【作者】 李秋云；

【导师】 施俊义；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 硕士

【摘要】 实验背景及目的乳腺癌是女性最常见的恶性肿瘤之一,好发于40-60岁之间的女性,近年来,随着人们生活方式及饮食习惯的改变,乳腺癌发病率呈逐年上升趋势,并且发病年龄逐渐呈低龄化改变。乳腺癌的发生是一个多因素、多突变累积的过程,至今发病机制尚未清楚。关于乳腺癌发生、发展、转移中相关蛋白过度表达以及激活一直是该领域研究的热点。Pak 1 (p21-activated kinase-1, p21激活激酶1)基因定位于人类染色体11q13.5-q14区,研究发现该基因的异常与乳腺癌和白血病等恶性肿瘤的发生发展密切相关。并对乳腺肿瘤细胞发生、存活、有丝分裂、细胞骨架再生重组、细胞迁移、以及激素非依赖性进展起重要作用。本实验采用实时荧光定量RT-PCR及免疫组化方法检测Pak1基因mRNA丰度及在乳腺癌组织中蛋白质含量。结合患者临床病理学资料,进一步了解其在肿瘤发生、发展中的分子机制及临床病理学意义。实验方法采用实时荧光定量PCR的方法,以SYBR Green I为荧光染料,GAPDH作为内参基因,相对定量出35例乳腺癌和10例乳腺良性病变、15例远癌正常小叶组织中Pak1基因mRNA的表达量,在转录水平分析Pak1的表达量与ER、PR、CerbB-2及腋窝淋巴结转移情况的相关性。另采用免疫组化Super Pic Ture法检测35例乳腺癌和15例远癌正常小叶组织标本中Pak1蛋白的表达,并在蛋白水平进行Pak1的表达与ER、PR、CerbB-2及淋巴结转移情况相关性分析。实验结果Pak1 mRNA和蛋白的相对表达量分别为：(1) Pak1mRNA在35例乳腺癌中相对表达量为4.76±0.11,在10例乳腺良性病变中相对表达量为2.66±0.10,15例正常小叶组织中相对表达量为0.94±0.06,。乳腺癌Pak1mRNA表达量明显高于乳腺良性病变,乳腺良性病变组织高于正常小叶组织,各组间的差异有显著性(P＜0.003)；(2)雌激素受体阳性的乳腺癌组织中Pak1的相对表达量为5.04±0.69,在雌激素受体阴性的乳腺癌组织中为4.59±0.57,两者间有显著差异(P=0.038)；(3)免疫组化染色结果中Pak1在雌激素受体阳性的乳腺癌组织中过度表达的阳性率为90.48%(19／21),雌激素受体阴性的乳腺癌组织中过度表达的阳性率为57.14%(8／14),两者间有显著差异。(P<0.05)；孕激素阳性的乳腺癌组织中过度表达的阳性率为86.96%(20／22),孕激素受体阴性的乳腺癌组织中过度表达的阳性率为53.85%(7／13),两者间有显著差异。(P＜0.05); (4)Pak1mRNA和蛋白表达与乳腺癌患者年龄、肿瘤大小、淋巴结转移情况及CerbB-2表达水平未见相关性(P>0.05)。实验结论Pak1作为癌基因参与了乳腺癌发生、发展及预后过程,Pak1的高表达是乳腺癌的普遍现象,并且与雌激素受体水平相关,也可能与孕激素受体相关,而与患者年龄、肿瘤大小、淋巴结转移情况、CerbB-2关系不密切。并在肿瘤细胞免疫逃逸、运动、血管发生和基因调节中发挥重要作用。因此,Pak1可能组成了肿瘤演进过程中信号传导的关键节点。实施对Pak1信号传导的干预必将诱导乳腺癌细胞凋亡、抑制增殖和抑制肿瘤血管生成。定量检测其水平,对乳腺癌的早期诊断、预后判断有一定价值。更多还原

