【作者】 杨琦；

【导师】 孙志熙；

【作者基本信息】 中国医科大学 ， 外科学， 2009， 硕士

【摘要】 前言表观遗传学被描述为没有DNA序列变化、可遗传的表达改变。研究表明表观遗传学改变在肿瘤发生中扮演一个重要角色,肿瘤发生中的主要表观遗传学改变是抑癌基因发生DNA异常甲基化和染色质中的组蛋白修饰。表遗传改变也是一个致癌事件,但是与遗传学改变不同的是,表遗传学的改变是可逆的。通过使用DNA甲基化酶抑制剂和组蛋白去乙酰化转移酶(HADC)抑制剂可以使表遗传静止的基因恢复活性,从而逆转肿瘤的恶性表型或延缓肿瘤的进展。表遗传修饰可逆的特性提示它可作为肿瘤患者进行治疗的靶标。DNA甲基化调节基表达及某些抑癌基因高甲基化失活与肿瘤发生相关的理论已得到广泛认同。DNA甲基化调节基因表达与组蛋白去乙酰化的作用密切相关。DNA甲基化可诱导局部组蛋白脱乙酰化,使染色质乙酰化水平降低,且甲基化的序列可募集甲基CpG结合蛋白(MeCP)与组蛋白去乙酰化酶(HADC)复合物(MeCP-HADC),发挥对基因表达的抑制作用。因DNA甲基化失表达的抑癌基因能否通过去甲基化制剂或/和HDAC抑制剂的作用重新表达为本次研究的目的。雌激素受体属于配体激活的核转录因子超家族的一员。分为二型,ERα和ERβ;ERα位于前列腺基底上皮细胞和基质间隙,ERβ位于前列腺腺体上皮间隙,目前认为雌激素对于前列腺上皮的作用是通过ERβ信号途径。Pasquali等研究表明在前列腺癌中没有发现ERβ表达。然而,Leav等报道ERβ在高级别发育异常中免疫活性减少,但在高级别肿瘤和转移性前列腺癌中重新出现,进而有人认为同乳腺癌一样,前列腺癌中即使有表达的ERβ可能是野生型ERβ的变异体,ERβ具有调节细胞生长抑制的作用,其发生变异突变可能导致抗雌激素治疗不敏感或使癌细胞更具侵袭性。而目前绝大多数免疫组化染色研究仅仅用一种ERβ抗体,难以区分其他各种ERβ亚型的蛋白表达情况。相关研究表明ERβ在大多数前列腺癌中表达缺失显示ERβ可能是潜在的肿瘤抑制基因,可能是治疗前列腺癌的理想目标。在前列腺癌细胞株与前列腺癌组织中广泛存在雌激素受体(ER)基因的甲基化,甲基化的程度与肿瘤病理级别呈正相关,与ER基因的表达呈负相关,虽然在前列腺增生组织中也存在ER甲基化,但明显低于前列腺肿瘤。在前列腺癌中,目前可能的一个使ER基因失活的原因就是ER基因启动区域的CpG岛甲基化,通过一些尚不明了的机制导致ER基因转录失活。为了研究前列腺癌细胞中雌激素受体β的表达情况与DNA甲基化酶抑制剂和HDAC抑制剂的作用是否有关联,本实验通过DNA甲基化酶抑制剂5-杂氮-2’-脱氧胞苷(5’-aza-2’-deoxycytidine,5-AZAC)和HDAC抑制剂曲古抑霉素A(Trichostatin A,TSA)作用于前列腺癌细胞系Du145、PC-3和22RV,检测细胞凋亡情况;应用荧光实时定量PCR检测细胞中雌激素受体β的表达情况,观察其是否上调,为前列腺癌的临床治疗提供基础。方法一.材料与主要试剂雄激素非依赖性前列腺癌细胞Du145、PC-3和雄激素依赖性前列腺癌细胞22RV购于中科院上海细胞库;5-杂氮-2’-脱氧胞苷(美国Sigma公司),曲古抑霉素A(美国Sigma公司),RT—PCR试剂盒(Takara公司),四甲基偶氮唑蓝(美国Sigma公司),二亚基亚矾(美国Sigma公司),Trizol(美国Gibco公司),SBRY-Green(天根),Master-mix(天根)。二.实验方法1.细胞培养Du145和22RV细胞以含10%胎牛血清的RPMI1640培养基,PC-3以含10%胎牛血清的HAM/F-12培养基于37℃、5%CO2培养箱中培养,每3～4d消化传代1次,取对数生长期的细胞进行实验。2.MTT检测5-AZAC和TSA对前列腺癌细胞的影响检测不同药物浓度的5-AZAC和TSA以及5-AZAC和TSA共同作用的前列腺癌细胞的细胞生存率。细胞生存率(%)=(实验组A值-空白组A值)/(阴性对照组A值-空白组A值)×100%。实验重复3次。3.实时荧光定量PCR检测5-AZAC和TSA作用后的前列腺癌细胞雌激素受体β的表达收集经不同药物处理或未处理的细胞,Trizol提取总RNA,应用TaKaRa公司的RT-PCR试剂盒和SBRY-Green检测雌激素受体β的表达。4.统计学分析所有数据采用SPSS11.6软件包进行处理,取p<0.05具有统计学意义。结果MTT检测发现5-AZAC和TSA处理后的前列腺癌细胞株生存率降低,在一定范围内随着剂量的增高,前列腺癌细胞生存率降低的越明显;5-AZAC和TSA联合作用时前列腺癌细胞的生存率最低。实时荧光定量PCR显示三种前列腺癌细胞经过5-AZAC和TSA作用后,mRNAERβ表达量增加,并且随着药物浓度的增大,mRNA ERβ表达量越多;5-AZAC和TSA联合作用mRNAERβ表达量比单独作用都多出1倍左右。结论5-AZAC和TSA对前列腺癌细胞的生长起抑制作用,在一定范围内随着剂量的增高,对前列腺癌细胞增殖的抑制作用越明显;TSA对细胞的抑制作用强于5-AZAC;5-AZAC和TSA联合作用时抑制作用强于单独药物作用。5-AZAC和TSA都可以诱导前列腺癌细胞的ERβ表达上调,从而抑制了前列腺癌细胞的生长。更多还原

【Abstract】 IntroductionEpigenetic events are defined as alterations in gene expression without changes in the DNA coding sequence that are heritable through cell division.Study shows that epigenetic changes in tumor happen to play an important role in tomorigenesis,main epigenetic changes in tomorigenes contain DNA methylation and histone modification.Epigenetic is a reversible carcinogenesis event that is different from genetic change.Static genes can reactivate by using DNA methyltransferase inhibitors