5种中药体外抗鼻咽癌作用筛选及石上柏提取物诱导鼻咽癌细胞凋亡机制的研究

发布时间：2018-03-23 23:05

本文选题：鼻咽癌　切入点：中药　出处：《广西医科大学》2010年硕士论文

【摘要】： 背景和目的鼻咽癌是我国南方常见的恶性肿瘤之一,目前以放、化疗治疗鼻咽癌,只能使5年生存率达到50%～70%,鼻咽癌放化疗耐受和治疗并发症严重影响治疗效果。寻找对鼻咽癌治疗更有效、毒副反应更小的药物,提高疗效是临床治疗迫切需要解决的问题。传统中医药是人们在生活、疾病中不断总结、筛选而得,经过临床实践和基础研究发现多种中草药有抗肿瘤作用,但是否有抗鼻咽癌作用仍有待研究。本课题采用体外抗鼻咽癌中药筛选模型,比较5种广西常见中药的抗鼻咽癌效果,筛选出一种较佳的抗鼻咽癌药物,并对其抗鼻咽癌作用机制进行研究。方法通过提取中药、血清药理学、体外MTT实验方法,比较白花蛇舌草、半枝莲、仙鹤草、石上柏、两面针,5种中药提取物和含药血清对人鼻咽癌CNE-1、CNE-2细胞生长的抑制率。用体外抗鼻咽癌作用较强的石上柏提取物,观察对小鼠的急性毒性,处理鼻咽癌细胞和正常人胚肺成纤维细胞株HFL-I后,MTT法检测抑制率,倒置相差显微镜观察细胞的形态学改变,碘化丙啶(PI)标记流式细胞术检测鼻咽癌细胞周期分布,Annexin V-FITC和PI标记流式细胞术检测细胞凋亡,免疫细胞化学检测凋亡相关蛋白Bax、Bcl-2的表达变化。结果石上柏的提取物和含药血清对CNE1、CNE2细胞生长均有明显抑制作用,有剂量-效应和时间-效应关系;高浓度的两面针提取物和其低浓度含药血清对CNE1、CNE2细胞生长有一定抑制作用;较高浓度的白花蛇舌草提取物、半枝莲提取物和仙鹤草提取物对CNE1、CNE2生长有一定的抑制作用,呈剂量-效应和时间-效应关系,但其含药血清对CNE1、CNE2生长未见抑制作用。 2.5mg/mL石上柏提取物可显著抑制鼻咽癌细胞的生长,而对正常人胚肺成纤维细胞株HFL-I细胞生长影响较小;小鼠经720g/kg石上柏提取物灌胃,7天内无死亡,肝肾功能和病理切片未见明显异常;石上柏提取物作用于鼻咽癌细胞后,细胞生长受抑,形态改变;流式细胞术检测细胞周期分布显示,石上柏提取物诱导鼻咽癌细胞S期阻滞;流式细胞术检测细胞凋亡显示,鼻咽癌细胞凋亡率随着石上柏提取物浓度的升高而逐渐升高;免疫细胞化学实验显示,石上柏提取物处理后的细胞与对照组细胞相比:Bax蛋白表达增强,Bcl-2蛋白表达减少。结论石上柏提取物和含药血清在体外能显著抑制人鼻咽癌细胞生长,体内和体外急性毒性较小;诱导鼻咽癌细胞S期阻滞,可能为细胞周期特异性药物,诱导鼻咽癌细胞凋亡,其作用机制与Bax、Bcl-2蛋白表达改变有关。
[Abstract]:Background and objective Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China. The 5-year survival rate can only reach 50% and 70%. The radiotherapy and chemotherapy tolerance and treatment complications of nasopharyngeal carcinoma (NPC) have a serious impact on the effectiveness of the treatment. Look for drugs that are more effective and less toxic in the treatment of nasopharyngeal carcinoma. To improve the curative effect is an urgent problem to be solved in clinical treatment. Traditional Chinese medicine is a kind of traditional Chinese medicine that has been continuously summarized and screened in people's lives and diseases. Through clinical practice and basic research, it has been found that many kinds of Chinese herbal medicines have anti-tumor effects. However, whether it has anti-nasopharyngeal carcinoma effect is still to be studied. In this paper, we compared the anti-nasopharyngeal carcinoma effect of five kinds of common traditional Chinese medicine in Guangxi by using the screening model of anti-nasopharyngeal medicine in vitro, and screened out a better anti-nasopharyngeal cancer drug. The mechanism of its anti-nasopharyngeal carcinoma was studied. Methods by extracting traditional Chinese medicine, serum pharmacology and MTT method in vitro, the comparison was made between Scutellaria chinensis, Scutellaria barbata, Herba Scutellariae and Cypress sclerotii. The inhibitory rate of five kinds of traditional Chinese medicine extracts and serum containing drugs on the growth of human nasopharyngeal carcinoma (NPC) CNE-1 CNE-2 cells was studied. The acute toxicity of the extracts of Cypress sclerotifolia, which has a strong effect on nasopharyngeal carcinoma, was observed in vitro. After HFL-I was treated with nasopharyngeal carcinoma cells and normal human embryonic lung fibroblasts, the inhibition rate was detected by MTT assay, and the morphological changes of the cells were observed by inverted phase contrast microscope. Annexin V-FITC and Pi labeled flow cytometry were used to detect apoptosis and immunocytochemistry to detect the expression of apoptosis-related protein Bcl-2. Results the extracts and serum of Phellodendron lanceolata significantly inhibited the growth of CNE 2 cells in a dose-effect and time-response relationship. The growth of CNE1CNE2 cells was inhibited by high concentration of Acanthopsis nitifolia extract and its low concentration serum, and the growth of CNE1CNE2 cells was inhibited by higher concentration extract of Herba Scutellariae, Herba Scutellariae and Herba Herba Sinensis. There was a dose-effect and time-effect relationship, but the growth of CNE _ 2 was not inhibited by its serum. The growth of nasopharyngeal carcinoma (NPC) cells was significantly inhibited by the extract of 2.5mg/mL, but had little effect on the growth of normal human embryonic lung fibroblast cell line HFL-I, and no death was found in mice fed with 720g/kg extract. There were no obvious abnormalities in liver and kidney function and pathological sections. The growth of nasopharyngeal carcinoma cells was inhibited and the morphology of nasopharyngeal carcinoma cells was changed after treated with the extract of Phellodendron sclerotifolia. Flow cytometry was used to detect the distribution of cell cycle. The apoptosis of nasopharyngeal carcinoma cells was detected by flow cytometry. The apoptosis rate of nasopharyngeal carcinoma cells increased with the increase of extract concentration. The immunocytochemical experiment showed that the apoptosis rate of nasopharyngeal carcinoma cells increased with the increase of extract concentration. Compared with the control group, the expression of Bcl 2 protein was significantly increased in the cells treated with the extract of Cypress sclerotii, and the expression of Bcl 2 protein was decreased. Conclusion the extracts of Phellodendron lanceolata and serum containing drugs can significantly inhibit the growth of human nasopharyngeal carcinoma cells in vitro and have little acute toxicity in vivo and in vitro, and induce S phase arrest of nasopharyngeal carcinoma cells, which may be cell cycle specific drugs. The mechanism of inducing apoptosis of nasopharyngeal carcinoma cells is related to the change of Baxon Bcl-2 protein expression.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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