【作者】 罗沙阳；

【导师】 汤恢焕；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 化疗和内分泌治疗是激素受体阳性的乳腺癌患者内科治疗的两个重要方面。理论上来说,雌激素能促进受体阳性的乳腺癌细胞更多的进入细胞周期的S期,而化疗药物则主要作用于癌细胞的S期,因此雌激素水平的高低势必会影响化疗药物的作用。探讨两者的相互关系,将有利于制定合理的化疗以及内分泌治疗方案与时机。为此,本实验进行了临床和体外细胞学的研究,通过了解雌激素对化疗药物引起的肿瘤细胞凋亡作用产生的影响来进一步的了解雌激素和化疗药物相互作用的关系。第一章雌激素水平和新辅助化疗后乳腺癌组织标本中凋亡相关因子Bcl-2及Caspase-3表达的关系目的研究不同年龄段雌激素受体阳性的乳腺癌患者体内雌激素浓度对新辅助化疗后乳腺癌组织标本中凋亡相关因子Bcl-2及Caspase-3表达的影响及相互关系。方法我院乳腺科2008年1月至2010年5月接受两周期CAF方案术前化疗的乳腺癌患者按年龄分为A,B,C三组(≤35岁,36-49岁和≥50岁),共36例患者,检测所有患者化疗前后血清雌二醇和化疗后乳腺癌标本凋亡相关因子Caspase-3及Bcl-2的表达,分析三组患者血清雌二醇水平的变化与caspase-3及Bcl-2表达的关系。结果三组中,通过两周期化疗后,血清中雌激素水平均出现不同程度的下降,而相对于A组和C组,B组中雌激素水平下降幅度更大,且三组中雌激素水平均存在差异；Caspase-3的表达在三组中无明显差异(p>0.05),但在B组中略高(66.67%对58.33%,50.00%)。Bcl-2的表达B组明显低于A组及C组(25.00%对66.67%,75.00%；p<0.05)。结论三组患者在化疗两周期后雌激素水平存在差异；而B组患者有较低的Bcl-2表达阳性率和略高的Caspase-3表达阳性率,这可能与雌激素水平相关。但本实验也试图进行两者相关性的分析,可能由于样本量偏少以及雌激素的标准差较大,没有得出两者有相关性的结论。第二章雌激素对阿霉素引起MCF-7凋亡作用的影响的体外细胞研究目的雌激素在体外实验中对阿霉素引起的人乳腺癌细胞株MCF-7凋亡和增殖的影响。方法用不含雌激素的胎牛血清培养MCF-7细胞48小时后分为3组。A组：加入5组不同浓度的雌激素及不含雌激素的对照组进行培养；B组：加入5组不同浓度的雌激素及不含雌激素的对照组进行培养同时加入阿霉素；C组：加入5组不同浓度的雌激素及不含雌激素的对照组进行培养同时加入阿霉素和雌激素拮抗剂他莫西芬。再采用Hoechst荧光染色法,PI染色及AnnexinV-FITC流式细胞仪检测分析各组细胞凋亡和细胞周期比例；用分光光度法检测各组凋亡相关基因Caspase-3产生的酶活性,用RT-PCR法检测各组凋亡相关基因Bcl-2及Bax的表达。结果A组细胞随着雌激素浓度的增高,细胞周期中S期的比例明细增高,Bcl-2基因表达也明显增高而Bax的表达降低；B组细胞中当雌激素浓度为10-9mmol/L时,细胞的凋亡率明显增高(p<0.05),Bcl-2基因表达仍随雌激素浓度的增高而增高,而Bax的表达仍逐渐降低；C组细胞总体的凋亡率无明显差异,细胞均大部分停留在G0/G1期,Bcl-2和Bax的表达也失去差异性。Capase-3的表达在这三组中均无明显差异(p>0.05)。结论10-91mmol/L浓度的雌激素可以增强阿霉素对MCF-7的杀伤作用,加入他莫西芬后完全阻断雌激素受体使得这个差异性消失,过低浓度的雌激素由于不能使更多的细胞进入细胞周期S期,导致阿霉素对MCF-7细胞的凋亡效应降低；而高浓度的雌激素抑制阿霉素对MCF-7细胞的凋亡作用可能与Bcl-2的表达增高Bax的表达降低有关。第三章雌激素对阿霉素引起MCF-7凋亡作用的影响的动物实验研究目的雌激素在MCF-7乳腺癌细胞株裸鼠移植瘤模型中对阿霉素诱导的MCF-7细胞凋亡抑制作用的影响。方法建立二十只裸鼠MCF-7乳腺癌细胞株移植瘤模型。然后分为四组。A组：吡柔比星1.5mg/kg尾静脉注射,苯甲酸雌二醇继续10mg/kg/2day腹腔注射,B组：吡柔比星1.5mg/kg尾静脉注射,苯甲酸雌二醇继续5m8/kg/2day腹腔注射,C组：吡柔比星1.5mg/kg尾静脉注射。D组：灭菌用水尾静脉注射。然后,绘制肿瘤生长曲线,计算肿瘤的最终重量,用RT-PCR方法检测Bcl-2表达水平。结果在肿瘤的生长曲线中,给药后第九天,增长趋势出现显著差异；A,B,C三组与对照组即D组相比肿瘤的生长逐渐减慢(p<0.05)；A,B两组与C组相比肿瘤生长明显减缓(p<0.05)；但是,A,B两组相比没有显着性差异(p>0.05)。在肿瘤的最终重量中；A,B,C三组与D组相比明显降低(p<0.05)；A、B两组与C组相比明显降低(p<0.05)；但是,A,B两组无显著性差异(p>0.05)。在Bcl-2的表达中；A,B两组Bcl-2基因相比D组表达明显增加(p<0.05),C组Bcl-2基因表达相比D组明显降低(p<0.05)；但是,A,B两组无显著性差异(p>0.05)。结论一定的雌激素剂量强度可以增加阿霉素的肿瘤抑制作用；一定的雌激素剂量强度也可以增加凋亡相关基因Bcl-2的表达。更多还原

【Abstract】 Chemotherapy and hormone therapy are important medical treatment in hormone receptor-positive breast cancer patients. Theoretically speaking, estrogen can promote receptor-positive breast cancer cells into the cell cycle S period, and chemotherapy drugs effect are just in S period, so estrogen level will definitely affect chemotherapy drugs role. Then discusses the relationship between them, will help us to formulate rational chemotherapy and hormone therapy solutions and opportunities. Therefore, we conducted clinical and in vitro cytological studies, through