【作者】 钱林华；

【导师】 曲芃芃；

【作者基本信息】 天津医科大学 ， 妇产科学， 2010， 硕士

【摘要】 目的：观察DNA甲基化转移酶(DNA methyltransferase,DNMT)抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-CdR)联合组蛋白去乙酰化酶(histone deacetylase,HDAC)抑制剂苯丁酸钠(Sodium 4-Phenylbutyrate,SPB)对荷卵巢癌细胞系SKOV3裸鼠的抗肿瘤增殖作用及其与上皮性钙黏素(epithelial cadherin,E-cad)表达变化的关系。方法：1.用人卵巢浆液性乳头状囊腺癌细胞系SKOV3建立卵巢癌裸鼠腹腔移植瘤模型,成瘤后随机分为4组：对照组、5-Aza-CdR组、SPB组和5-Aza-CdR+SPB组。2.观察5-Aza-CdR,SPB以及5-Aza-CdR+SPB对裸鼠腹腔移植瘤的抑制作用及对裸鼠的副作用。3.免疫组化法检测腹腔移植瘤中DNMT1、HDAC1以及E-cad的表达变化情况。4.应用甲基化特异性PCR(methylation specific polymerase chain reaction,MSP)检测各组移植瘤E-cad基因启动子区5’CpG岛甲基化状况,以凝胶成像系统对结果进行分析。5.RT-PCR法检测各组移植瘤E-cad mRNA的表达变化情况。6. Western印迹方法检测各组移植瘤E-cad蛋白的表达变化情况。结果：1.比较4组裸鼠在治疗前后的体重变化,发现对照组裸鼠体重有所减轻,与治疗组相比无统计学差异(P>0.05)；各组裸鼠体重在治疗前后均无统计学差异(P>0.05)。2.与对照组相比,治疗组中裸鼠腹围增长量较小,腹水量较少,差异显著(P<0.05),而与单用药组相比,联合组裸鼠腹围增长量较小,腹水量较少,差异显著(P<0.05),两组单用药组之间腹水量无统计学差异(P>0.05)。3.与对照组相比,治疗组裸鼠瘤重明显减轻,差异显著(P<0.05),与单用药组相比,联合组瘤重明显较轻,差异显著,(P<0.05)；而单用药组间瘤重无统计学差异(P>0.05)。联合组比单用药组裸鼠腹腔移植瘤抑瘤率高,差异显著(P<0.05)。4.与对照组相比,各治疗组腹腔肿瘤负荷分级要低,差异显著(P<0.05),而联合组与单用药组相比,联合组腹腔肿瘤负荷分级要低,差异显著(P<0.05),两单用药组之间无统计学差异(P>0.05)。5.与对照组相比,治疗组中肝转移减少,差异显著(P<0.05),而各给药组问肝转移无统计学差异(P>0.05),肺、肾、胰、脾转移情况各组间无统计学差异(P>0.05)6.在移植瘤组织中,DNMT1和HDAC1呈高表达,5-Aza-CdR能抑制DNMT1的表达,SPB能抑制HDAC1的表达,与对照组相比,差异显著(P<0.05),联合应用5-Aza-CdR和SPS对DNMT1和HDAC1的抑制未见协同作用。7.MSP检测发现对照组、SPB组移植瘤中E-cad基因高甲基化,5-Aza-CdR能够诱导甲基化的E-cad基因去甲基化,联合应用5-Aza-CdR和SPB可更大程度上诱导甲基化的E-cad基因去甲基化。8.RT-PCR检测发现对照组、SPB组移植瘤中甲基化的E-cad基因表达缺失或者下调,而经过5-Aza-CdR治疗后,mRNA恢复表达或者表达增强。联合应用5-Aza-CdR和SPS可更大程度上恢复mRNA的表达。9.免疫组化和Western-blotting检测发现对照组、SPB组移植瘤中甲基化的E-cad蛋白表达缺失或者下调,而经过5-Aza-CdR治疗后,E-cad蛋白恢复表达或者表达增强。联合应用5-Aza-CdR和SPB可更大程度上恢复E-cad蛋白的表达。结论：1.5-Aza-CdR和SPB对荷卵巢癌细胞系SKOV3裸鼠都有抑制增殖和诱导凋亡作用,而联合用药组抑制增殖和诱导凋亡作用明显增强；联合用药组比单用药组裸鼠腹腔移植瘤抑瘤率高。2.5-Aza-CdR和SPB都无明显的毒副作用,联合用药组也未发现明显的毒副作用3.5-Aza-CdR能够降低E-cad基因启动子区的甲基化,可恢复其表达；联合应用5-Aza-CdR和SPB可产生协同效应,显著增强甲基化的E-cad基因的表达,降低细胞侵袭、转移力,对于卵巢癌的治疗有着重要的作用。更多还原

