【作者】 刘予生；

【导师】 陈建中；

【作者基本信息】 郑州大学 ， 外科学， 2010， 硕士

【摘要】 背景与目的乳腺癌是威胁女性生命和健康的恶性疾病之一,乳腺癌术后化学治疗有效地改善了患者的预后,是乳腺癌综合治疗的重要措施,恶性肿瘤具有较强异质性,对各种化疗药物存在明显个体差异,肿瘤药敏试验通过检测癌细胞对化疗药物的敏感性,直接获得试验数据,指导临床个性化选择药物。由于肿瘤药敏试验开展的局限性,建立乳腺癌基因型与各种抗肿瘤药物的敏感性关系,间接根据肿瘤药敏指导临床选药,为乳腺癌个性化化疗提供了一种方法。乳腺癌基因型分为Lumi nalA型：ER(+).PR(+).C-erbB-2(-), LuminalB型：ER(+).PR(+).C-erbB-2(+),Basal-like型：ER(-).PR(-).C-erbB-2(-)和ERBB2(+)型：ER(-).PR(-).C-erbB-2(+).目前,肿瘤药敏试验在乳腺癌药物选择方面进行过一些研究,但是尚无ER、PR、C-erbB-2与抗肿瘤药物的敏感性和化疗方案关系的类似研究。ER是细胞高特异性糖蛋白,可与细胞配体紧密结合,较低数量的配体即可激活ER通路,产生作用。ER通过诱导雌激素作用的基因转录促使癌细胞增殖。PR是雌激素作用的终产物,PR的合成须有雌激素启动,ER水平是影响PR表达的重要因素,其阳性程度越高,PR表达就越多,ER活性及其通路是PR表达的关键。C-erbB-2是一种原癌基因,其在细胞生长因子信号传导通路及控制正常细胞生长和分裂中起决定性作用,生长因子与细胞表面C-erbB-2受体结合后,活化胞内酪氨酸蛋白激酶,引起酪氨酸自身磷酸化,进而引发瀑布式连锁反应,信号经细胞膜和细胞间质传至细胞核,激活C-erbB-2基因,增强细胞有丝分裂,调控细胞生长,促进肿瘤增殖及分化,其过度表达提示C-erbB-2基因无限扩增,进而引起C-erbB-2基因复制转录增加,从而导致C-erbB-2mRNA翻译水平提高和C-erbB-2受体合成增加及过表达,通过与配体结合,诱使信号传导通路激活,引发肿瘤细胞无限分裂和生长。C-erbB-2也是耐药基因,其阳性率越高,癌细胞耐药性越强,疗效越差。由于乳腺癌基因分型可更好地反映肿瘤的生物学行为,且其基因表型可影响化疗药物的敏感性,因此,通过肿瘤药敏试验建立ER、PR、C-erbB-2与抗肿瘤药物的敏感性关系,进而指导临床个性化选择药物,可使化学治疗更有针对性。材料与方法1标本收集2008年7月至2009年12月在郑州大学第二附属医院乳腺科入院手术治疗的69例乳腺癌标本,术中经快速冰冻病理证实为乳腺癌,行乳腺癌根治术或保乳术后标本进一步常规病理检查,并检测ER、PR、C-erbB-2。肿块标本收集后,立即送到实验室进行如下处理,避免标本细菌污染,①在15ml培养管中用RMPI-1640培养液冲洗标本3次；②加含100U/ml青、链霉素的RMPI-1640培养液适量浸泡标本20min,完成上述处理后,在无菌原则下,剪碎标本,进行下步处理。2研究方法把标本制成单细胞悬液,检测细胞活性度和计数,调整细胞浓度,设立试验组、空白对照组和细胞对照组,各种药物稀释成试验用药液后,加药液于96孔细胞培养板上,培养四天后,加入0.5%MTT经过培养和溶解后,在酶标仪上检测吸光度值,计算各孔细胞抑制率。3评价标准以5PPC溶液浓度作为药物敏感性评价浓度,其评价标准为：高敏感组肿瘤细胞抑制率>50%,敏感组肿瘤细胞抑制率为30%-50%,耐药组肿瘤细胞抑制率<30%。4统计学方法本试验所有数据采用SPSS17.0统计软件进行处理,计量资料以均数±标准差(x±s)表示,进行方差齐性检验和正态性检验,符合条件多组比较采用单因素方差分析(one-way ANOVA),组间两两比较采用最小显著性差异(LSD)法,P<0.05为差异有统计学意义。结果氟尿嘧啶在ER(+)、PR(+)、C-erbB-2(-), ER(+)、PR(+)、C-erbB-2(+), ER(-)、PR(-)、C-erbB-2(-)和ER(-)、PR(-)、C-erbB-2(+)四组标本中,细胞抑制率没有体现出统计学差异(P>0.05),这可能与各组收集标本例数较少有关,甲氨喋呤在ER(+)、PR(+)、C-erbB-2(-)型细胞抑制率高于ER(+)、PR(+)、C-erbB-2(+)型,ER(-)、PR(-)、C-erbB-2(-)型和ER(-)、PR(-)、C-erbB-2(+)型(P<0.05)；阿霉素在ER(+)、PR(+)、C-erbB-2(-)型细胞抑制率高于ER(-)、PR(-)、C-erbB-2(-)和ER(-)、PR(-)、C-erbB-2(+)型(P<0.05)；表阿霉素在ER(+)、PR(+)、C-erbB-2(-)型细胞抑制率高于ER(-)、PR(-)、C-erbB-2(+)型(P<0.05)；紫杉醇在ER(+)、PR(+)、C-erbB-2(-)型细胞抑制率高于ER(+)、PR(+)、C-erbB-2(+)型,ER(-)、PR(-)、C-erbB-2(-)型和ER(-)、PR(-)、C-erbB-2(+)型(P<0.05)。结论1.乳腺癌基因型与化疗敏感性存在关系。ER(+)、PR(+)、C-erbB-2(-)、ER(+). PR(+)、C-erbB-2(+)和ER(-)、PR(-)、C-erbB-2(-)三型在各种抗肿瘤药物的细胞抑制率高低顺序一致：PTX>EPI>ADM>5-FU>MTX；ER(-)、PR(-)、C-erbB-2(+)型在各种抗肿瘤药物的细胞抑制率高低为：PTX>ADM>EPI> 5-FU>MTX。2.不同基因型的乳腺癌对同一种化疗药的细胞抑制率不同,提示化疗的获益不同。紫杉醇、阿霉素、表阿霉素、氟尿嘧啶和甲氨蝶呤在ER(+)、PR(+)、C-erbB-2(-)型细胞抑制率均最高,在ER(-)、PR(-)、C-erbB-2(-)型细胞抑制率均最低。乳腺癌的化疗应考虑其基因型的不同,选择敏感药物,争取最大获益,实现乳腺癌治疗个体化。更多还原

【Abstract】 Background and ObjectivesBreast cancer is one of malignant disease which poses a great threat to women’s life and health. Adjuvant chemotherapy for the treatment of breast cancer is an effective method in improving the prognosis, which is an important measure for comprehensive treatments for breast cancer. malignant tumors have strong heterogeneity. Various chemotherapy have obviously individual differences in medicines, The sensitivity assay for anticancer drugs may examine the sensitivity of cancer cells to chemotherapeutic agents, which can directly acquire experimental data. This can guide the application of drugs for clinical