【作者】 韩金薇；

【导师】 王小玲； 刘月平；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2012， 硕士

【摘要】 目的：我国是食管癌的高发国家，根据调查研究显示,食管癌死亡率仅次于肺癌、胃癌、肝癌，居全国第4位。肿瘤的发生、发展是物理、化学、生物等环境因素与遗传因素共同作用的结果，是一个多因素、多阶段、累积渐进的过程。而在肿瘤细胞局部浸润、无限增殖及远处转移的背后，细胞信号转导异常与其表现出了一定的关系。因此进一步对与其相关的信号转导通路及临床抗肿瘤药物作用机制的研究，仍为食管癌治疗研究的目标。细胞外信号调节激酶(ERK1/2)是丝裂原活化蛋白激酶((mitogen-activated protein kinase,MAPK)家族中的重要成员之一，其中，Ras/Raf/MEK/ERK是ERK信号转导通路的主要途径。而其中，MEK是ERK信号通路的关键蛋白，分为MEK1和MEK2两种亚型,其作用是磷酸化并激活下游底物ERK。MEK1/2广泛存在于各种生物体中，在细胞癌变过程中发挥重要作用，其可被多种丝裂原信号及癌基因产物所激活从而成为P-MEK1/2，进而转导胞外信号至细胞核内，进一步参与调节细胞的增长、分化、凋亡等多种生理活动，并在细胞的恶性转化过程中发挥重要作用。国内外学者研究发现，基质金属蛋白酶(MMPS)是降解细胞外基质蛋白的最主要的蛋白水解酶，它在肿瘤的发生、发展过程中具有核心作用。大量研究表明，MMPS具有广泛的底物特异性，通过降解细胞外基质成分，促进肿瘤侵袭及转移，并且还可以通过新生血管生成等促进肿瘤的生长和扩散。其中，MMP-2与MMP-9是基质金属蛋白酶家族中分布最广、研究较清楚的主要成员。研究发现，MMP-2、MMP-9在多种实体瘤中（包括肺癌、乳腺癌、宫颈癌、结肠癌、骨肉瘤、乳头状甲状腺癌）均呈现高表达。另有学者研究认为，在纤维肉瘤细胞HT1080中，抑制ERK磷酸化，MMP-2、MMP-9的表达进而受到抑制，表明MMP-2和MMP-9的表达依赖ERK信号通路中ERK的磷酸化。而本实验中MEK又为ERK信号通路的重要成员。本实验通过研究不同药物浓度的Bufalin(0、10、25、50、100nmol/L)作用于体外培养的食管癌细胞株(TE-13)后，并观察对MMP-2、MMP-9以及MEK1/2及其活化形式(P-MEK1/2)表达的影响，进一步探讨MEK信号通路与基质金属蛋白酶(MMPS)在食管癌发生中的相互作用关系，为MEK信号通路成为食管癌临床治疗的新靶点提供必要的实验依据。方法：常规培养的人食管癌细胞TE-13经不同浓度的Bufalin（蟾蜍灵）进行处理后，通过MTT方法检测细胞药物毒性，采用逆转录聚合酶链反应RT-PCR方法对细胞中MMP-2、MMP-9mRNA的表达进行分析，采用蛋白印记Western-blot及免疫细胞化学方法对食管癌细胞进行MEK1/2、P-MEK1/2蛋白的相对定量检测，分析药物作用后其表达水平的变化。统计分析采用SPSS13.0软件，数据采用单因素方差分析。结果：1MTT检测结果不同浓度的蟾蜍素（对照组，10nM/L，25nM/L，50nM/L，100nM/L，200nM/L，400nM/L，800nM/L）干预24h后平均OD值（0.991、0.869、0.735、0.561、0.499、0.371、0.265、0.258），蟾蜍素在100nM/L及其以下的药物浓度作用下，细胞存活率大于50%，即TD0为100nM/L。2RT-PCR结果显示：MMP-2mRNA的表达量在食管癌细胞TE-13中随着Bufalin药物浓度的升高而逐渐降低，100nmol/L时表达量最低。相对表达量对照组为（0.772±0.010），10、25、50、100nmol/L分别为（0.725±0.019、0.663±0.015、0.519±0.019、0.582±0.019），各浓度与对照组相比，差异具有显著的统计学意义（P<0.05）。MMP-9mRNA的表达量随着Bufalin药物浓度的升高而呈现逐渐降低的趋势，并且100nmol/L时表达量最低。相对表达量对照组为（0.783±0.013），10、25、50、100nmol/L分别为（0.740±0.010、0.703±0.006、0.531±0.010、0.601±0.017），与对照组相比，差异具有显著的统计学意义（P<0.05），结果与MMP-2mRNA相一致。3Western-blot结果显示：MEK1/2的表达量随着Bufalin浓度的升高没有显著变化，相对表达量对照组、10、25、50、100nmol/L分别为（0.724±0.005、0.729±0.008、0.736±0.006、0.736±0.015、0.743±0.006），各组间差异没有统计学意义（P＞0.05）。P-MEK1/2表达量随着Bufalin浓度的升高而呈降低趋势，100nmol/L时表达量最低。相对表达量对照组为（0.710±0.006），10、25、50、100nmol/L分别为（0.676±0.010、0.656±0.010、0.521±0.021、0.599±0.020），各组与对照组相比，差异具有显著的统计学意义（P<0.05）。说明Bufalin可以抑制MEK1/2的活化形式(P-MEK1/2)，而对MEK1/2没有显著影响。4免疫细胞化学检测结果显示：MEK1/2的阳性表达率随着Bufalin浓度的升高没有显著变化，对照组、10、25、50、100nmol/L分别76.58、76.47、76.04、75.40、74.98，差异没有统计学意义（P＞0.05）。P-MEK1/2阳性细胞随着Bufalin浓度的升高而减少，棕黄色颗粒密度减低，阳性率分别为80.33、68.41、67.30、52.62、27.00，与对照组相比，差异具有统计学意义（P<0.05）。结论：1在食管癌细胞TE-13中，随着Bufalin药物浓度的升高，MEK1/2蛋白的表达水平没有显著变化，表明不能被Bufalin有效的抑制。2MMP-2、MMP-9mRNA及P-MEK1/2蛋白在食管癌细胞TE-13中的表达能够被Bufalin有效的抑制，且随着药物浓度的升高，表达逐渐减弱，呈现药物剂量依赖性。表明Bufalin治疗食管癌的机制可能与下调MMP-2、MMP-9mRNA及P-MEK1/2蛋白的表达有关。3在食管癌中，MMP-2、MMP-9与P-MEK1/2的表达呈现正相关，二者联合有可能成为食管癌治疗的新靶点。更多还原

