您现在的位置:网站首页答辩论文医学论文中医论文

p38MAPK信号分子与uPA在乳腺癌中表达的相关性研究

  • 简介:(医学所硕士毕业论文 页数:34 字数:14710) 中文论著 摘要 p38MAPK信号分子与uPA在乳腺癌中表达的相关性研究 乳腺癌是妇女最常见的恶性肿瘤,其发病率呈逐年上升趋势,且常发生浸润和转移,因此与乳腺癌侵袭转移有关的因子成为当前研究重点之一。至今...
    • 请与管理员联系购买资料 QQ:5739126
  • 论文简介
  • 相关论文
  • 论文下载

(医学所硕士毕业论文  页数:34  字数:14710)

中文论著 摘要
p38MAPK信号分子与uPA在乳腺癌中表达的相关性研究 

乳腺癌是妇女最常见的恶性肿瘤,其发病率呈逐年上升趋势,且常发生浸润和转移,因此与乳腺癌侵袭转移有关的因子成为当前研究重点之一。至今许多研究结果表明uPA在多种肿瘤组织中过表达,在肿瘤侵袭转移过程中发挥了重要的作用。但迄今为此的研究对其表达的分子调控机制研究较少。p38MAPK是丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)家族的重要成员,最近,p38MAPK信号通路因以正性调节的方式参与肿瘤的侵袭转移过程而倍受关注。为此,本研究检测了p-p38及uPA在乳腺癌组织及细胞系中的表达情况,并进一步在体外研究p38MAPK通路对乳腺癌细胞uPA蛋白表达的影响,为p38MAPK抑制剂应用乳腺癌治疗提供相应的理论依据。
材料与方法
1. 材料
60例乳腺癌组织蜡块取自中国医科大学第一临床学院病理科,为2001年~2003年手术切除标本。依据WHO2003年所推荐的乳腺癌组织学分类标准进行分类,60例均为浸润性导管癌。人乳腺癌细胞系MDA-MB-231及MCF-7由中国医科大学病理学教研室已存的细胞复苏而得。
2.试剂
DMEM及RPMI 1640培养基购自Gibco公司,第一抗体分别为鼠抗人p-p38单克隆抗体及兔抗人uPA多克隆抗体。p38MAPK特异性抑制剂SB203580购自美国Calbiochem公司。
3.实验方法
3.1 免疫组织化学(S-P)法:采用链霉素抗生物素蛋白—过氧化物酶法(S-P法)。PBS代替一抗作为阴性对照,用已知阳性片作阳性对照。结果判定:每张切片在光镜下随机选取5个视野,每个视野计数100个瘤细胞,着色强度分为无色为0分,浅黄色和黄色为1分,棕黄色为2分。阳性细胞数分为<10%为0分,10-50%为1分,>50%为2分,两项得分相乘结果≥2记为阳性。
3.2 Western 印迹法:收集细胞加入RIPA buffer裂解液,超声处理,高速低温离心提取总蛋白,用Bradford法进行蛋白浓度测定。12%SDS-PAGE电泳,转印,封闭后加入相应一抗、二抗并行DAB显色,观察拍照,进行灰度值测定。
4. 统计学分析
应用SPSS10.0统计学软件,行χ2检验和Pearson相关分析,确定P<0.05有统计学意义。
结 果
1.p-p38和uPA蛋白在乳腺癌组织中的表达及相关性分析
免疫组织化学结果显示,p-p38蛋白主要位于乳腺癌细胞核内,呈棕黄色颗粒,阳性率为56.7%(34/60),uPA蛋白主要分布于乳腺癌细胞浆,呈棕黄色颗粒,阳性率为60.0%(36/60)。p-p38表达阳性的34例乳腺癌中uPA表达阳性者25例; p-p38表达阴性的26例乳腺癌中uPA表达阴性者15例。统计学分析显示,两者之间表达存在正相关(r=0.316,P<0.05)。
2.乳腺癌组织中p-p38、uPA蛋白表达与临床病理特征的关系
p-p38及uPA蛋白表达与乳腺癌淋巴结转移及临床分期有关(P<0.05),而与患者的年龄、肿瘤大小无明显相关性(P>0.05)。
3. 两种转移性不同的乳腺癌细胞中p-p38及uPA蛋白表达
Western印迹法检测结果显示,高转移性的MDA-MB-231细胞中p-p38及uPA蛋白表达水平高于低转移性的MCF-7细胞。
4. 用SB203580阻断p38MAPK信号通路后乳腺癌细胞uPA蛋白表达的变化
加入SB203580后,乳腺癌MDA-MB-231细胞uPA蛋白表达水平降低,且表达水平随着SB203580浓度升高而降低。
讨 论
MAPK(mitogen-activated protein kinase),即丝裂原活化蛋白激酶,是细胞内广泛存在的丝/苏氨酸蛋白激酶超家族,将细胞外信号传至细胞内,参与细胞的生长、分化、增殖的调节及恶性转化,与肿瘤的发生、发展有关。p38MAPK是MAPK家族的重要成员,多种炎症因子和生长因子及应激反应可使p38MAPK的酪氨酸和苏氨酸双磷酸化,从而激活p38MAPK,p38MAPK信号通路参与调节细胞生长、凋亡、运动及侵袭表型,在细胞迁移、肿瘤细胞侵袭转移方面具有重要的调控功能。尿激酶型纤溶酶原激活剂(uPA)是一种丝氨酸蛋白水解酶,能激活纤溶酶原转变为纤溶酶对细胞迁移和肿瘤侵袭转移中细胞外基质的降解有重要作用。
对于uPA表达与信号传导通路的关系目前研究较少。近来研究发现,p38MAPK信号通路参与胃癌、肺癌、淋巴瘤及乳腺癌等细胞uPA的表达调控。本研究显示60例乳腺癌组织中p-p38及uPA表达与乳腺癌的临床分期及淋巴结转移有关,且高转移性乳腺癌MDA-MB-231细胞中p-p38、uPA蛋白表达水平高于低转移性的MCF-7,提示p-p38、uPA过表达可能与乳腺癌的恶性进展及侵袭转移有关。免疫组化结果显示,乳腺癌组织中p-p38和uPA表达存在正相关,提示uPA蛋白表达可能受p38MAPK信号通路的调控。因此,我们又进一步在体外研究了乳腺癌细胞中p38MAPK信号通路与uPA蛋白表达的关系。Western印迹显示,SB203580以浓度依赖方式降低乳腺癌MDA-MB-231细胞 uPA蛋白表达,证明乳腺癌细胞uPA蛋白表达可能通过p38MAPK信号通路来调控。
综上所述,p38MAPK信号通路通过上调uPA的表达促进乳腺癌的恶性进展及侵袭转移,但其确切机制有待于进一步研究。目前以细胞信号传导分子为靶点的MAPK信号传导通路抑制剂成为肿瘤治疗的新靶点,我们的实验结果可为p38MAPK信号通路作为乳腺癌治疗的靶点提供相应的理论依据。
结 论
1.p-p38、uPA蛋白表达与乳腺癌临床分期及淋巴结转移有关,并在高转移性乳腺癌细胞系中表达高于低转移性细胞系。
2.p-p38和uPA蛋白在乳腺癌中表达存在正相关。
3.p38MAPK特异性抑制剂SB203580以浓度依赖方式降低乳腺癌细胞uPA蛋白表达水平。
关 键 词
p38MAPK; p-p38; uPA; 乳腺癌


