【作者】 丁波泥；

【导师】 陈道瑾；

【作者基本信息】 中南大学 ， 外科学， 2008， 博士

【摘要】 乳腺癌是严重危害女性健康的最常见的肿瘤,发病率在逐年上升。大量的实验与临床研究发现雌激素在乳腺癌的发生发展中起着重要作用。在乳腺癌人群中,大约2/3乳腺癌患者表达雌激素受体(Estrogen Receptor,ER),此类患者肿瘤细胞生长具有雌激素依赖性,其作用机制是雌激素通过与其受体结合,作用于乳腺上皮,促使乳腺肿瘤的发生与发展。临床上对于ER阳性乳腺癌患者常采取抗雌激素的内分泌治疗,该疗法与联合化疗疗效相当,且成本低,毒副反应小,口服方便。但是,临床上仍有1/3的原发乳腺癌患者缺乏ER表达,此类患者对内分泌治疗反应差。ERα基因定位于染色体6q24-27上,其表达的ERα蛋白常作为一个重要分子标志物用于评估乳腺癌治疗的激素依赖性和预测内分泌治疗效果。研究发现ERα阴性乳腺癌细胞缺乏ERα蛋白的表达不是由于ERα基因内部发生突变,因此推测ERα基因的失转录是导致激素抵抗的可能机制。文献报道ERα阴性乳腺癌ER基因启动子CpG岛(富含胞嘧啶和鸟嘌呤)呈高甲基化,因为基因启动子CpG岛的甲基化可直接抑制基因转录,因此ERα蛋白的失表达很可能是由于雌激素受体(ER)基因启动子CpG岛异常的高甲基化所致。本研究初步探讨了具有中国特色的抗肿瘤中药天花粉蛋白(Trichosanthin,TCS)对MDA-MB-231和MCF-7乳腺癌细胞系的生长抑制作用,并进一步研究其诱导雌激素受体(ER)阴性人乳腺癌细胞株MDA-MB-231的ERα基因去甲基化的可能性及ERα基因表达情况和功能状态,以期阐明乳腺癌中ERα基因的甲基化情况,以及通过改变ERα基因的甲基化而为乳腺癌治疗提供新的方法。具体实验研究设计如下:(1)以MDA-MB-231和MCF-7乳腺癌细胞作为研究对象,TCS作用于乳腺癌细胞株,观察其对癌细胞生物学行为的影响。(2)检测MDA-MB-231和MCF-7乳腺癌细胞中ERα的甲基化状态、ERαmRNA和蛋白表达,研究它们之间的相关性,初步探讨ERα在乳腺癌中表达沉默的机制。(3)以TCS作用于MDA-MB-231乳腺癌细胞,并以去甲基化药物5-氮杂胞苷(5-azacytidine,5-Aza-C)为阳性对照,观察上述指标的变化及细胞对内分泌治疗药物他莫昔芬(Tamoxifen,TAM)的敏感性,探讨TCS的去甲基化效应。(4)最后,在裸鼠体内实验中,研究TCS的抗乳腺癌效果及其在体内应用的安全性,并研究其对ERα表达的影响,以证明TCS的抑瘤作用和去甲基化在ERα表达下调中的作用。第一章TCS对MDA-MB-231和MCF-7乳腺癌细胞的影响目的:探讨TCS对MDA-MB-231和MCF-7乳腺癌细胞生物学行为的影响以及可能的量效关系。方法:(1)MDA-MB-231和MCF-7乳腺癌细胞株常规培养,用TCS处理细胞,光学显微镜下观察药物处理前后乳腺癌细胞的形态学改变;(2)MTT法检测TCS处理MDA-MB-231和MCF-7乳腺癌细胞前后细胞的增殖情况;(3)TCS(90μg/ml)处理MDA-MB-231和MCF-7乳腺癌细胞48小时后,用流式细胞仪检测用药前后细胞周期时相变化和细胞凋亡率。结果:(1)TCS处理MDA-MB-231和MCF-7乳腺癌细胞后细胞形态发生了变化,部分细胞出现变性坏死;(2)四唑盐比色法(MTT法)显示随着TCS作用时间延长,浓度增大,细胞的生长受到明显的抑制(p＜0.05);(3)流式细胞仪检测发现,TCS使MDA-MB-231和MCF-7乳腺癌细胞阻滞于G0/G1期,细胞不能通过G1/S限制点进入S期进行复制,S期细胞比例明显减少。流式细胞仪检测到凋亡峰。结论:TCS可抑制MDA-MB-231和MCF-7乳腺癌细胞增殖,并呈时间和浓度依赖性;TCS可诱导MDA-MB-231和MCF-7乳腺癌细胞的凋亡,使细胞阻滞在G0/G1期。第二章TCS对MDA-MB-231乳腺癌细胞ERα基因表达及甲基化的影响目的:探讨乳腺癌ERα基因表达缺失与ERα基因高甲基化的相关性,并研究TCS对ERα阴性乳腺癌细胞株MDA-MB-231 ERα基因表达和CpG岛甲基化的影响及细胞经TCS处理后,可否恢复对TAM的敏感性。方法:选用ERα阴性人乳腺癌细胞株MDA-MB-231为研究对象,ERα阳性人乳腺癌细胞株MCF-7为阳性对照,分别用去甲基化剂5-Aza-C(90μg/ml)和抗肿瘤中药TCS(5μM)处理MDA-MB-231乳腺癌细胞,并进行以下研究:(1)RT-PCR法检测细胞ERαmRNA的表达情况;(2)甲基化特异性PCR(methylation specific PCR,MSP)方法检测细胞ERα基因5′CpG岛甲基化的情况;(3)Western blot方法检测细胞ERα蛋白的表达;(4)MTT法检测MDA-MB-231乳腺癌细胞经TCS和5-Aza-C处理后,雌激素拮抗剂TAM对肿瘤细胞生长的影响。结果:(1)RT-PCR法显示ERα阴性人乳腺癌细胞株MDA-MB-231ERαmRNA无表达,ERα阳性人乳腺癌细胞株MCF-7 ERαmRNA表达呈强阳性,5-Aza-C和TCS处理后的MDA-MB-231细胞ERαmRNA恢复表达;(2)MSP鉴定ERα甲基化状态:MDA-MB-231细胞仅甲基化引物扩增出特异PCR条带;MCF-7细胞仅非甲基化引物扩增出特异PCR条带;5-Aza-C和TCS处理后的MDA-MB-231细胞仅非甲基化引物扩增出特异PCR条带;(3)Western-blot方法分析显示MDA-MB-23细胞ERα蛋白不表达;MCF-7细胞ERα蛋白表达呈强阳性;5-Aza-C和TCS作用MDA-MB-231细胞后ERα蛋白恢复了表达;(4)MTT检测TCS和5-Aza-C处理ERα阴性MDA-MB-231细胞后,TAM恢复了对肿瘤细胞的生长抑制作用,且TCS处理组抑制作用比5-Aza-C组强(p＜0.05)。