曲古抑菌素A联合顺铂对肺腺癌A549凋亡的影响

发布时间：2018-05-24 05:24

本文选题：曲古抑素A + 顺铂　；参考：《山东大学》2015年博士论文

【摘要】：表观遗传学是遗传学分支学科,指在不改变DNA序列的前提下,基因或者蛋白表达发生变化,并可以在细胞的生长和传代过程中稳定传递,主要包括组蛋白的共价修饰,DNA甲基化,基因沉默,染色质重塑,以及非编码RNA编辑等机制。表观遗传学与肿瘤的发生和转移有密切联系,肿瘤中存在表观遗传方式的异常。表观遗传学通过影响肿瘤抑制基因及其表达,促进肿瘤发生。组蛋白组成了核小体,它可以被多种化合物所修饰,如磷酸化、乙酰化和甲基化等,称为共价修饰,组蛋白的这些共价修饰作用主要发生在其N2末端,其中最为重要的是组蛋白乙酰化,组蛋白乙酰化会对肿瘤细胞的染色体结构以及基因的表达产生影响,从而使肿瘤的治疗更加乐观。组蛋白乙酰化和去乙酰化在细胞信号转导、分裂、凋亡、细胞内的DNA修复、以及及染色体组装等方面起着重要作用。各种抗肿瘤药物有着不同的作用特点,不同类型的肿瘤使用不同的抗肿瘤药物,其作用机制各不相同,但多种针对实体瘤或血液系统肿瘤的抗肿瘤药物均可与组蛋白去乙酰化酶抑制剂联合,起到协同作用,研究发现,抗肿瘤药物与组蛋白去乙酰化酶抑制剂联合后,比单药使用抗肿瘤药物组生存率高,对肿瘤细胞杀伤作用显著,并且其疗效有统计学差异。组蛋白去乙酰化酶抑制剂在恶性肿瘤的治疗作用已受到广泛认可。组蛋白去乙酰化酶抑制剂目前常常用来与DNA甲基转移酶抑制剂、拓扑异构酶抑制剂、激素、化疗药物如紫杉醇及铂类等、酪氨酸激酶受体阻滞剂等药物联合使用。目前在肺癌的研究中,组蛋白去乙酰化酶抑制剂联合铂类药物的报道较少,为研究组蛋白去乙酰化酶抑制剂与顺铂联合用药,对肺癌细胞的影响,并对其作用机制进一步探索,设计了本次的实验。目的：研究曲古抑菌素A (trichostatin A,TSA)联合顺铂(cisplatin)对肺腺癌细胞株A549凋亡的影响,以及药物处理后A549细胞中cFLIP、caspase-8蛋白的表达情况。方法：1、使用PRMI1640培养液培养肺腺癌A549细胞株,将细胞分为四组：1)、对照组2)、TSA250nmol/L处理组3)、Cisplatin 10μg/mL处理组4)、TSA250nmol/L+ Cisplatin 10μg/m L联合处理组,药物处理24h后,使用光学显微镜观察各个组细胞形态学的变化。2、使用四甲基偶氮唑蓝(MTT)比色法测定细胞的抑制率,将细胞分为10组：1)、空白对照组2)、TSA 250nmol/L处理组3) Cisplatin 5.3μg/mL处理组4)TSA250nmol/L+ Cisplatin 5.3μg/mL处理组5) Cisplatin 9μg/m处理组6)TSA250nmol/L+Cisplatin 9μg/mL处理组7) Cisplatin 13μg/mL处理组8)TSA250nmol/L+ Cisplatin 13μg/mL处理组9) Cisplatin 20μg/mL处理组10)TSA250nmol/L+ Cisplatin 20μg/mL处理组。测定不同浓度顺铂处理细胞后,对细胞抑制率,以及不同浓度顺铂联合TSA处理细胞后,对细胞的抑制率。3、按照1中的分组方法将细胞分为4组,使用Hoechst33258染色法显示不同处理组细胞凋亡的特点。4、按照1中的分组方法将细胞分为四组,使用Western blot法检测cFLIP、 Pro-caspase-8及Caspase-8蛋白表达的变化。实验数据使用SPSS 15软件进行χ2检验、t检验,p0.05表示差异有统计学意义。结果：1、光学显微镜下观察四个组药物处理后细胞形态的变化①对照组：细胞排列规则,紧密贴壁,细胞边界清晰,未看到悬浮死细胞。②TSA250nmol/L处理组：细胞变大,有些细胞呈并指状,细胞间隙增大,可见少量悬浮死细胞。③Cisplatin 10μg/mL处理组：细胞比对照组变小、变圆,发生皱缩,可见较多死细胞。④TSA250nmol/L+ cisplatin 10μg/mL联合处理组：与前三组相比,细胞密度明显下降,细胞发生皱缩,可见大量死细胞悬浮。2、四甲基偶氮唑盐(MTT法)检测不同浓度顺铂对细胞的抑制作用,以及不同浓度顺铂联合TSA对细胞的抑制作用使用不同浓度的顺铂处理细胞,发现顺铂对细胞的抑制作用与顺铂的浓度呈正相关。随着顺铂浓度的增加,细胞凋亡率增加。当联合TSA后,各组对细胞的抑制率均升高。Cisplatin 5.3μg/mL、TSA250nmol/L+ Cisplatin 5.3μg/mL细胞抑制率分别为13.68%、26.42%。Cisplatin 9μg/mL、TSA250nmol/L+ Cisplatin 9μg/mL细胞抑制率分别为28.73%、46.72%。Cisplatin 13μg/mL、TSA250nmol/L+ Cisplatin 13μg/mL细胞抑制率分别为44.58%、49.65%。Cisplatin 20μg/mL、TSA250nmol/L+ Cisplatin 20μg/mL细胞抑制率分别为51.12%、61.92%。顺铂与TSA联合对细胞的抑制明显高于单药组(p0.05)。3、Hoechst33258染色法观察各组使用药物处理后细胞凋亡的特点各个组药物处理24h后,使用Hoeschst33258染色法观察细胞的凋亡特点,相比于对照组,TSA及Cisplatin药物单药处理组凋亡细胞增多,出现染色质凝集,核染色荧光增强,联合用药上述现象更加明显,荧光度增强,并可以观察到核裂解及凋亡小体。4、TSA联合Cisplatin对cFLIP蛋白表达量的影响使用TSA 250nmol/L联合Cisplatin 10μg/mL处理细胞24h后,单药处理组cFLIP蛋白表达量减少,相比对照组及单药处理组,联合用药组cFLIP蛋白表达明显减少。测定各个处理组蛋白灰度值,结果显示灰度值差异有统计学意义。5、TSA联合Cisplatin对caspase-8及其前体表达的影响TSA 250nmol/L联合Cisplatin 10μg/mL处理细胞24h后,单药处理组Pro-caspase-8表达减少,caspase-8蛋白表达增多,与对照组与单药处理组相比,联合用药组Pro-caspase-8明显减少,caspase-8蛋白明显增多,测定四个处理组蛋白灰度值,结果显示其蛋白表达差异有统计学意义。结论：TSA联合顺铂能够通过影响Caspase-相关信号通路增加A549细胞株的凋亡,TSA和顺铂联合具有协同作用。
[Abstract]:Epigenetics, a branch of genetics, refers to changes in gene or protein expression without changing the DNA sequence, and can be transferred steadily during cell growth and generation, mainly including covalent modification of histone, DNA methylation, gene silencing, chromatin remodeling, and non coded RNA editing. Epigenetic inheritance. It is closely related to the occurrence and metastasis of tumor. Epigenetics is abnormal in the tumor. Epigenetics promotes the occurrence of tumor by affecting the tumor suppressor gene and its expression. The histone is composed of nucleosomes, which can be modified by a variety of compounds, such as phosphorylation, acetylation and methylation, called covalent modification, histone These covalent modifications