personalization. Due to the limitations of tumor chemosensitivity assay carried on, establishing relationships of breast cancer genotype and anticancer drugs, we can indirectly select medicines based on clinical cancer susceptibility, this can provides a way to personalized chemotherapy for breast cancer.Genotype is divided into LuminalA of breast cancer:ER(+), PR(+), C-erbB-2 (-),and LuminalB:ER (+), PR (+), C-erbB-2 (+), Basal-like:ER (-), PR (-), C-erbB-2 (-) and ERBB2 (+):ER (-), PR (-), C-erbB-2 (+). At present, tumor chemosensitivity assay was carried on some study in breast cancer, but no related studies of ER, PR, C-erbB-2 and anticancer drugs sensitivity in chemotherapy for breast cancer.ER can be closely integrated with the cell ligand, which is highly specific glycoprotein cells. A small quantity of ligand can activate ER pathway effectively. ER depend on estrogen effect to induce gene transcription and promote proliferation of tumor cells.PR is end products, which can be affected by estrogen. Estrogen promotes the synthesis of PR. ER expression levels are important factors which affect PR expression, the higher the positive degree of ER, the more PR expression, activity and the pathway of ER is a key factor for PR expression.C-erbB-2 is a proto-oncogene, which plays a decisive role in growth factor signaling pathways and controls normal cell growth and differentiation. Growth factors and C-erbB-2 receptor on the cell surface can activate intracellular tyrosine kinase, and then cause phosphorylation of tyrosine itself, which may keep triggering a cascade of chain reactions. The signals through the cell membrane and the stromal cells spreads into the nucleus, which activate C-erbB-2 gene, accelerate mitosis, regulate cell growth and promote the proliferation and differentiation of malignant tumor, its overexpression prompts C-erbB-2 gene amplification infinitely, and then promotes C-erbB-2 gene transcription, increases replication, and keeps enhancing C-erbB-2mRNA translation level and C-erbB-2 receptors synthesis and overexpression, this can induce signaling pathways activated through ligand binding, keep triggering the growth and differentiation of tumor cell infinitely. C-erbB-2 is also a drug-resisted gene. The higher the positive rate of C-erbB-2, the stronger the cancer cells resists. Because genotyping of breast cancer may affect the biological behavior of tumors and the sensitivity of chemotherapy. Therefore, depending on test of tumor susceptibility, establishing relationship of ER, PR, C-erbB-2 and the anti-tumor drugs, and then guided the selection of clinical personalized medicine. Chemotherapy will be more focused treatment.Materials and Methods1 SpecimenBreast surgical treatment of 69 hospitalized patients were collected from July 2008 to December 2009 at the Second Affiliated Hospital of Zhengzhou University. Intraoperative frozen section and routine pathological examination confirmed breast cancer after radical mastectomy or breast conserving surgery. We will detect ER, PR, C-erbB-2 simultaneously. Tumor specimens were immediately sent to the laboratory for the following treatment to avoid specimens contamination,①We use culture medium of RMPI-1640 