结论:乳腺癌ERα基因表达缺失与ERα基因高甲基化有关,TCS可逆转ERα的甲基化,使ERαmRNA和蛋白重新表达,并可恢复对内分泌治疗药物TAM的敏感性。第三章TCS对ERα阴性乳腺癌裸鼠移植瘤生长及ERα表达的影响目的:研究TCS对ERα阴性乳腺癌裸鼠移植瘤生长及移植瘤内ERα基因表达和CpG岛甲基化的影响。方法:(1)将MDA-MB-231乳腺癌细胞悬液接种于20只BALB/C(nu/nu)雌性裸鼠(SPF级)。18只裸鼠成瘤后将其分为三组:对照组、TCS组和盐水组,每组6只。分别于移植瘤旁注射TCS(0.4mg/kg)、盐水0.2ml,隔日注射一次,共7次;(2)检测裸鼠移植瘤体积和瘤重,计算肿瘤生长抑制率,检测各组裸鼠肝肾功能和血常规;(3)RT-PCR检测TCS对裸鼠乳腺癌移植瘤ERαmRNA表达的影响;(4)MSP检测TCS对裸鼠乳腺癌移植瘤ERα甲基化状态的影响;(5)Western blot检测TCS对裸鼠乳腺癌移植瘤组织ERα蛋白表达的影响。结果:(1)细胞接种裸鼠成瘤成功率为90%(18/20);(2)TCS组裸鼠移植瘤体积和瘤重较对照组明显缩小(P＜0.05);(3)TCS对荷瘤裸鼠肾功能、血液系统无明显影响(P＞0.05),可引起谷丙转氨酶轻度升高(P＜0.05);(4)RT-PCR结果显示TCS治疗组裸鼠乳腺癌移植瘤ERαmRNA重新表达,而对照组和盐水组则无ERαmRNA表达;(5)裸鼠移植瘤模型的对照组和盐水组仅甲基化引物扩增出特异PCR条带,而TCS组仅非甲基化引物扩增出特异PCR条带;(6)治疗组乳腺癌组织可见ERα蛋白表达,对照组和盐水组ERα蛋白表达阴性。结论:TCS可抑制乳腺癌肿瘤生长且在体内应用有一定的安全性;体内实验进一步证实TCS可以逆转ERα基因的高甲基化,使ERαmRNA和蛋白重新表达。更多还原

【Abstract】 Breast cancer is the most common cancer in women,of which the morbidity increases year by year.Many researches presented estrogen is important in breast cancer.Two thirds of primary breast cancer have the expression of estrogen receptor（ER） and the growth of tumor cell is hormonal dependent.The function of estrogen depends on binding with its receptor and promoting the progression of breast tumor.The clinical therapy of breast cancer patients（ER positive） usually includes endocrinotherapy,which is equal to chemotherapy.However,one third breast cancer patients（ER negative） are not sensitive to endocrine therapy.Estrogen receptor alpha（ERα） gene is located on the chromosome 6q24-27.The proteinum expressed by ERαgene is usually an important molecule marker to evaluate hormonal dependence and predicts the effect of endocrine therapy on breast cancer.Current researches report the mechanism of ERαgene expression silencing is not due to gene mutation, but probably associate with the genetic transcription.Because of the 5’CpG island methylation in the core promotor of ERαgene,the genetic transcription is suppressed which induce to the silence of ERαgene.Trichosanthin（TCS） is a kind of traditional Chinese medicine,which we choose as experimental drug of demethylation.The purpose is to investigate the the effect of TCS on proliferation of MDA-MB-231 and MCF-7 breast cancer cell,the demethylating effect on ERαgene in MDA-MB-231 breast cancer cell,the relationship between demethylation and gene expression;meanwhile,to explore a new therapeutic strategy for breast cancer by demethylating treatment.The design of the study include:（1） Investigate the effect of TCS acting on breast cancer cell MDA-MB-231 and