丹参酮ⅡA对人食管癌细胞系Eca-109增殖抑制作用及机制研究

发布时间：2018-01-08 12:07

本文关键词：丹参酮ⅡA对人食管癌细胞系Eca-109增殖抑制作用及机制研究　出处：《河南中医学院》2015年硕士论文　论文类型：学位论文

更多相关文章： 食管癌 TanⅡA 细胞周期 细胞凋亡

【摘要】：研究背景据报道,在我国每年死于食管癌的人数约为21万人,男性人数多于女性,40岁以上的发病率高,它严重威胁着我国人民的健康和生命[1]。其治疗方法主要有手术切除、肿瘤的放疗和化疗等,单一的任何一种方法都不是理想的,因此多采用综合治疗,在手术切除后配合药物化疗。现在的化疗新药层出不穷,但因化疗产生的副反应仍然较大,并且耐药性不断增加,病人的的5年存活率依然很低。因此,利用我国的优势,研究中药及其提取的有效成分在肿瘤治疗方面的作用是以后发展的方向之一。本研究探讨了食管癌可能的发生机制及中药丹参中的挥发油-丹参酮ⅡA(TanⅡA)对人食管癌细胞株Eca-109的作用。TanⅡA是中药丹参的主要成分。丹参酮在临床上已被广泛应用于非肿瘤领域,主要用来治疗心血管疾病,在抗菌消炎等[1]方面也有显著疗效。近年来,学者对丹参酮IIA的抗肿瘤活性产生了极大兴趣,并进行了多种肿瘤细胞的体内外试验,但对食管癌的抗癌作用却鲜见报道。目的通过对人食管癌Eca-109细胞的培养,用不同浓度的TanⅡA干预其生长,运用现代分子生物学技术,探讨TanⅡA促进Eca-109细胞凋亡的机制,为TanⅡA更深入的研究做准备。方法1.MTT法:通过培养人食管癌Eca-109细胞,用不同剂量的TanⅡA作用于Eca-109细胞,通过四甲基偶氮唑盐法(MTT法)测定各组的吸光值(OD值),并计算出各组细胞的增殖抑制率,绘制增殖抑制率曲线。2.显微形态观察:药物组细胞和正常组细胞培养24小时后,在显微镜下观察正常细胞和药物干预细胞的生长状态和形态变化。3.细胞荧光观察:药物组细胞和正常组细胞培养24小时后,应用AO/EB染色,在荧光显微镜下观察两组细胞的形态变化。4.流式细胞术:采用140μg/ml、160μg/ml的TanⅡA干预细胞生长24和48小时,设置阴性对照,用流式检测细胞周期和细胞凋亡。5.免疫蛋白印迹法:采用140μg/ml、160μg/ml的TanⅡA干预细胞生长24小时,提取细胞的总蛋白,用BCA法进行蛋白定量,采用蛋白免疫印迹技术(western blot)检测Caspase-3、Caspase-9、Cyclin A和CDK2的表达,以β-actin作为内参,用BIO-RAD软件分析条带的灰度值。结果1.MTT检测结果显示:正常组细胞活力明显高于药物组,并且在一定的范围内,随药物的浓度增加,细胞的活力下降,最高的抑制率可达66.164%,与阴性对照组相比较,差异有统计学意义(P㩳0.05)。2.显微镜观察显示:经过TanⅡA处理后的细胞与正常组细胞相比较,药物组细胞有明显的凋亡形态的改变,出现细胞漂浮,细胞核固缩,细胞膜结构破坏出现“小泡”现象。3.细胞荧光观察结果显示:与正常组细胞相比较,药物组细胞出现核染色质着绿色或红色并呈固缩状、圆珠状。4.流式结果表明:1)140μg/ml TanⅡA干预Eca-109细胞24小时后,结果显示S期的细胞数升高,达到54.47%;同时G0/G1期的细胞数下降,由51.317%降至41.787%,与阴性对照相比,差异具有统计学意义(P㩳0.05)。2)采用140μg/ml和160μg/ml的TanⅡA培养细胞24小时,结果显示药物组凋亡指数随着药物浓度的增加,凋亡指数增大,由阴性的8.319增加到TanⅡA140、160μg/ml组的31.982和52.937。用药组和阴性对照组相比较,差异具有统计学意义(P㩳0.01)。5.Western blot结果显示:药物培养24小时后,细胞内Cyclin A、CDK2表达量降低;Caspase-3、Caspase-9的表达量上升,和正常组相比较,差异具有统计学意义(P㩳0.05)。结论1.TanⅡA可以抑制人食管癌细胞的增殖活力,并且这种抑制作用随药物浓度的增加会更明显。2.在TanⅡA作用于食管癌细胞系Eca-109的过程中,随着药物浓度的升高,细胞在S期的比例逐渐升高,S期检查点的调控因子Cyclin A和CDK2的相对表达量降低。3.TanⅡA抑制人食管癌Eca-109细胞的增殖并促进其凋亡的机制可能与通过下调Cyclin A和CDK2,将细胞阻滞于S期和上调Caspase-9和Caspase-3促进细胞凋亡增加有关。
[Abstract]:According to reports in the background, the number of China's annual died of esophageal cancer is about 210 thousand people, the number of men more than women over the age of 40, the incidence rate is high, it is a serious threat to our health and life of the people [1]. the main treatments include surgical resection, radiotherapy and chemotherapy of tumor, any single method are not ideal, so we adopt comprehensive treatment, after surgical removal combined with chemotherapy. Chemotherapy drugs now because chemotherapy side effects emerge in an endless stream, the resistance is still large, and increasing the patient's 5 year survival rate is still very low. Therefore, the use of our advantages, effect of effective components Chinese herbal medicine and its extract in the treatment of cancer is one of the future direction of development. This study explores the possible occurrence of esophageal cancer and mechanism of volatile oil of Salvia miltiorrhiza in tanshinone A (Tan A) on human esophageal carcinoma fine The role of Eca-109 in cell line.Tan II A is the main component of Salvia miltiorrhiza. Tanshinone in clinical practice has been widely used in the field of non tumor, mainly used in the treatment of cardiovascular diseases, anti inflammatory [1] also had significant effect. In recent years, the antitumor activity of tanshinone IIA have generated great interest, and in vivo test of tumor cells to anticancer effect on esophageal cancer, but is rarely reported. The culture of human esophageal carcinoma Eca-109 cells, the growth of the intervention with different concentrations of Tan II A, using modern molecular biology technology, discuss the mechanism of Eca-109 Tan II A promote cell apoptosis, prepare for study of Tan A deeply. 