【作者】 王元虎；

【导师】 何俊琳；

【作者基本信息】 重庆医科大学 ， 遗传学， 2010， 硕士

【摘要】 绒毛膜癌(chorionic carcinoma,CH)简称绒癌,是一种极为常见的、高度恶性的妊娠性滋养细胞肿瘤( gestational trophoblastic neoplasia,GTN),其临床治疗以化疗为主,治愈率可达90%。然而,近年来难治性、多药耐药性绒癌的增多,使临床治愈率大大降低,因此寻找安全、有效且毒副作用小的抗绒癌药物,首要解决的问题就是阐明绒癌发生的机制。绒癌发病机制较为复杂,遗传学研究显示多种原癌基因、抑癌基因、细胞转录因子的表达异常以及激素水平异常与其发病相关[1,2]。目前研究发现,由表观遗传,如DNA甲基化异常引发的基因转录异常可能在绒癌的发生机制中发挥重要作用,但是其机制并不明了。DNA甲基化是一个逆转的修饰过程,且该逆转可直接恢复肿瘤细胞中某些抑癌基因的功能[3-6]。因此,从控制DNA甲基化的关键酶的表达角度去探讨绒癌的增殖、侵袭和凋亡等绒癌发生中的重要事件,可为临床上认识并寻找新的治疗策略提供实验依据。本研究采用不同浓度(1、4、8、16、32μmol/L)DNA甲基化酶抑制剂5-Aza-CdR处理绒毛膜癌JEG-3细胞,应用Real Time PCR及Western印迹检测5-Aza-CdR处理细胞后DNA甲基化转移酶(DNAmethyltransferases,Dnmts)、DNA去甲基化酶(Methyl-DpG binding domain protein 2,Mbd2)mRNA及其蛋白的表达变化。进一步研究其对细胞生长特征的影响,包括细胞形态学变化、细胞增殖水平变化、细胞凋亡及细胞侵袭能力的改变。研究发现:(1) Real Time PCR检测Dnmt1、Dnmt3a mRNA的表达显示,5-Aza-CdR可降低Dnmt1、Dnmt3a mRNA的表达,且呈剂量依赖关系,与对照组相比差异显著(P <0.05)。而Dnmt3b、Mbd2 mRNA表达水平几乎未受到影响,与对照组相比,无统计学意义(P >0.05)。(2) Western-blotting结果显示,5-Aza-CdR可明显降低Dnmt1、Dnmt3a mRNA的表达水平,并呈剂量依赖关系(P <0.05);Dnmt3b、Mbd2蛋白表达差异不明显(P >0.05)。(3)用5-Aza-CdR处理JEG-3细胞48 h后,显微镜观察发现细胞明显皱缩、细胞核聚集成块,死亡漂浮细胞明显增多,细胞长势较差。(4) MTT实验结果显示,不同浓度的5-Aza-CdR均可抑制JEG-3细胞增殖,并呈剂量和时间依赖关系。(5)流式细胞术检测结果显示,随着5-Aza-CdR浓度的增加,JEG-3细胞凋亡率显著增加(P <0.01)。5-Aza-CdR可促进细胞凋亡。(6) Transwell小室检测5-Aza-CdR对JEG-3细胞侵袭能力表明10μmol/L的5-Aza-CdR可显著降低细胞的侵袭能力(P <0.01)。综上,5-Aza-CdR可抑制Dnmt1、Dnmt3a mRNA和蛋白的表达。而不影响Dnmt3b、Mbd2mRNA和蛋白的表达。5-Aza-CdR可致JEG-3细胞皱缩、细胞核聚集成块,可明显抑制细胞增殖,促进其凋亡,降低细胞的侵袭力。Dnmt1、Dnmt3a可能在绒癌的增殖、侵袭和凋亡中发挥着重要作用。更多还原

【Abstract】 horionic carcinoma called choriocarcinoma for short,is an extremely common and highly malignant gestational trophoblastic neoplasia. Chemotherapy is the main clinical therapy , the cure rate can reach to 90%. However,in recent years,with the increase of multidrug-resistant chorioepithelioma which is difficult to cure, the clinic cure rate is decreased significantly.So before seeking for anti-chorioepithelioma drug,which is safe,effective,and with small toxic and side-effect,the most important thing is to clarify the mechanism of occurrence of choriocarcinoma.The pathological mechanism of chorioepielioma is complicate.The studies on genetic have shown a variety of oncogenes, tumor suppressor genes, abnormal expression of cellular transcription factors, and abnormal hormone levels associated with this disease [1,2].Present researches find the abnormal transcription of genes can induce by epigenetic, such as DNA methylation abnormalities which may be play an important role in the pathogenesis of choriocarcinoma.But it is not clear at present.DNA methylation is a reversible modification process,and the reversal of tumor cells can directly restore the function of certain tumor suppressor genes[3-6].Therefore,to study on point of view in DNA methyltransferase,cell proliferation,invasion and apoptosis can provide some fundamental experimental evidence for new strategy of the chorioepielioma treatment.We treated the choriocarcinoma JEG-3 cells with different concentrations(1,4,8,16,32μmol/L)of the DNA methyltransferase inhibitor 5-Aza-CdR.Then we used Real Time PCR and Western blotting to detect the change of mRNA and protein of DNA methyltransferases,DNA demethylase.Further more changes of cell morphology were observed under the microscope,and cell proliferation was detected by MTT.Flow Cytometry was used to detected JEG-3 cell apoptosis,and Transwell chamber measured the invasion of JEG-3 cells.Real Time PCR 5-Aza-CdR could significantly reduce the Dnmt1,Dnmt3a expression levels in a dose dependent manner,compared with the control degree of significant difference(P<0.05),and it could not affect the expression of (P> 0.05).(2) Western blotting results showed the same results as the Real Time PCR.5-Aza-CdR could significantly reduce the (P<0.05),and it could not affect the expression of (P>0.05).(3) After treatment JEG-3 cells with 5-Aza-CdR for 48 h,we observed by the microscope that the cells showed shrinkage,nuclear poly integrated block.Floating dead cells were significantly increased and cells grew badly.(4) MTT experiment results showed that different concentrations of 5-Aza-CdR could inhibit the proliferation of JEG-3 cells in a dose and time dependent manner.(5) The cell apoptosis was dected by flow cytometry.We found that the apoptosis rate was significantly increased with the multiplicative concentration of 5-Aza-CdR(P<0.01).(6) The invasion of JEG-3 was tested by Transwell small chamber. 5-Aza-CdR treatment led to depression of the cell invasiveness.There was a significant difference compared with the control group(P<0.01).5-Aza-CdR can inhibit the Dnmt1,Dnmt3a mRNA and protein expression remarkably,but it does not affect the Dnmt3b,Mbd2 mRNA and protein expression.5-Aza-CdR can also induce JEG-3 cell shrinkage,nuclear poly blocks,inhibit the cell proliferation and induce apoptosis and reduce the cell invasiveness.Dnmt1,Dnmt3a may play an important role in choriocarcinoma proliferation,invasion and apoptosis.更多还原