【Abstract】 Objective:To evaluate the effect of inhibiting growth of the tumor on nude mice models of SKOV3 after given 5-Aza-CdR and SPB and its relationship with the expression of epithelial cadherin.Methods:1.To establish the ascite implantation nude mice models of SKOV3.The tumor-bearing nude mice were randomly divided into four groups:control group,5-Aza-CdR group,SPB group,and 5-Aza-CdR plus SPB group.2.Observe the effect of inhibiting carcinoma and adverse effects to the nude mice after given 5-Aza-CdR and SPB.3.The DNMT1,HDAC and E-cad expression in xenograft tumors were analyzed by immunohistochemical staining.4. The CpG island methylation status of E-cadherin promoter region in xenograft tumors were analyzed by MSP.5.The mRNA expressions of E-cad was analyzed by RT-PCR.6.The protein expressions of E-cad was analyzed by Western blotting.Results:1.Comparison the weight changes of four groups before and after treatment,the weight of control group was loss,compared to the treating-group,there is no significant difference in weight with the treating-group,and no significant difference in weight among four groups before and after treatment(P>0.05).2.Compared to the control group, there is significant difference in increment of abdominal circumference and the volumn of ascites with the treating-group(P<0.05). Compared to the Single drug group, there is significant difference in increment of abdominal circumference and the volumn of ascites with the Combination group(P<0.05);Compared two groups of single-drug group, there is no significant difference in increment of abdominal circumference and the volumn of ascites(P>0.05).3.Compared to the control group, there is significant difference in Tumor weight with the treating-group(P<0.05);Compared to the Single drug group,there is significant difference in Tumor weight with the Combination group(P<0.05); Compared two groups of single-drug group, there is no significant difference in Tumor weight(P>0.05).There is significant difference in the inhibiting tumor rate between single-drug group and Combination group(P<0.05).4. Compared to the control group, there is significant difference in levels of tumor load with the treating-group(P<0.05);Compared to the Single drug group, there is significant difference in evels of tumor load with the Combination group(P<0.05); Compared two groups of single-drug group, there is no significant difference in evels of tumor load(P>0.05).There is significant difference in the inhibiting tumor rate between single-drug group and Combination group(P<0.05).5.Compared to the control group,there is significant difference in the rate of liver tranplantation with the treating-group(P<0.05), and no significant difference in the rate of liver transplantation among the treating-groups.(P>0.05).There is no significant difference in the rate of Lung, kidney, pancreas and spleen transplantation among the four groups.6.Immunohistochemistry detection:DNMT1 protein and HDACl protein was high expressed in xenograft tumors;5-Aza-CdR can inhibit the expression of DNMT1,SPB can inhibit the expression of HDAC1;Compared to the control group,there is significant difference in the the expression of DNMTl protein and HDAC1 protein with the treating-group(P<0.05);The combining 5-Aza-CdR with SPB have no obvious synergistic effect on the inhibition of DNMT1 and HDAC1.7.MSP detection:E-cad gene was hypermethylated in xenograft tumors of control group and SPB group;5-Aza-CdR induced the demethylation of E-cad gene and the methylation of E-cad in xenograft tumors were reversed after 5-Aza-CdR treatment.The combination of 5-5-Aza-CdR and SPB dramatically enhance the effect of demethylation.8.RT-PCR detection:E-cad mRNA was expressed in xenograft tumors after 5-Aza-CdR treatment,but it was undetectable or express weakly before the treatment.The combination of 5-Aza-CdR and SPB dramatically up-regulate expression of mRNA in xenograft tumors. 9.Immunohistochemistry and western-blotting detection:E-cad protein was expressed in xenograft tumors after 5-Aza-CdR treatment,but it was undetectable or express weakly before the treatment.The combination of 5-Aza-CdR and SPB dramatically up-regulate xpression of E-cad protein in xenograft tumors.Conclusions:1.Both 5-Aza-CdR and SPB can inhibit proliferation and induce apoptosis to the implantion tumor on nude mice models of SKOV3,Combined treatment group inhibited proliferation and induced apoptosis significantly increased;The Combination group is better than the Single drug group in the rate of inhibition.2.5-Aza-CdR and SPB are no obvious side effects, the combination group also found no obvious side effects.3.5-Aza-CdR can cut down the methylation of E-cadherin promoter region and recover the expression of E-cadherin;5-Aza-CdR and SPB can synergistically re-express E-cadherin gene and weaken the invasiveness of SKOV3 cell,which plays an important part in treatment of ovarian cancer.更多还原