and histone deacetylase inhibitors,and thus reverse the malignant phenotype of the tumor or slow down the progress of tumor.The reversible characteristics of epigenetic modification hints it can be used as treatment targets for tumor patients.DNA methylation-based regulation of expression and certain tumor suppressor gene hypermethylation and inactivation of tumor-related theory has been widely recognized. DNA methylation in regulating gene expression is closely related to histone acetylation. DNA methylation can induce local histone deacetylation,so that the level of chromatin acetylation reduced.Methylation sequences can collect methy CpG binding protein (MeCP) and histone deacetylase(HADC) complex(MeCP-HADC),exert inhibitory effects on gene expression.If the tumor suppressor gene that inactivated by loss of DNA methylation expression can reexpress by demethylation agents and / or HDAC inhibitors is the purpose of this study.Estrogen receptor belongs to the ligand-activated nuclear transcription factor superfamily.Estrogen receptor is divided into two types,ERa and ERβ;ERa is located in the basement of prostate epithelial cells and stromal space,ERβis located in the prostate gland epithelial gap,the role of estrogen is considered by the prostatic epithelial signaling pathways through ERβ.Pasquali research shows that prostate cancer is found no expression of ERβ.However,Leav reports ERβdecrease in high-level dysplasia,but reemerge in prostate cancer and metastatic tumor.Prostate cancer is considered,like breast cancer,even if there is the expression of Erβ,that are maybe the wild-type ERβvariants.ERβcan regulate cell growth and its mutation may cause anti-estrogen therapy is not sensitive or cause a more invasive cancer.At present, there is only one ER8 antibody in the vast majority of immunohistochemical staining,it is difficult to distinguish subtypes of ERβprotein expression.Related study shows that loss of ERβexpression in the majority of prostate cancer reveals ERβmay be a potential tumor suppressor gene,and ERβis probably the desired objectives treatment of prostate cancer.In prostate cancer cell lines and prostate cancer,there is a wide range of estrogen receptor(ER) gene methylation,the degree of methylation was positively correlated with tumor pathological level,and was negatively correlated with ER expression. Although ER methylation exists in benign prostatic hyperplasia organizations,it was significantly lower than that of prostate tumor.In prostate cancer,at present,a possible reason to make ER gene inactivate is that the ER gene promoter region CpG island methylation,through some mechanism that is not yet known causes inactivation of ER gene transcription.In order to study the association of estrogen receptorβexpression in prostate cancer and DNA methylase inhibitors,HDAC inhibitors,we use DNA methylase inhibitor,5 -aza-2 ’- deoxycytidine(5-AZAC) and HDAC inhibitors Trichostatin A(TSA) acts on prostate cancer cell line Du145,PC-3 and 22RV,detect cell apoptosis;detect of estrogen receptorβexpression by fluorescence real-time quantitative PCR,provide a basis for clinical treatmentMethod1.Materials Androgen-independent prostate cancer cells Dul45,PC-3 and androgen independent prostate cancer 22RV purchased from cell bank of China Shanhai Cell Institute;5 - aza-2 ’- deoxycytidine(Sigma USA),Trichostatin A(Sigma USA), RT-PCR kit(Takara Company),MTT(Sigma USA),Dimethyl sulfoxide(Sigma USA), Trizol(USA Gibco Company),SBRY-Green(Tiangen),Master-mix(Tiangen).2.Experimental methods (l)Cell CultureDU145,and 22RV cells were maintained in RPMI-1640 medium,PC-3 cell was maintained in HAM/F-12 medium,supplemented with 10%fetal bovine serum(FBS) at 37℃in a humidified,5%CO2 incubator.The cell was passaged every three or four days.The cell on logarithmic growth phase was used to make the study.(2)The effects of 5-AZA and TSAon prostate cell lines by MTTProstate cells were treated with different concentrations of 5-AZA and TSA,and then method of MTT was used to detect the inhibition rate.Survival rate=(A of treated group-A of blank group)/(A of control group- A of blank group)×100%.(3)Detection of ERβexpression by fluorescence real-time quantitative PCRCells were harvested,and total RNA was isolated using Trizol reagent,detection of ERβby RT-PCR kit(Takara Company) and SBRY-Green.(4)Statistic analysisAll the data were analyzed by SPSS11.6 and when the value of p was less than 0.05,it was statistical significance.Result1.The survival rate of prostate cancer cell line after 5-AZAC and TSA treatment decreased by MTT;At a certain range,as the dose increased the survival rate of prostate cancer cells reduce more;prostate cancer treated by association of 5-AZAC and TSA has the lowest survival rate.2.Real-time quantitative PCR showed that after 5-AZAC and TSA treatmenr,mRNA ERβexpression of three types of prostate cancer increased;as drug dose increase, mRNA ERβexpress more;mRNA ERβexpression of prostate cancer treated by association of 5-AZAC and TSA is the most.Conclusion1.5-AZAC and TSA inhibit the growth of prostate cancer cells,in a certain range,as the dose increased,inhibitory effect of prostate cancer cell proliferation was more obvious;TSA had strong inhibitory effect on cells than 5-AZAC;inhibitory effects of association of 5-AZAC and TSA was more obvious than single drug effects.2.5-AZAC and TSA can induce ERβexpression of prostate cancer cells,thereby inhibiting the growth of prostate cancer cells.更多还原