MCF-7;（2） Measure the demethylation of ERαmRNA and protein.Analyze the relationship between ERαmRNA expression and methylation;（3） Explore the demethylating effect of TCS on MDA-MB-231 breast cancer cell and the sensitivity to endocrine therapy;（4） Study the suppressive effect of TCS on transplanted human breast cancer in nude mice and the security in vivo.The PartⅠObjective:Investigate the effects of TCS on proliferation,apoptosis of MDA-MB-231 and MCF-7 breast cancer cell.Methods:（1） Human breast cancer cell lines MDA-MB-231 and MCF-7 were growing at 37℃and 5%CO₂.The morphological change of cells were observed by optical microscope after treated with TCS（90μg/ml） for 72 hours;（2） MTT was performed for the proliferation of MDA-MB-231 and MCF-7 cell after treated with TCS;（3） The period and apoptosis rate of MDA-MB-231 and MCF-7 cell were tested by Flow Cytometry method（FCM） after treated with TCS（90μg/ml） for 48 hours.Results:（1） The cellular morphology changed after treated with TCS.Denaturation and necrosis occurred in some cells;（2） TCS could inhibit the growth of MDA-MB-231 and MCF-7 cell by MTT test;（3） TCS could suspend MDA-MB-231 and MCF-7 cell generation at G0/G1 period.It also induced MDA-MB-231 and MCF-7 cell apoptosis by FCM test.Conclusion:TCS could inhibit MDA-MB-231 and MCF-7 cell proliferation in a time- and concentration-dependent manner.TCS could induce MDA-MB -231 and MCF-7 cell apoptosis and suspend the cell generation at G0/G1 period.The PartⅡObjective:Explore the relationship between ERαgene expression silencing and hypermethylation.Study the effect of TCS on ERαgene expression and 5’CpG island methylation in MDA-MB-231 breast cancer cell.Test the possibility that TCS may induce MDA-MB-231 breast cancer cell sensitive to endocrine therapy. Methods:We chose MDA-MB-231 breast cancer cell（ERαnegative） and MCF-7 breast cancer cell（ERαpositive）.MDA-MB-231 cell was treated with TCS（90μg/ml） or 5-Aza-C（5μM）.（1） The ERαmRNA level of the cells was measured by RT-PCR;（2） The 5’CpG island methylation of ERαgene was tested by Methylation Specific PCR（MSP） test;（3） Western blot method was performed for ERαprotein in the cells;（4） MTT method tested whether Tamoxifen could induce the growth inhibition of MDA-MB-231 cell after treated with TCS or 5-Aza-C.Results:（1） The expression of ERαmRNA in MDA-MB-231 cell was silencing by RT-PCR test,while the expression of ERαmRNA in MCF-7 cell was positive.The re-expression of ERαmRNA in MDA-MB-231 cell treated with TCS or 5-Aza-C was shown by