the methods of tumor cell apoptosis, this experiment analyzed the effect of estrogen to chemotherapy drugs and their interaction relationship.Part Ⅰ The relationship between estrogen level and apoptosis factors Bcl-2, Caspase-3expression in breast tissue samples after neoadjuvant chemotherapy.Objective:To evaluate the relationship between estrogen level and apoptosis factors Bcl-2and Caspase-3expression in the different age range breast cancer patients after neoadjuvant chemotherapy. Methods:From January2008to2010May36breast cancer patients in Xiangya hospital breast department accepted two cycles CAF neoadjuvant chemotherapy were divided into A,B and C three groups according to age≤35,36-49, and≥50years old, detected their serum estrogen levels and apoptosis factor Caspase-3, Bcl-2expression, analyzed the relationship between the estrogen levels and Caspase-3, Bcl-2expression in the three groups.Results:Through two cycles of chemotherapy, the estrogen levels were all decreased, but it seemed that decreased more in B group compared with A and B group and the three groups had difference in estrogen level(p<0.05). The Caspase-3expression had no difference in three groups(p>0.05), but looked like higher in B group (66.67%to58.33%and50.00%). Bcl-2expression rate is obviously lower in B group compared with the other two group (25.00%to66.67%and75.00%)Conclusion:The estrogen levels had obvious difference in the three groups. At the same time the lower expression of Bcl-2and the feasible higher expression Caspase-3were in B group. That means Caspase-3and Bcl-2expression may affect by the estrogen levels though we have analyzed the relationship between them, had no significance result because of the number of the patient and the considerable standard deviation.Part Ⅱ The vitro cell analysis of estrogen effect on adriamycin induced MCF-7apoptosis.Objective:Estrogen effect on adriamycin induced MCF-7apoptosis and proliferation.Methods:MCF-7cells were cultured in steroid depleted fetal calf serum after48hours and then divided into three groups. Group A:added different concentrations of estrogen, Group B:added different concentrations of estrogen and adriamycin; Group C:added different concentrations of estrogen, adriamycin and estrogen antagonists tamoxifen. Then, used Hoechst fluorescence staining, PI staining, AnnexinV-FITC staining followed streaming cell instrument detection, we analyzed the apoptosis and proliferation conditions and each cell cycle ratio in three groups. More over, this experiment used spectrophotometric method to detect apoptosis gene Caspase-3and RT-PCR method to detect apoptosis gene Bcl-2expression in three groups.Results:In group A MCF-7cells had increased rate to S period as estrogen concentration increased, Bcl-2gene expression also significantly increased