【Abstract】 Background and ObjectBreast cancer is one of the most common malignancy of women, mainly between the ages of 40-60.With the improvement of people’s standard of living and changes of lifestyle and the structure of diet, the incidence of breast cancer has increased year by year, and the age of onset has inclined to get younger. The occurrence of breast cancer is a multi-factor, multi-mutation accumulation process. So far the pathogenesis remains unknown. Researches with regard to the occurrence, development and metastasis-associated protein over-expression and activation of breast cancer have been hot in this field.Pakl (p21-activated kinase-1, p21 activated kinase 1) gene is located in human chromosome 11q13.5-q14 area, the study found that the gene abnormalities and breast cancer and leukemia and other malignant tumors were closely related to the occurrence and development (1-2) of the disease. For breast tumor cells, survival, mitosis, cytoskeleton regeneration reorganization, cell migration, as well as progress in hormone-dependent, it plays an important role. In this study, real-time fluorescent quantitative RT-PCR and immunohistochemistry were used to detect pakl gene mRNA abundance and protein content in breast cancer tissues. Studies of patients with clinical and pathological data will provide a better understanding of their tumor occurrence and development of the molecular mechanisms and clinical pathological significance.MethodsThe mRNA expression of pakl gene were detected in 35 cases of breast carcinoma and 10 cases of benign mammary lesions and 15cases of normal lobular tissue by fluorescent real-timg RT-PCR, which take SYBR Green I as fluorescent dye and house-keeping gene as intra-control. Ststistial analysis was carried out to analyze the correlation between Pakl gene expression and clinical significance in these breast cancer patients on transcriptional level. The protein expression of Pakl were detected in 35 cases of breast carcinoma and 15cases of normal lobular tissue by immunohistochemcal study. Stastistial analysis was carried out to analyze the correlation between Pakl gene expression and clinical significance in these breast cancer patients on protein level.ResultsThe relative expression level of PAK1 mRNA and protein are:(1)The relative expression of Pakl mRNA were 0.94±0.06 in 15cases of normal lobular tissue; the relative expression of Pakl mRNA were 2.66±0.10 in 10 cases of benign mammary lesions, in contrast to 35 cases of breast carcinoma; the relative expression of Pakl mRNA were 4.76±0.11, which was significantly higher than the other groups(P<0.003). (2)The relative expression of Pakl mRNA were 4.59±0.57 in 14 speciments of ER-negative, in contrast to 21 speciments of ER-positive; the relative expression of Pakl mRNA were 5.04±0.69,which was significantly higher than the other groups(P=0.038). (3) Immunohistochemical staining results:Pakl gene was overexpression in 8 speciments of 14 cases of ER-negative(57.14%), in contrast to 19 speciments of 21 cases of ER-positive(90.48%), which was significantly higher than the other groups (P＜0.05); Pakl gene was overexpression in 7 speciments of 13 cases of PR-negative (53.85%), in contrast to 20 speciments of 22cases of PR-positive(86.96%), which was significantly higher than the other group(P<0.05). (4)Neither mRNA nor protein expression of Pakl gene correlated with the age of patients, size of tumor, Lymph node metastasis and the level of CerbB-2(P>0.05).ConclusionsAs an oncogene,Pakl participated in the carcinogenesis,development and prognosis of breast carcinoma. Pakl over expression is a common phenomenon in breast cancer, and may be related to the level of ER and PR of breast carcinoma;but was not associated with the age of patients,size of tumor, Lymph node metastasis, the level of CerbB-2. Pakl in the tumor cell plays an important role in immune escape, exercise, angiogenesis and gene regulation. Therefore,Pakl is a key point where signal conduction pathway with progression of breast carcinoma.To enforce the intervention on the signal conduction with Pakl will certainly to induce the apotosis in breast cancer cell,inhibiting proliferation,and inhibiting tumor angiogenensis.It is valuable for the early diagnosis and prognosis judgement in breast carcinoma through detected the level of Pakl quantity.更多还原