】： 喉癌是头颈部常见恶性肿瘤之一,过去的20年中,多种治疗方法的实施,并没有使患者的生存率明显升高。紫杉醇是目前临床上常用化疗药物,联合其他的化疗药物或作为放疗增敏剂,广泛的用于晚期肿瘤的治疗,疗效显著,近年来,有报道紫杉醇用于晚期头颈肿瘤化疗,可延长患者的生存期。研究报道紫杉醇能诱导多种癌细胞发生凋亡,抑制肿瘤的生长。但紫杉醇在喉癌的临床应用和基础研究报道均较少。TWIST,属于碱性螺旋-环-螺旋转录因子家族,在多种实体肿瘤中过表达,参与肿瘤的侵袭、转移,并与肿瘤预后和患者生存有关。TWIST的过高表达,在肿瘤进展的过程中,不仅能够促进肿瘤转移,而且还与肿瘤细胞对化疗药物耐药的产生有关。TWIST在肿瘤转移和多药耐药方面存在调控作用,其具体机制还不是很清楚。本研究,针对转录因子TWIST在人喉癌细胞系Hep-2中的表达变化及其与紫杉醇诱导Hep-2细胞凋亡的关系进行初步研究,为探讨喉癌化疗敏感性及耐药机制提供理论依据。目的： 1.探讨TWIST在喉癌Hep-2细胞中的表达； 2.初步探讨TWIST在喉癌Hep-2细胞中的表达与紫杉醇诱导Hep-2细胞凋亡的关系。方法：以人喉癌Hep-2细胞为实验材料,以1×10-9、5×10-9、10×-9、25×10-9mol/L的紫杉醇作用于Hep-2细胞,采用倒置相差显微镜观察其形态变化,四甲基偶氮唑蓝(MTT)比色法观察不同浓度紫杉醇对Hep-2细胞的相对存活率的影响,吖啶橙细胞化学染色观察紫杉醇对Hep-2细胞凋亡的影响,流式细胞术检测Hep-2细胞的凋亡率,RT-PCR和Western-blotting分别检测紫杉醇作用后的Hep-2细胞中TWIST mRNA和蛋白的表达变化情况。结果： 1. TWIST在喉癌Hep-2细胞中存在表达 2.倒置相差显微镜下和吖啶橙细胞化学染色可见细胞凋亡的形态学改变,显示加药组细胞染色质固缩,细胞核聚集呈现块状、新月状,出现凋亡小体。 3.MTT显示紫杉醇对Hep-2细胞的生长抑制作用呈时间和浓度依赖性。 4.流式细胞术检测10×10-9mol/L紫杉醇作用24 h、48 h及72 h Hep-2细胞的凋亡率分别为22.6%±5.30%、38.7%±7.90%、52.4%±14.27%,显著高于对照组9.85%±5.83%,差异具有统计学意义(F=12.621,P＜0.05)。 5.在10×10-99mol/L紫杉醇作用的Hep-2细胞中,RT-PCR显示TWIST mRNA在24 h、48 h及72 h的表达呈降低趋势。与对照组相比,TWIST mRNA分别降低了16.70%±5.83%、46.85%±2.74%、76.87%±2.45%,差异具有统计学意义(F=10.407,P0.05)。 6.在10×10-9mol/L紫杉醇作用的Hep-2细胞中,Western-blotting检测结果显不,TWIST蛋白在24 h、48 h及72 h的表达亦呈降低趋势。与对照组相比,TWIST蛋白分别降低了16.44%±4.95%、33.56%±2.00%、69.62%±5.69%,差异具有统计学意义.(F=18.013,P0.05)。结论： 1. TWIST在喉癌细胞系Hep-2细胞表达 2. TWIST能够参与紫杉醇诱导Hep-2细胞发生凋亡的过程,TWIST表达变化是紫杉醇诱导Hep-2细胞发生凋亡的机制之一。 3.TWIST作为与肿瘤耐药相关一个新的指标,不仅有助于解释肿瘤耐药的机制,而且有可能成为以后肿瘤基因治疗的新靶点。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.65

【参考文献】