英文论著摘要
Correlation between Expression of p38MAPK
Signaling Molecule and uPA in Breast Cancer
ABSTRACT
INTRODUCTION
Breast cancer is the most common malignancy in women and its incidence rate is increasing year by year. Breast cancer invasion and metastasis related-factor has currently become one of investigation emphases, because breast cancer often generate invasion and metastasis. Up to now, a growing body of evidence has showed that the overexpression of urokinase-type plasminogen activator (uPA) is detected in various malignant tumors and plays a key role in tumor invasion and metastasis. However, less studies demonstrated the molecular regulation mechanisms for uPA expression. p38MAPK is one important member of mitogen-activated protein kinases (MAPKs). Recently, attention has been paid that p38MAPK signaling pathway is involved in the process of tumor invasion and metastasis by positively regulating mechanism. Therefore, we investigated the expression of p-p38 and uPA protein in breast cancer tissues and cells and the effect of p38MAPK signaling pathway on the expression of uPA in breast cancer cells. Our studies suggested that the inhibitor of p38MAPK might be a potential therapeutic target for anti-breast cancer treatment.
METERAL AND METHODS
1. Samples
A total of 60 paraffin-embedded breast cancer tissues were obtained from the surgical resection at the pathological department of the First Affiliated Hospital of China Medical University between the year 2001 to 2003. According to the World Health Organization breast carcinoma histological classification criteria(2003), All specimens were invasive ductal carcinomas. Human breast cancer cells MDA-MB-231 and MCF-7 were obtained from pathological department of China Medical University.
2. Reagents
DMEM and RPMI 1640 were purchased from Gibco . The first antibodies were mouse monoclonal antibody to phosphorylated-p38 and rabbit polyclonal antibody to uPA. SB203580, an specific inhibitor of p38MAPK was purchased from Calbiochem.
3. Methods
3.1 Immunohistochemistry (S-P): IHC was performed according to the indirect streptavidin-biotin-hyperoxidase method, as manufacture protocol. For the negative controls, the primary antibody was omitted by PBS, but all incubation steps were identical. Previously identified strongly staining tumor tissue sections were used as positive controls. We used an intensity-adjusted scoring system to evaluate immunostaining indices. The sections were scanned by light microscope. Five fields were randomly selected and 100 tumor cells in each field were counted. The staining intensity was graded 0-2, corresponding to weak, moderately strong, and intense staining, respectively. With respect to the invasive cell count, <10% positive cells were scored as 0, 10-50% were scored as 1, and >50% were scored as 2. By multiplying these two factors, an immunoreactive score ≧2 was positive.
3.2 Western blot: Cells were lysed in RIPA buffer, then were ultrasounded and centrifuged at low temperature. Protein concentration was measured by Bradford method. Cell lysates were electrophoresed in 12% polyacrylamide SDS gel and transferred onto polyvinylidene difluride membranes , Protein bands were blocked and incubated with the first and second antibody and visualized with DAB kit. Protein contents were calculated by densitometry.
4. Statistical analysis
All statistical calculations were carried out using the SPSS 10.0 statistical software. The results were compared using chi-squared test and Pearson test. P<0.05 was considered statistically significant.
RESULTS
1. Correlation between p-p38 and uPA protein expression in breast cancer tissues:
Immunohistochemical results showed that p-p38 protein was observed in nucleus of breast cancer cells and stained darkly and its positive rate is 56.7%(34/60). uPA protein was in cytoplasm of breast cancer cells and stained darkly and its positive rate is 60.0%(36/60). Twenty-five uPA protein expressed positively in 34 cases of breast cancers which were positively expressed of p-p38 protein , and 15 cases were negatively expressed of uPA protein in 26 cases of breast cancers with p-p38 negative expression .Statistical analysis suggested a positive relationship between expression of p-p38 and uPA(r=0.316,P<0.05).