and Bax gene expression decreased; in group B when estrogen concentration was10-9mmol/L, cell apoptosis rate was higher than any other concertration(p<0.05), Bcl-2gene expression still increased and Bax gene expression decreased as estrogen concentration increased; in group C cells apoptosis was obvious, but no difference was found in each estrogen concentration, and cells were most stayed in G0/G1period, Bcl-2and Bax expression also lose difference. Capase-3expression had no significant differences in all three groups(p>0.05). Conclusion:10-9mmol/L concentration of estrogen can strengthen adriamycin apoptosis role in MCF-7, when tamoxifen blocked the estrogen receptor, this phenomenon disappeared. The possible reason was lower concentration of estrogen can not make enough cells into the cell cycle S period and influenced the adriamycin effect and higher levels of estrogen inhibition of adriamycin in MCF-7cell apoptosis role maybe had relevant with Bcl-2and Bax expression.Part Ⅲ The analysis of estrogen effect on adriamycin induced MCF-7apoptosis through animal experimentObjective:Estrogen effect on adriamycin induced MCF-7apoptosis inhibition on nude mice of breast cancer xenograft(MCF-7)Methods:Twenty nude mice models of MCF-7breast cancer xenograft(MCF-7) were established, then divided into four groups, each group had five mice. Group A:Pirarubicin1.5mg/kg by tail vein injection and continued Estradiol benzoate10mg/kg/2day by Intraperitoneal injection, Group B:Pirarubicin1.5mg/kg by tail vein injection and continued Estradiol benzoate5mg/kg/2day by Intraperitoneal injection, Group C:Pirarubicin1.5mg/kg by tail vein injection. Group D:Sterilized water. Then, Drew the tumor growth curve and calculated the tumor final weight, used RT-PCR method to detect apoptosis gene Bcl-2expression in four groups. Results:In tumor growth curve, after the administration of ninth day, the growth trends appeared significant differences; A, B, C group of tumor growth slowed down gradually compared with control group (group D)(p<0.05); the tumor growth slowed down significantly on A and B groups compared with C group(p<0.05); but A and B had no significant difference (p>0.05). In tumor final weight; A, B, C group significantly reduced compared with the control group (group D)(p<0.05); A and B groups reduced more than in C group; but A, B groups had no significant difference (p>0.05). In Bcl-2expression; A, B Bcl-2mRNA expression was significantly increased (p<0.05), group C Bcl-2mRNA expression was significantly reduced (p<0.05) compared with the control group (group D); but A, B groups had no significant difference (p>0.05).Conclusion:A certain dose intensity of estrogen can enhance tumor inhibition by adriamycin; apoptosis related gene Bcl-2increased by the estrogen dose intensity.更多还原