mainly occur at the end of the N2, the most important of which is histone acetylation. Histone acetylation will affect the chromosome structure and gene expression of tumor cells, thus making the cancer treatment more optimistic. Histone acetylation and deacetylation in cell signal transduction, division, apoptosis, cells. DNA repair, and chromosomal assembly play an important role. Various antitumor drugs have different characteristics, different types of tumor use different antitumor drugs, their mechanisms are different, but a variety of antitumor drugs against solid tumors or blood system tumors can be inhibited with histone deacetylase. As a synergistic effect, the study found that the combination of antitumor drugs and histone deacetylase inhibitors has a higher survival rate than the single drug use antitumor drug group and has a significant effect on tumor cells, and its efficacy is statistically different. Histone deacetylase inhibitors have been widely recognized in the treatment of malignant tumors. Histone deacetylase inhibitors are currently used in combination with DNA methyltransferase inhibitors, topoisomerase inhibitors, hormones, chemotherapeutic drugs such as Taxol and platinum, tyrosine kinase receptor blockers and other drugs. In the present study of lung cancer, histone deacetylase inhibitors combined with platinum drugs are reported to be more effective. For the purpose of studying the effect of trichostatin A (TSA) combined with cisplatin (cisplatin) on the apoptosis of lung adenocarcinoma cell line A549, and the A549 fine after treatment, the effect of the combination of protein deacetylase inhibitor and cisplatin on lung cancer cells was further explored. The expression of cFLIP and caspase-8 protein in the cell. Methods: 1, the A549 cell lines of lung adenocarcinoma were cultured with PRMI1640 culture, and the cells were divided into four groups: 1), 2 in the control group, 3 in the TSA250nmol/L treatment group, 4 in the Cisplatin 10 g/mL treatment group, and TSA250nmol/L+ Cisplatin 10 micron g/m L combined treatment group. After the drug processing 24h, the optical microscope observation of each group was used. The cell morphology changes of the group.2, using the four methyl azazolium blue (MTT) colorimetric assay to determine the cell inhibition rate, the cells were divided into 10 groups: 1), the blank control group 2), the TSA 250nmol/L treatment group 3) Cisplatin 5.3 mu g/mL treatment group 4) TSA250nmol/L+ Cisplatin 5.3 micron rational group 5) Cisplatin 9 mu g/m processing group 6) TSA250nmol/L+Cisplatin 9 Mu L treatment group 7) Cisplatin 13 mu g/mL treatment group 8) TSA250nmol/L+ Cisplatin 13 g/mL treatment group 9) Cisplatin 20 g/mL treatment group 10) TSA250nmol/L+ Cisplatin 20 mu g/mL treatment group. After the determination of cisplatin treated cells with different concentrations, the inhibitory rate of cisplatin, and the inhibition rate of cisplatin with TSA treated cells, 1. In the group, the cells were divided into 4 groups. Hoechst33258 staining was used to show the characteristics of cell apoptosis in different treatment groups.4. The cells were divided into four groups according to the group method of 1. The changes of cFLIP, Pro-caspase-8 and Caspase-8 protein expression were detected by Western blot method. The experiment number was tested by SPSS 15 software for chi 2 test, t test, P0.05. The difference was statistically significant. Results: 1, the morphological changes of the four groups were observed under the optical microscope (1) the control group: the cells were arranged regularly, the cells were closely adhered, the cell boundaries were clear, and the dead cells were not seen. TSA250nmol/L treatment group: the cells became larger, some cells showed and finger like, the space gap was enlarged, and a small amount could be seen. Suspension of dead cells. (3) Cisplatin 10 mu g/mL treatment group: cells were smaller, round, crinkle and more dead cells than the control group. (4) TSA250nmol/L+ cisplatin 10 mu g/mL combined treatment group: compared with the first three groups, cell density decreased significantly, cell shrinkage, large number of dead cells suspended.2, four methyl azazolium salt (MTT method) detection The inhibitory effects of cisplatin on cells and the inhibition of cisplatin with different concentrations of cisplatin combined with TSA on cells were treated with cisplatin at different concentrations. It was found that the inhibition of cisplatin to cisplatin was positively correlated with the concentration of cisplatin. With the increase of cisplatin concentration, the rate of cell apoptosis increased. When combined with TSA, the inhibition of cells was inhibited. The rate of.Cisplatin 5.3 mu g/mL, TSA250nmol/L+ Cisplatin 5.3 g/mL cell inhibition rate were 13.68%, 26.42%.Cisplatin 9 g/mL, TSA250nmol/L+ Cisplatin 9 micron g/mL cell inhibition rate was 28.73%, 46.72%.Cisplatin 13 micron g/mL, 13 micron cell inhibition rate was 44.58%, 20 micron, respectively. The inhibitory rate of TSA250nmol/L+ Cisplatin 20 mu g/mL cells was 51.12% respectively. The inhibition of 61.92%. cisplatin and TSA was significantly higher than that of single drug group (P0.05).3. Hoechst33258 staining method observed the characteristics of cell apoptosis after the use of drug treatment in each group. After the treatment of 24h, the apoptotic characteristics of the cells were observed by Hoeschst33258 staining. Compared with the control group, the number of apoptotic cells in the TSA and Cisplatin drug treatment group increased, the chromatin agglutination and the nuclear staining were enhanced. The above phenomenon was more obvious, the fluorophore was enhanced, and the nuclear fragmentation and the apoptotic body.4 could be observed. The effect of TSA combined with Cisplatin on the expression of cFLIP protein was combined with TSA 250nmol/L combined Cisplatin 1. After 0 mu g/mL treatment of 24h, the expression of cFLIP protein in the single drug treatment group decreased. Compared with the control group and the single drug treatment group, the expression of cFLIP protein in the combination group decreased obviously. The gray value of each processing group was measured, the results showed that the difference of gray value was statistically significant.5, TSA combined with Cisplatin on the expression of Caspase-8 and its precursors in TSA 250nmo. After l/L combined with Cisplatin 10 mu g/mL to treat 24h, the expression of Pro-caspase-8 in the single drug treatment group decreased and the expression of caspase-8 protein increased. Compared with the control group, the Pro-caspase-8 of the combined drug group decreased obviously, the caspase-8 protein increased obviously, and the gray value of the four treated group proteins was measured. The results showed that the protein expression was different. Conclusion: TSA combined with cisplatin can increase the apoptosis of A549 cell line by affecting Caspase- related signaling pathway. TSA and cisplatin have synergistic effect.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

【参考文献】