2. Relationship between p-p38 and uPA protein expression and clinicalpathological characteristics:
There was significant correlation between the level of uPA ﹑ p-p38 expression and lymph node status and clinical stage(P<0.05).The level of uPA and p-p38 was not significantly related to patients’age and tumor size(P>0.05).
3. Expression of p-p38 and uPA protein in two differently metastatic breast cancer cells:
Western blot analysis indicated that the expression of p-p38 and uPA in the highly metastatic MDA-MB-231 cells was higher than that in lowly metastatic MCF-7 cells.
4. Change of uPA protein expression after p38MAPK signaling pathway was blocked by SB203580:
The expression level of uPA protein in breast cancer MDA-MB-231 cells decreased after SB203580,an specific inhibitor of p38MAPK was added into the culture fluid of MDA-MB-231 cells .SB203580 concentration-dependently reduced uPA protein expression.
DISCUSSION
The mitogen-activated protein kinases (MAPKs) are serine/ threonine kinases, which generally exist in various cells. MAPKs have been shown to transduce extracellular signals into endocells and be involved in cell proliferation, differentiation and malignant transformation and play important roles in tumor generation and development. p38MAPK is one important member of mitogen-activated protein kinase(MAPK) family. The p38MAPK undergoes phosphorylation at both tyrosine and threonine sites and can be activated by a wide spectrum of stimuli, including inflammatory cytokines, growth factors and cellular stress. p38MAPK signaling pathway has been implicated in cell growth, apoptosis, motion and invasive phaenotype and mediates cell migration, tumor invasion and metastasis. Urokinase plasmitogen activator (uPA) is a serine protease and initiates the conversion of plasminogen to plasmin. These proteases confer on the cells the ability to degrade the extracellular matrix . uPA has been clearly demonstrated to be essential in the maintenance of invasive and metastatic phenotypes.
At present, less studies demonstrated the relationship between uPA expression and signal transduction pathway. Recent researches have indicated that p38MAPK signaling pathway participates in regulating uPA expression of gastric, lung, leukoma and breast cancer cells. Our studies suggested that there was significant correlation between the level of uPA ﹑ p-p38 expression and lymph node status and clinical stage of breast cancer and the expression of p-p38 and uPA in the highly metastatic MDA-MB-231 cells was higher than that in lowly metastatic MCF-7 cells. Our results hinted that overexpression of p-p38 and uPA might be related to malignant progression, invasion and metastasis of breast cancer. Immunohistochemical results showed an positive relationship between expression of p-p38 and uPA and suggested uPA protein expression might be regulated by p38MAPK signaling pathway. In further study, we investigated the relationship between uPA expression and p38MAPK signaling pathway. Western blot analysis showed that SB203580 decreased uPA protein expression in a concentration-dependent manner in breast cancer MDA-MB-231 cells. Thereby demonstrating that p38MAPK signaling pathway might be involved in regulating uPA expression.
Together, p38MAPK signaling pathway promoted breast cancer malignant progression , invasion and metastasis. However, the precise mechanisms remain unknown. Currently, an inhibitor of MAPK signaling pathway has represented a novel target for cancer intervention strategies. Our study suggests that p38MAPK signaling pathway may be potential therapeutic target for breast cancer treatment.
CONCLUSION
1. There was correlation between the level of uPA ﹑ p-p38 expression and lymph node status and clinical stage, and the expression of p-p38 and uPA in the highly metastatic MDA-MB-231 cells was higher than that in lowly metastatic MCF-7 cells.
2. There was a positive relationship between expression of p-p38 and uPA
3. SB203580 , an specific inhibitor of p38MAPK concentration-dependently reduced uPA protein expression.
KEYWORDS
p38MAPK; phospho-p38; uPA; breast cancer