【Abstract】 Objective: China is a country of high incidence of esophageal cancer, thesurvey data show that the mortality rate of esophageal cancer ranked fouth inChina, after only to lung cancer, stomath cancer and liver cancer. Theoccurrence and developpment of tumer is a result of the role of genetic andenvironmental factors，and is a multi-factor, multi-stage, accumulated gradualprocess. Behind the local infiltration, unlimited proliferaton, distant metastasisin the tumor cells, the abnormal of cell sigal transduction show a certainrelationship. Therefore, the further study of the mechanism of action ofclinical anticancer drugs and associated with signal transduction pathways, isstill the goal in the treatment of esophageal cancer.Extracellular signal-regulated kinase (ERK) is one of the importantmembers of mitogen activated protein kinase (MAPK) family. In the ERKsignal transduction pathway, the Ras/Raf/MEK/ERK is the main way. Amongthem, the MEK is the key protein of the ERK signal transduction pathway andit is divided into two subtypes, MEK1and MEK2. The role of MEK is tophosphorylate and activate ERK, which is the downstream substrates of MEK.MEK1/2widely exists in a variety of organisms, and plays an important role inthe process of carcinogenesis in the cell. MEK1/2can also be activated intothe state of phosphorylation (P-MEK1/2) by variety of mitogen-activatedsignaling and oncogene products. P-MEK1/2transducts signal to the nucleus,involves in regulating cell proliferation, differentiation, apoptosis and otherphysiological processes, and plays an important role in malignanttransformation of cell.Domestic and foreign scholars have found that matrix metalloproteinase（MMPS）is the main protelytic enzymes to degradate the extracellular matrixproteins, and it has a central role in the process of the occurrence and developpment of tumer. Numerous studies show that MMPS has a broadsubstrate specificity, it can promote the invasion and metastasis of tumor bydegradating the extracellular matrix components. Also, it can promote thegrowth and spread of tumor through angiogenesis. Among them, MMP-2andMMP-9are the main members of most widely distributed and clearer researthin MMPS family. Studies have found that MMP-2and MMP-9showed a highexpression in a variety of solid tumors, including lung cancer, breast cancer,cervical cancer, colon cancer, osreosarcoma and papillary thyroid carcinoma.Some scholars think that the expression of MMP-2and MMP-9dependents onthe phosphorylation of ERK in the ERK signaling pathway in fibrosarcomacells HT1080. We can inhibit the expression of MMP-2and MMP-9byinhibiting the phosphorylation of ERK. In this experiment, MEK is animportant member in ERK signaling pathway.In this experiment, by investigating Bufalin with different concentrationroles in esophageal carcinoma cell line in vitro (TE-13), we observe the effectof Bufalin on the expression of MMP-2, MMP-9, MEK1/2and its activatedform (P-MEK1/2). And to further explore the interaction between MEKsignaling pathway and matrix metalloproteinases (MMPS) in esophagealcancer. It may provide experimental evidence for whether MEK signalingpathway is a new target for treating esophageal carcinoma.Methods: After dealing the esophageal cancer cell lines TE-13withBufalin of different concentration, we detected the expression of MMP-2andMMP-9by transcriptase-polymerase chain reaction RT-PCR and theexpression of MEK1/2and