1.MTT method by cultured human esophageal cancer Eca-109 cells, with different doses of Tan II A in Eca-109 cells by four methyl thiazolyl tetrazolium method (MTT method) were measured by light absorption value (OD), And calculate the inhibition rate of cell proliferation, rendering the proliferation rate of observation of.2. morphology curve: drug group and normal group cells cultured for 24 hours, to observe the growth state of normal cells and drug intervention cells and morphological changes of.3. cell fluorescence microscope observation: drug group and normal group cells cultured for 24 hours the application, AO/EB staining to observe the morphological changes in two groups of.4. cells under the fluorescence microscope and flow cytometry using 140 g/ml Tan II A 160 g/ml intervention on cell growth of 24 and 48 hours, set the negative control, using flow cytometry to detect the cell cycle and apoptosis of.5. cells by Western blot: 140 g/ml, Tan II A 160 g/ml intervention on cell growth in 24 hours, the total protein extracted from cells, proteins were quantified by BCA assay, Western blot technique (Western blot) Caspase-9, Cyclin detection of Caspase-3, A and CDK2 The expression of beta -actin as reference, using BIO-RAD software to analyze the gray value of bands. 1.MTT results showed that: in normal group, the cell viability was significantly higher than that of the drug group, and in a certain range, with the drug concentration increased, cell viability decreased, the highest inhibition rate reached 66.164%, compared with the negative control group, the difference was statistically significant (P? 0.05).2. microscope observation showed that: after Tan II A treated cells compared with normal cells, the drug group cells have obvious apoptotic morphology changes, cell floating, karyopyknosis, "vesicles" phenomenon of.3. cell fluorescence observation results show that the damage of the cell membrane structure: compared with normal group cells, drug group cells showed nuclear chromatin with green or red and showed pyknosis, bead like.4. flow cytometry results showed that: 1) 140 g/ml Tan II A Eca-109 cells 24 hours after intervention, the results showed that S The number of cells increased, reaching 54.47%; at the same time, the number of cells in G0/G1 phase decreased from 51.317% to 41.787%, compared with the negative control, the difference was statistically significant (P.2)? 0.05) by 140 g/ml and 160 g/ml Tan II A cells cultured for 24 hours. The results showed that the drug group with apoptosis index the increase of drug concentration, the apoptosis index increased, increased from 8.319 to Tan II A140160 negative g/ml group 31.982 and 52.937. group and negative control group compared the difference was statistically significant (P? 0.01).5.Western blot results showed that the drug culture after 24 hours, Cyclin A cells, CDK2 expression was decreased Caspase-3; the expression of Caspase-9 was increased, compared with normal group, the difference was statistically significant (P? 0.05). Conclusion 1.Tan II A can inhibit human esophageal cancer cell proliferation, and this inhibition effect will be more obvious in.2. Tan II with the increase of drug concentration In the process of esophageal cancer cell line Eca-109 A, with the increase of drug concentration, the cells gradually increased in the proportion of S phase, S phase checkpoint regulation factor Cyclin A and CDK2 decreased the relative expression mechanism and promote the apoptosis of.3.Tan II A inhibited the proliferation of Eca-109 cells may be related to downregulation by Cyclin A and CDK2, arrest the cells in S and upregulation of Caspase-9 and Caspase-3 to promote the increase of cell apoptosis.

【学位授予单位】：河南中医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

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