【作者】 郑一琼；

【导师】 李荣； 李席如；

【作者基本信息】 中国人民解放军军医进修学院 ， 普通外科学， 2009， 博士

【摘要】 新辅助化疗(Neoadjuvant chemotherapy,NCT)已经成为乳腺癌综合治疗手段中一个重要的组成部分,然而,大约有27%左右的患者不能从新辅助化疗当中真正获益。寻找能够预测化疗反应性的生物学因子,可以为临床给不同患者施行个体化治疗提供必要的参考依据。该研究针对一个新发现的抑癌基因——FHL1(four and a half LIM domains 1,FHL1)进行了临床与基础研究,以期发现FHL1在乳腺癌紫杉联合蒽环类药物新辅助化疗中的预测价值,为临床提供一个新的化疗反应预测性指标。本研究共分三个部分进行。第一部分,以2006年至2008年在解放军总医院普通外科接受紫杉联合蒽环类药物新辅助化疗的164名患者为研究对象,回顾性分析他们的临床及病理资料,共获得67名患者化疗前、后完整配对的组织切片,为其进行FHL1免疫组化染色,运用秩和检验分析化疗前、后FHL1表达水平的改变,以及化疗前FHL1表达水平与患者临床、病理缓解率的相关性。结果发现,FHL1表达水平在化疗后明显下调;化疗前FHL1表达水平越高,化疗有效性越低。第二部分,通过构建FHL1过量表达稳定转染乳腺癌细胞株(MCF-7-FHL1)和空白载体细胞株(MCF-7-control),采用生长曲线(结晶紫)方法,检测外源性FHL1对于不同药物化疗敏感性的影响;利用内源性表达少量FHL1的MCF-7细胞株和不表达FHL1的T47D细胞株,检测内源性FHL1对于不同药物化疗敏感性的影响。结果发现,无论是内源性还是外源性FHL1表达,对乳腺癌细胞株的化疗敏感性均未见明确影响。同时发现,多西他赛(Docetaxel)对乳腺癌细胞的生长抑制作用较吡柔比星(THP)弱,亦即,乳腺癌细胞对Docetaxel的药物敏感性较之对THP的敏感性低。第三部分,将MCF-7-FHL1细胞株用空白对照、Docetaxel单药、FHP单药以及Docetaxel+THP联合用药四种处理方式处理后,采用Western Blot方法分析各组细胞中FHL1表达水平的改变。结果发现,多西他赛在使FHL1表达下调中起关键性作用。综上所述,FHL1作为一个抑癌基因,其表达在乳腺癌新辅助化疗中具有重要的预测价值。乳腺癌肿瘤组织内的FHL1表达越高,化疗有效率越低,FHL1表达阴性的患者,临床有效率可达100%。此外,Docetaxel可以降低肿瘤组织中FHL1的表达,可能增加肿瘤扩散和转移的机会。更多还原

【Abstract】 Neoadjuvant chemotherapy(NCT)has been an important component of multimodality treatment for Breast cancer.However,about 27%of the patients did not benefit from this clinical treatment and,predictive factors of response were not defined yet.This study was designed to evaluate the value of the four and a half LIM domains 1(FHL1)to predict response in stageⅡandⅢbreast cancer patients treated with taxane and anthracycline combination as neoadjuvant setting.FHL1 was regard as a cancer suppressor gene in most human tumor.This study was divided into three part.Firt part of all,164 patients received preoperative Pirarubicin(THP)(50 mg/m2)in i.v.infusion in D1 and combination with Docetaxel(75 mg/m2)in D2 every 3 weeks after core needle biopsy.Their clinical and pathological material were retrospectively analyzed,and slides of 67 patients have been got matched at last.Clinical and pathologic response rates were measured after preoperative therapy.Expression level of FHL1 was investigated in immunohistochemistry (IHC)stains.We evaluated the response rate to neoadjuvant chemotherapy and the predictive significance of immunohistochemical(FHL1)outcomes.As a result, FHL1 protein expression decreased after chemotherapy,overexpression was associated with lower clinical response rates.The second part,FLAG-tagged FHL1 and empty vector was transfected into breast cancer cells,MCF-7,and the stably transfected cell strains was constructed. MCF-7 cells transfected with FHL1 and empty vector both were diveded into four groups,and then respectively cultured in regular medium,and gave different chemotherapy agents seperately.At specified times,cell numbers were determined by crystal violet assay.Values shown are mean±SD of triplicate measurements and have been repeated 3 times with similar results.Results showed that,both Docetaxel and THP could suppress the growth of cells with or without FHL1-overexpression,and Docetaxel led lower decrease in the number of alive cells than THP.FHL1 had not significantly affect on the response to different chemotherapy agents of the cells.The third part,cells stably transfected with FHL1 were treated with four different agent regimen,include blank control.Proteins extracted from above cells lines were analyzed by Western blot with anti-FHL1.The densitometric quantitation of FHL1 bands normalized to GAPDH.The results showed that Docetaxel was the main contributor to decrease the expression of FHL1.In sum,as a tumor suppressor,expression of FHL1 in breast cancer was an important predictive factor in neoadjuvant chemotherapy.And higher level of FHL1 in breast tumor means lower clinical respons to chemotherapy.Docetaxel could decrease the expression of FHL1 in breast tumor,may promote anchorage-independent cell growth and migration.更多还原