P-MEK1/2using Western-blot andImmunocytochemistry methods. And further more to detect the expression ofdrug effects. Statistical analysis used SPSS13.0software, date usesingle-factor analysis of variance.Results：1RT-PCR results showed that in esophageal cancer cells TE-13, theexpression level of MMP-2mRNA gradually decreased with the Bufalinconcentration increasing and100nmol/L was the lowest. The relative expression level of MMP-2mRNA with different concentration Bufalin(10、25、50、100nmol/L) were0.725±0.019、0.663±0.015、0.519±0.019、0.582±0.019, compared with0.772±0.010of control group. Differences werestatistically significant (P＜0.05).The expression level of MMP-9mRNA also gradually decreased with theBufalin concentration increasing and100nmol/L was the lowest. The relativeexpression level of MMP-9mRNA with different concentration Bufalin(10、25、50、100nmol/L) were0.740±0.010、0.703±0.006、0.531±0.010、0.601±0.017, compared with0.783±0.013of control group. Differences werestatistically significant (P＜0.05). The results was consistent withMMP-2mRNA.2Western-blot results showed that the expression level of MEK1/2proteinhad no significant change with the Bufalin concentration increasing. Therelative expression level of MEK1/2with different concentration Bufalin(10、25、50、100nmol/L) were0.729±0.008、0.736±0.006、0.736±0.015、0.743±0.006, compared with0.724±0.005of control group. The differencebetween the groups was not statistically significant (P＞0.05).The expression level of P-MEK1/2protein gradually decreased with theBufalin concentration increasing and100nmol/L was the lowest. The relativeexpression level of P-MEK1/2with different concentration Bufalin(10、25、50、100nmol/L) were0.676±0.010、0.656±0.010、0.521±0.021、0.599±0.020, compared with0.710±0.006of control group. Differences werestatistically significant (P＜0.05). It explain Bufalin can inhibit the activationof MEK1/2(P-MEK1/2), and the inhibition was dose-dependent.3The Immunocytochemistry method showed that the positive expressionrate of MEK1/2had no significant change with the Bufalin concentrationincreasing. The difference between the groups was not statistically significant(P＞0.05).With the concentration of Bufalin increased, the number of positive cellsgradually reduced, the density of brown-yellow particles also graduallyreduced. Differences were statistically significant (P＜0.05). Conclusion:1In esophageal cancer cells TE-13, the expression of MEK1/2protein hadno siginificant change with the Bufalin concentration increasing, showingthat it can not be inhibited by Bufalin.2MMP-2、MMP-9mRNA and P-MEK1/2protein expression in esophagealcancer cells TE-13can be effectively inhibited by Bufalin. And theexpression level gradually decreased with the Bufalin concentrationincreasing, showing dose-dependent. This study indicates the treatingmechanism of Bufalin may be related with down-regulation ofMMP-2,MMP-9mRNA and P-MEK1/2protein.3In esophageal cancer, the expression between MMP-2、MMP-9and P-MEK1/2have a positive correlation. Thus, their combination may becomea new target for esophageal cancer treatment.更多还原