to wash specimens three times in 15ml culture tubes;②We add with amount of RMPI-1640 involved 100U/ml penicillin and streptomycin to immerse specimens at twenty minutes, after completion of the deal, under the principle in sterile, clipping specimens to process the next step.2 Research methodsThe specimens were made into single cell suspension, then we examine cells degrees and counting, adjust the cell concentration, establish the experimental group, control group and cell control group. Various drugs are diluted to experimental liquids. We may add the liquids to 96 holes training board to cultivate specimens four days later, and add again 0.5% MTT cultivating and dissolved. We axamine absorbency in the microplate reader to calculate inhibiting rate of tumor cells in every hole.3 Evaluation Criteria5PPC solution serves as the concentration of drug sensitivity. Criteria of evaluation are that high sensitivity of tumor inhibiting rate is above 50%, sensitivity of tumor inhibiting rate is between 30%-50%, inhibiting rate of tumor cells of resistance is below 30%.4 Statistic analysisIn this study, All the data were expressed by (x±s). The statistical software SSPS17.0 for windows was exploited to analyze data. Which was processed the test of homogeneity of variance and normality, Deviation and difference were compared using the one way ANOVA and LSD test between each group, The different significance is judged by P<0.05.ResultsThe four specimens of ER(+), PR(+), C-erbB-2(-), ER(+), PR(+), C-erbB-2(+), ER(-), PR(-), C-erbB-2(-) and ER(-), PR(-), C-erbB-2(+) do not reflect the significant difference in fluorouracil(P>0.05). The ER(+), PR(+), C-erbB-2(-) have an advantage over ER(+), PR(+), C-erbB-2(+), ER(-), PR(-), C-erbB-2(-) and ER(-), PR(-), C-erbB-2(+) in methotrexate(P<0.05). The ER(+), PR(+), C-erbB-2(-) have an advantage over ER(-), PR(-), C-erbB-2(-) and ER(-), PR(-), C-erbB-2(+) in adriamycin(P<0.05); The ER(+), PR(+), C-erbB-2(-) have an advantage over ER(-), PR(-), C-erbB-2(+) in epirubicin (P<0.05). The ER(+), PR(+), C-erbB-2(-) have an advantage over ER(+), PR(+), C-erbB-2(+), ER(-), PR(-), C-erbB-2(-) and ER(-), PR(-), C-erbB-2(+) in paclitaxel(P<0.05).Conclusions1. The experiments shows that genotypes are correlate with sensitivity of chemotherapy in breast cancer. ER(+), PR(+), C-erbB-2(-), ER(+), PR(+), C-erbB-2(+) and ER(-), PR(-), C-erbB-2(-) three types of cells have the same orders in various inhibiting rates in anticancer drugs:PTX> EPI> ADM> 5-FU> MTX. ER(-), PR(-), C-erbB-2(+) cells has a different inhibiting effect on a variety of anticancer, its sequence follows as:PTX> ADM> EPI> 5-FU> MTX.2. Different genotypes of breast cancer have different inhibiting rates to the same chemotherapeutic drug, which suggests that they have different chemotherapeutic benefit. Paclitaxel, doxorubicin, epirubicin, fluorouracil and methotrexate have the highest inhibiting effect in the ER(+), PR (+), C-erbB-2(-). All five drugs have the lowest inhibiting effect in the ER(-), PR(-), C-erbB-2(-). Based on the different genotypes, we choose sensitive drugs so as to obtain maximum benefit of treatment and achieve individualized treatment for breast cancer.更多还原