组蛋白去乙酰化酶抑制剂SAHA联合紫杉醇抑制宫颈癌HeLa细胞的体外实验研究

发布时间：2018-06-29 23:31

  本文选题：宫颈癌 + SAHA　； 参考：《深圳大学》2017年硕士论文

【摘要】：宫颈癌(cervical cancer)是女性宫颈上皮细胞癌变而形成的肿瘤,是全球女性最常见的恶性肿瘤之一,发病率位居恶性肿瘤第二位。同时,宫颈癌的发病率在中国居于第一的位置,死亡率位居第二。有研究资料显示,宫颈癌的发病率正在以每年2%-3%的速度迅速增长。发达国家宫颈癌的防治经验表明,通过对易发人群的密切筛查和早期预防,宫颈癌的发病率可以降低70%-90%。因此,研究宫颈癌的发病的分子机制,筛查和防治宫颈癌的发生,是世界宫颈癌研究和防治中最为艰巨和紧迫的课题。目前宫颈癌的临床治疗方法主要以手术治疗辅以化学治疗疗和放射治疗及分子靶向治疗,虽然近几十年来宫颈癌的临床治疗取得了长足进步,早期患者采用手术或放疗,能够取得较好的疗效,但对于中晚期宫颈癌患者,手术操作难度较大。并且,传统的化疗药物在杀伤肿瘤细胞的同时,也杀伤大量的正常细胞,使患者产生较严重的不良反应。同时,随着化疗的继续,化疗药物的耐药性问题也常使化疗效果难以达到预期。因此在宫颈癌的传统治疗模式中,发展更有效的治疗药物与方法成为新的研究课题。本课题以人宫颈癌HeLa细胞为研究对象,研究组蛋白去乙酰化酶抑制剂SAHA联合紫杉醇对宫颈癌He La细胞的体外杀伤效果。MTT法检测宫颈癌HeLa细胞增殖情况,分别用SAHA、紫杉醇、SAHA+紫衫醇处理HeLa细胞24h、48h后,发现相较于SAHA(53.62±3.66%、34.74±3.03%)和紫杉醇(74.07±5.32%、70.88±3.09%)单独使用组,SAHA与紫衫醇联合使用(45.73±4.02%、22.98±3.86%)组的HeLa细胞存活率更低,能够显著抑制He La细胞的增殖。采用SPSS16.0计算SAHA增敏前后紫杉醇对He La细胞IC50的影响,结果显示SAHA增敏后,紫杉醇对He La细胞24h的半数致死剂量由(18.023±1.256)nM降低到(8.887±0.949)nM,48h的半数致死剂量由(12.119±1.083)n M降低到(3.994±0.601)nM,SAHA能够显著降低紫杉醇对HeLa细胞的IC50(P0.01);利用流式细胞术检测He La细胞的凋亡率,结果显示SAHA组、紫杉醇组及SAHA与紫杉醇联合用药组的HeLa细胞的凋亡率分别为(6.44±0.86)%,(7.23±1.49)%,(16.22±3.38)%,联合用药组HeLa细胞的凋亡率分别高于紫杉醇组、SAHA组(P0.05,P0.05),SAHA与紫杉醇联合可以显著增加HeLa细胞的凋亡率,提高诱导HeLa细胞凋亡的能力;利用流式细胞术检测HeLa细胞周期,发现单独SAHA处理24h的HeLa细胞主要处于G0/G1期(90.07±1.39)%,S期(3.88±1.47)%,单独紫杉醇的HeLa细胞主要处于G0/G1期(33.48±6.64)%,S期(40.77±4.43)%,SAHA联合紫杉醇的HeLa细胞主要处于G0/G1期(84.22±2.07)%,S期(11.67±1.28)%,提示SAHA与紫杉醇联合使用能够抑制HeLa细胞有丝分裂过程中的DNA合成和复制;利用流式细胞仪检测HeLa细胞中活性氧(ROS)水平,对照组、SAHA、紫杉醇和SAHA+紫杉醇组处理24h后的He La细胞的ROS水平分别为(1.65±0.84)%、(35.19±4.23)%、(27.26±3.74)%和(44.04±10.77)%,SAHA+紫杉醇组中ROS水平明显升高,且相较于紫杉醇组具有统计学意义(P0.05);免疫荧光法标记HeLa细胞微管和DNA,发现SAHA和紫杉醇联合处理24h后的HeLa细胞的微管呈短小纤细,并向细胞核聚集;RT-PCR检测P27抑癌基因mRNA的表达情况,发现紫杉醇组、SAHA组以及SAHA和紫杉醇联合用药组的HeLa细胞中抑癌基因p27基因的mRNA相对表达量分别为0.978±0.117,5.845±0.548和10.601±0.673,联合用药组相对表达量均高于紫杉醇组、SAHA组,具有统计学意义(P0.001,P0.001);Western blot检测凋亡相关蛋白(caspase-3和caspase-9)和乙酰化组蛋白(Ac-H4)的表达情况,发现SAHA+紫杉醇能够激活caspase-3和caspase-9蛋白,增强组蛋白H4的乙酰化水平。综上所述,在体外培养条件下,SAHA与紫杉醇联合作用于宫颈癌HeLa细胞时,通过激活caspase蛋白途径、提高细胞内活性氧ROS水平、上调p27抑癌基因的表达、增强组蛋白H4的乙酰化水平和影响微丝微管的结构和功能,从而诱导细胞凋亡,抑制细胞增殖,阻滞细胞周期,最终在增强抗肿瘤的能力的同时减轻化疗药物的副作用。
[Abstract]:Cervical cancer (cervical cancer) is a tumor formed by canceration of cervical epithelial cells in women. It is one of the most common malignant tumors in women all over the world. The incidence of cancer is the second most malignant tumor. At the same time, the incidence of cervical cancer is the first in China and the mortality rate ranks second. The rate of 2%-3% is increasing rapidly in the year. The experience of prevention and control of cervical cancer in developed countries shows that the incidence of cervical cancer can be reduced by close screening and early prevention. The molecular mechanism of the study of cervical cancer, screening and preventing the occurrence of cervical cancer are the most arduous in the research and prevention of cervical cancer in the world. At present, the clinical treatment of cervical cancer is mainly based on surgical treatment, chemotherapy, radiotherapy and molecular targeting therapy. Although the clinical treatment of cervical cancer has made great progress in recent decades, early patients with surgery or radiotherapy can achieve better curative effect, but for patients with middle and late cervical cancer, hand It is difficult to operate, and the traditional chemotherapeutic drugs kill the tumor cells and kill a large number of normal cells, so that the patients produce more serious adverse reactions. At the same time, with the continuation of chemotherapy, the drug resistance of chemotherapeutic drugs often makes the effect of chemotherapy difficult to achieve expectations. More effective therapeutic drugs and methods have become a new research topic. In this study, human cervical cancer HeLa cells were used to study the killing effect of histone deacetylase inhibitor SAHA combined with taxol on cervical cancer He La cells in vitro.MTT assay for the proliferation of cervical cancer HeLa cells, respectively, SAHA, paclitaxel, and SAHA+ After 24h and 48h, HeLa cells were found to be compared to SAHA (53.62 + 3.66%, 34.74 + 3.03%) and paclitaxel (74.07 + 5.32%, 70.88 + 3.09%) alone. The survival rate of HeLa cells in the group of SAHA and the combined use of (45.73 + 4.02%, 22.98 + 3.86%) group was lower, and the proliferation of He La cells could be suppressed significantly. SPSS16.0 calculation of paclitaxel before and after SAHA sensitization to SAHA was used. The effect of IC50 in He La cells showed that after SAHA sensitization, the median lethal dose of paclitaxel to He La cell 24h decreased from (18.023 + 1.256) nM to (8.887 + 0.949) nM, and the median lethal dose of 48h decreased from (12.119 + 1.083) n M to (3.994 + 0.601). The apoptosis rate of He La cells was measured. The results showed that the apoptosis rate of HeLa cells in group SAHA, paclitaxel group and SAHA and paclitaxel group was (6.44 + 0.86)%, (7.23 + 1.49)%, (16.22 + 3.38)%. The apoptosis rate of HeLa cells in combination group was higher than that of paclitaxel group, SAHA group (P0.05, P0.05), SAHA and paclitaxel could significantly increase HeLa. The apoptotic rate of cells increased the ability to induce apoptosis of HeLa cells, and HeLa cell cycle was detected by flow cytometry. The HeLa cells treated with single SAHA for 24h were mainly in G0/G1 phase (90.07 + 1.39)%, S phase (3.88 + 1.47)%, and HeLa cells of taxol alone in G0/G1 (33.48 + 6.64)%, S (40.77. 4.43)%, SAHA with paclitaxel HeL A cells were mainly in phase G0/G1 (84.22 + 2.07)% and S phase (11.67 + 1.28)%. The combination of SAHA and taxol could inhibit the synthesis and replication of DNA during the mitosis of HeLa cells, and the level of reactive oxygen (ROS) in HeLa cells was detected by flow cytometry. The control group, SAHA, paclitaxel and SAHA+ paclitaxel group treated 24h He cells The level of (1.65 + 0.84)%, (35.19 + 4.23)%, (27.26 + 3.74)% and (44.04 + 10.77)%, ROS level in SAHA+ paclitaxel group increased significantly, and compared with paclitaxel group (P0.05). The immunofluorescence method labeled HeLa cell microtubules and DNA, and the microtubules of HeLa cells after SAHA and paclitaxel combined with 24h were small and thin. The expression of P27 suppressor gene mRNA was detected by RT-PCR. The relative expression of the mRNA relative expression of the tumor suppressor gene p27 gene in the paclitaxel group, the SAHA group and the SAHA and paclitaxel group HeLa cells was 0.978 + 0.117,5.845 + 0.548 and 10.601 + 0.673 respectively. The relative expression of the combined drug group was higher than that of the paclitaxel group, the SAHA group, It was statistically significant (P0.001, P0.001); Western blot detected the expression of apoptosis related proteins (caspase-3 and caspase-9) and acetylation histone (Ac-H4). It was found that SAHA+ paclitaxel could activate caspase-3 and caspase-9 proteins and enhance the level of acetylation of histone H4. Under the conditions of culture, SAHA and taxol were combined in vitro. When used for cervical cancer HeLa cells, by activating the caspase protein pathway, increasing the intracellular reactive oxygen ROS level, up regulation of the expression of p27 suppressor gene, enhancing the level of acetylation of histone H4 and influencing the structure and function of microtubule microtubule, inducing cell apoptosis, inhibiting cell proliferation, blocking cell cycle, and finally enhancing the ability of anti-tumor. It also alleviated the side effects of chemotherapeutic drugs.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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