目 录

一、摘要
1.中文论著摘要…………………………………………………()
2.英文论著摘要………………………………………… ()
二、英文缩略语…………………………………………………()
三、论 文
1. 前 言…………………………………… ()
2. 实验材料与方法…………………………… ()
3. 实验结果(附图表)……………………… ()
4. 讨 论…………………………………… ()
5. 结 论…………………………………… ()
6.本研究创新性的自我评价………………………………… ()
7.参考文献…………………………………………()
四、附 录
1. 综 述………………………………………… ()
2. 在学期间科研成绩…………………………………………()
3. 致 谢……………………………………… ()
4. 个人简介………………………………………()

前 言
乳腺癌是妇女最常见的恶性肿瘤,其发病率呈逐年上升趋势,且常发生浸润和转移。因此,与乳腺癌侵袭转移有关的相关因子成为当前研究的重点之一。侵袭和转移是恶性肿瘤的重要生物学特征,是肿瘤细胞与宿主细胞、细胞外基质之间一系列复杂的、多步骤、多因素相互作用的动态过程,涉及到细胞外基质的降解,肿瘤新生血管和新生淋巴管的形成,而侵袭转移相关基因的调节涉及复杂的机制及多条信号传递途径。近年来,较多研究发现MAPK信号通路与肿瘤恶性演进有关,并介导多种转移相关基因表达调控。
丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)属丝氨酸/苏氨酸激酶,是细胞内重要的信号转导系统,是各种信号途径的汇总点。MAPK有多个亚家族,其中较为确定的,在细胞功能中发挥重要作用的有细胞外信号调节激酶(Extracellular signal-regulated protein kinase, ERK)、C-Jun氨基末端激酶(C-Jun N-terminal kinase, JNK)、大丝裂原活化蛋白激酶(big MAP kinase, BMK)和p38MAPK。多种生长因子、炎症因子和应激反应可激活p38MAPK,p38MAPK激活后可使多种转录因子活化,影响基因转录、蛋白合成,细胞能动性和细胞骨架结构改变,在细胞侵袭、转移方面具有重要的调控功能。uPA是一种丝氨酸蛋白水解酶,激活纤溶酶原转变为纤溶酶,对细胞迁移和肿瘤侵袭转移中细胞外基质降解有重要作用。迄今许多研究证实uPA在多种肿瘤中过表达,在肿瘤的浸润转移中扮演重要角色。但对于其表达与信号通路的关系目前研究较少。为此,本研究的目的是检测乳腺癌中磷酸化p38MAPK和uPA蛋白表达并分析其相关性及与乳腺癌病理特征的关系,探讨p38MAPK通路对乳腺癌uPA蛋白表达的影响。为p38MAPK抑制剂应用于乳腺癌治疗提供相应的理论依据。

查看评论 已有0位网友发表了看法
  • 验证码: