组蛋白去乙酰化酶抑制剂SAHA抑制RD细胞增殖和诱导其凋亡的实验研究

发布时间：2018-03-03 02:13

  本文选题：横纹肌肉瘤　切入点：HDAC　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：横纹肌肉瘤(rhabdomyosarcoma,RMS)是临床上儿童常见的软组织肉瘤,起源于骨骼肌细胞,约占小儿恶性实体瘤的10%左右,占儿童期所有软组织肉瘤50%~70%。随着在临床上综合治疗的应用,其预后已有了明显提高,5年总生存率达到33%~50%,但仍有超过50%的患者死于肿瘤复发或远处转移进展。在我国,儿童横纹肌肉瘤的患者人口基数大,总体治疗效果虽然有所改善,但是其远期治愈率并不令人满意。目前对于RMS的发病机制有了许多进展,对于其综合治疗方法的改善有一定的帮助。但是对于复发的患者,目前主要依靠施行化疗,但化疗的毒副反应则限制了其在儿童患者的使用。因此目前迫切需要治疗横纹肌肉瘤的新型药物出现。研究报道肿瘤细胞中组蛋白往往呈低乙酰化水平,并且组蛋白去乙酰化酶(HDAC)活性异常造成的组蛋白乙酰化状态失衡与肿瘤的发生发展有密切关系。HDAC抑制剂SAHA能可逆性地逆转组蛋白乙酰化状态,从而调节基因表达、促进凋亡、细胞周期的阻滞、抗血管生成、DNA的损伤和修复以及减弱肿瘤细胞的侵袭和迁移。因此SAHA有望成为治疗横纹肌肉瘤的一种新型靶向药物。因此本课题的目的是研究SAHA对人横纹肌肉瘤RD细胞增殖、细胞周期、凋亡、自噬和迁移能力的影响,并探索其诱导凋亡的机制。方法:不同浓度SAHA作用于RD细胞,MTT法检测对细胞增殖作用的影响,western blot检测ERK信号通路蛋白表达;PI染色法分析细胞周期;Annexin V-FITC/PI双染法检测细胞凋亡,DAPI染色法验证细胞凋亡,然后进行以下实验初步探讨诱导凋亡的机制:用JC-1探针检测线粒体膜电位的变化;western blot检测凋亡相关蛋白、线粒体途径相关蛋白的变化;MDC染色法检测细胞自噬,western blot检测自噬相关的蛋白的表达;细胞划痕检测细胞迁移能力;western blot检测细胞组蛋白H4的乙酰化水平。结果:1.saha抑制了rd细胞的增殖,并且具有时间和浓度依赖性。saha作用于rd细胞24h、48h、72h的半数抑制率ic50值分别为8.503μmol//l、3.965μmol/l和1.921μmol/l;与rd细胞增殖相关erk信号通路的变化是:erk蛋白磷酸化水平下降,而总erk蛋白水平没有显著的变化;2.流式细胞术结果显示saha使rd细胞发生g2/m期阻滞;3.annexinv-fitc/pi双染法结果显示saha能够诱导rd细胞凋亡;dapi染色法实验表明,随着saha浓度的增加,凋亡细胞的数量随之增加,jc-1流式细胞术结果显示,随着saha浓度的增高,rd细胞线粒体膜电位水平逐渐降低;westernblot结果显示:随着saha浓度的增高,凋亡相关蛋白caspase-3和parp-1表达量增高,抗凋亡蛋白bcl-2表达量逐渐减低,促凋亡蛋白bax表达量逐渐增加,细胞色素c和caspase-9的表达也均有所增加;4.saha使rd细胞发生自噬:mdc染色结果显示随着saha浓度的增高,细胞内的高亮点状绿色荧光的斑点数量逐渐增多,表明细胞发生自噬。westernblot结果显示:随着saha浓度的增高,p62蛋白表达逐渐减少,beclin1蛋白表达水平逐渐增高,lc3-Ⅱ蛋白的表达也显著增高。5.saha抑制rd细胞的迁移:细胞划痕实验结果显示:随着saha浓度的增高,划痕愈合率逐渐降低。6.westernblot结果显示:随着saha浓度的增高,组蛋白h4的乙酰化水平逐渐增高。结论:1.hdac抑制剂saha抑制rd细胞的增殖,使rd细胞发生g2/m期阻滞,该抑制作用具有时间和浓度依赖性;2.hdac抑制剂saha诱导rd细胞的凋亡,介导凋亡的机制可能为依赖于线粒体途径和具有caspase依赖性;3.hdac抑制剂saha诱导rd细胞自噬;4.hdac抑制剂saha抑制rd细胞的迁移能力。
[Abstract]:Rhabdomyosarcoma (rhabdomyosarcoma, RMS) is a clinically common soft tissue sarcoma, originated in skeletal muscle cells, accounting for about 10% of pediatric malignant solid tumor in childhood, accounting for all soft tissue sarcoma 50%~70%. with the application of comprehensive treatment in clinic, its prognosis has been improved obviously, 5 year survival rate of 33%~50%. But there are still more than 50% of the patients died of tumor recurrence or distant metastasis. In our country, children with rhabdomyosarcoma of the large population, the treatment effect was improved, but the long-term cure rate is not satisfactory. The pathogenesis of RMS has a lot of progress, for the comprehensive treatment can be improved help. But for patients who relapse, currently rely mainly on the implementation of chemotherapy, but the toxicity of chemotherapy is limited by its use in pediatric patients. So there is an urgent need for treatment The new drug research group reported rhabdomyosarcoma. Tumor cells often showed low protein acetylation, and histone deacetylase (HDAC) activity in the occurrence and development of abnormal histone acetylation imbalance and tumor is closely related to the.HDAC inhibitor SAHA can reversibly reverse histone acetylation, thereby regulating gene the expression of apoptosis, cell cycle arrest, anti angiogenesis, DNA damage and repair and weaken the invasion and migration of tumor cells. Therefore, SAHA is expected to become a new target for the treatment of rhabdomyosarcoma to drugs. So the purpose of this paper is to study the proliferation of SAHA on human rhabdomyosarcoma RD cells cell cycle, apoptosis, autophagy effect and migration, and to explore the mechanism of apoptosis. Methods: different concentrations of SAHA in RD cells. The effect of MTT method to detect cell proliferation, weste Expression of ERK signaling pathway protein RN blot; cell cycle analysis of PI staining method; Annexin V-FITC/PI method to detect cell apoptosis, apoptotic cells verified by DAPI staining, then the following experiments: preliminary study on the mechanism of apoptosis induced by changes in mitochondrial membrane potential detection JC-1 probe; Western blot detection of apoptosis related protein, mitochondrial pathway related changes protein; autophagy was detected by MDC staining, the expression of Western blot was used to detect the protein; cell scratch detection cell migration; acetylation level of histone H4 Western were detected by blot. Results: 1.saha inhibited the proliferation of RD cells, and has a time and concentration dependent effect of.Saha on RD 24h cells. 48h 72h, the half inhibition rate of IC50 was 8.503 mol//l, 3.965 mol/l and 1.921 mol/l; and RD cell proliferation and ERK signal pathway changes: ERK protein phosphorylation The level of decline, but no significant changes in the total protein level of ERK 2.; flow cytometry showed that RD Saha cells g2/m phase arrest; 3.annexinv-fitc/pi staining showed that Saha could induce Rd cell apoptosis; DAPI staining showed that the experiment, with the increase of SAHA concentration, the number of apoptotic cells increased JC-1 flow cytometry showed that with the increase of SAHA concentration, the level of RD cells mitochondrial membrane potential gradually decreased; Westernblot results showed that with the increase of SAHA concentration, apoptosis related protein caspase-3 and PARP-1 expression and anti apoptosis protein bcl-2 expression reduced gradually, the pro apoptotic protein Bax expression increased gradually, the increased expression of cytochrome c and caspase-9 are 4.saha; the autophagy in RD cells: MDC staining results show that with the increase of SAHA concentration, the number of spots of green fluorescent cells within the bright dot Gradually increased, indicating that cell autophagy.Westernblot results showed that with the increase of SAHA concentration, p62 protein expression decreased, Beclin1 protein expression gradually increased, the expression of lc3- protein also increased significantly inhibited Rd cell migration of.5.saha: the experimental results show that the node cell scratch with the increasing of SAHA concentration, the wound healing rate decreased.6.westernblot the results showed that with the increase of SAHA concentration, the acetylation level of histone H4 increased. Conclusion: 1.hdac inhibitor Saha inhibited the proliferation of RD cells, the g2/m phase arrest in RD cells, the inhibition is time and concentration dependent manner; the apoptosis of RD cells induced by 2.hdac inhibitor Saha mediated apoptosis, the possible mechanism for depending on the mitochondrial pathway and dependent on caspase; 3.hdac inhibitor Saha induces autophagy in RD cells; the migration ability of 4.hdac inhibitor Saha inhibited Rd cells.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738.7
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本文编号：1559090