RT-PCR; （2） Methylation of ERαgene was found in MDA-MB-231 cell with MSP technique.Demethylation of ERαgene was found in MCF-7 cell and MDA-MB-231 cell treated with TCS or 5-Aza-C;（3） The expression of ERαprotein was negative in MDA-MB-231 cell,but positive in MCF-7 cell determined by western blot method,and re-expressed when treated with TCS or 5-Aza-C;（4） Tamoxifen could inhibit the proliferation of MDA-MB-231 cell treated with TCS efficiently through MTT method and the suppressive effect of TCS was stronger than 5-Aza-C（p＜0.05）.Conclusion:ERαgene expression silencing was related to the hypermethylation of ERαgene.TCS could induce the re-expression of ERαmRNA and protein in MDA-MB-231 cell by demethylating ERαgene.TCS could induce MDA-MB-231 breast cancer cell sensitive to Tamoxifen.The PartⅢObjective:Study the suppressive effect of TCS on transplanted human breast cancer in nude mice and effect on the expression of ERα.Methods:（1） 20 nude mice were transplanted with human breast cancer MDA-MB-231 cell to construct tumor models.Once the tumor model in nude mice was established,18 nude mice were devided into 3 groups:control group（without treatment）,TCS group and saline group. TCS（0.4mg/kg） and sodium chloride were injected around the tumor.A total of 7 injections were given,once every 2 days;（2） Observations were made on tumor suppression rate,tumor volume and weight,liver and renal function,blood routine test;（3） The ERαmRNA level in transplanted neuplasma was measured by RT-PCR assays;（4） The methylation of ERαgene in transplanted neuplasma was performed by MSP test;（5） Immunohistochemistry and western blot were used to detemine the expression of ERαprotein in the 3 groups.Results:（1） The subcutaneous tumor model in nude mice was successfully established（18/20）;（2） The tumor volume and weight in TCS group decreased（p＜0.05）;（3） Renal function and blood system were normal（P＞0.05） in TCS group.SGPT in TCS group was a little higher than control group（P＜0.05）;（4） The re-expression of ERαmRNA was shown by RT-PCR in TCS group;（5） Demethylation of ERαgene was found in transplanted neuplasma treated with TCS by MSP test;（6） The expression,of ERαprotein was found in transplanted neuplasma treated with TCS by immunohistochemistry and western blot test.Conclusions:TCS could effectively suppress human breast cancer cells growth rate in subcutaneous tumor of nude mice and it was relatively safe to use it.TCS could induce the re-expression of ERαmRNA and protein in vivo by demethylating ERαgene.更多还原