【作者】 郑湘予；

【导师】 陈奎生； 李晟磊；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 浸润和转移是恶性肿瘤的重要特征,也是恶性肿瘤难以治愈、导致患者死亡的关键问题。趋化因子(chemokines)是一大类不同类型细胞分泌的具有趋化作用的细胞因子,是一单链小分子蛋白质超家族,分为CC、CXC、XC和CX3C四个家族,相应趋化因子受体依据结合的趋化因子类型不同,划分为CCR、CXCR、XCR和CX3CR。趋化因子受体不仅高表达于免疫细胞,而且在某些肿瘤细胞也存在高表达。Muller首先阐述了趋化因子受体与乳癌转移的关系,提示趋化因子受体与肿瘤转移密切相关。趋化因子受体在多种恶性肿瘤细胞的生长、侵袭和转移过程中都发挥重要的作用。因此,趋化因子受体已成为研究抗肿瘤药物的极具吸引力的新靶点。CXCR4 (chemokine CXC4 receptor)不是肿瘤特异性标记物,但其在很多肿瘤中都高表达。很多研究表明CXCR4在肿瘤的发生、浸润、转移、黏附及分泌过程中发挥着重要的作用。肿瘤转移首先需要突破细胞外基质(extra cellular matrix, ECM)和基底膜(basement membrance, BM)组成的屏障,其主要成分之一是结构蛋白,包括胶原、层粘素、纤维结合束和玻璃体结合束等,其相应的水解酶基质金属蛋白酶(matrix matalloproteinases, MMPs)等在肿瘤侵袭转移过程中具有不可忽视的作用。MMPs是一个大家族,其成员中MMP-2 (matrix matalloproteinases-2)和MMP-9 (matrix matalloproteinases-9)与肿瘤的侵袭转移最为密切,研究表明,MMP-9在肿瘤细胞介导的细胞外基质降解过程中起关键作用。目前有关CXCR4在食管鳞癌中表达情况的研究甚少,国内目前尚未见报道,CXCR4与食管鳞癌的关系亦尚不十分明确。为进一步探讨CXCR4和MMP-9基因与食管鳞癌发生和浸润转移的关系,寻求早期检测食管鳞癌浸润和转移的指标及寻找抑制食管鳞癌发生和浸润转移的有效方法,本研究采用原位杂交和免疫组化SP (streptavdin peroxidase)法联合检测食管鳞癌组织中CXCR4、MMP-9 mRNA和蛋白的表达水平,分析CXCR4和MMP-9与食管鳞癌浸润、转移之间的关系以及两者之间的关系。材料与方法1.采用免疫组织化学方法检测62例食管鳞癌组织、31例癌旁中-重度不典型增生组织及62例正常食管粘膜组织中CXCR4及MMP-9蛋白的表达情况。2.采用原位杂交方法检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管粘膜组织中CXCR4和MMP-9 mRNA的表达情况。3.统计学处理:应用SPSS13.0软件处理,采用x2检验,检验水准α=0.05。结果1.食管鳞癌组织中CXCR4蛋白(59.7%)和CXCR4 mRNA (74.2%)阳性表达率明显高于癌旁组织(35.5%,40.9%)和正常食管粘膜组织(25.8%,24.0%),三组间CXCR4蛋白及mRNA表达率相比较,差异有统计学意义(P<0.05)。2.浸润深度达纤维膜组CXCR4蛋白(68.2%)和CXCR4 mRNA (85.4)阳性表达率明显高于浸润深度达深肌层(57.1%,64.3%)及浅肌层组(14.3%,28.6%),三组间CXCR4蛋白及mRNA表达率相比较,差异有统计学意义(P<0.05)。3.随着组织学分级的升高,CXCR4 mRNA (77.3%,72.0%及73.3%)及CXCR4蛋白(63.6%,56.0%及60.0%)阳性表达率无明显变化,差异无统计学意义(P>0.05)。4.有淋巴结转移的癌组织CXCR4蛋白(85.0%)和CXCR4 mRNA (95.0%)阳性表达率明显高于无淋巴结转移的癌组织(47.6%,64.3%),二者差异有统计学意义(P<0.05)。5.食管鳞癌组织中MMP-9 mRNA (72.6%)和MMP-9蛋白(80.6%)阳性表达率明显高于癌旁不典型增生组织(51.6%,58.1%),和正常食管粘膜组织(41.9%,27.4%),三组间MMP-9 mRNA和MMP-9蛋白表达率相比较,差异有统计学意义(P<0.05)。6.浸润深度达纤维膜组MMP-9 mRNA (80.5%)和MMP-9蛋白(92.7%)阳性表达率明显高于浸润深度达深肌层(71.4%,71.4%)及浅肌层组(28.6%,28.6%),三组间MMP-9 mRNA和MMP-9蛋白表达率相比较,差异有统计学意义(P<0.05)。7.随着组织学分级的升高,MMP-9 mRNA(53.5%,68.0%及90.9%)和MMP-9蛋白(46.7%,88.0%及95.5%)阳性表达率逐渐升高,三组相比组间差异有统计学意义(P<0.05)。8.有淋巴结转移的癌组织MMP-9 mRNA (95.0%)和MMP-9蛋白(100.0%)阳性表达率明显高于无淋巴结转移的癌组织(61.9%及71.4%),二者差异有统计学意义(P<0.05)。9.食管鳞癌组织中CXCR4及MMP-9mRNA和蛋白阳性表达呈正相关关系(P<0.05)。结论1. CXCR4及MMP-9可能在食管鳞癌发生及侵袭转移过程起促进作用。2. CXCR4和MMP-9蛋白表达呈正相关关系,提示二者在食管鳞癌的发展过程中具有某种协同作用。3. CXCR4和MMP-9 mRNA表达和蛋白表达均具有一致性。更多还原

【Abstract】 Backgroud and ObjectiveInfiltration and metastasis are not only the major characteristic of the malignant tumor, but also the key to its hard curing and the resulting death of patients. Chemotatic factor is cell factor having chemotaxis through cell secretion by different groups of a macro-category, and it is a haplo-chain micromolecule (8-10kD) protein superfamily, including CC, CXC, XC and CX3C four families respectively. Chemotatic factor acceptor accordingly is divided as CCR, CXCR, XCR and CX3CR according to the connection with different categories. Chemotatic factor acceptor is over-expression not only on immunocell, but on some tumor cells. Muller stated firstly the relationship of chemotatic factor acceptor and breast cancer metastasis in 2001, and discovered that it has been close correlation with tumor metastasis. Moreover, Chemotatic factor acceptor also was found to play very important effects in the growth, invasion and metastasis of multi-species malignant tumors. Therefore, it has become the new attracting target in the research of antitumor drug. CXCR4 is not tumor specificity marker, but it shows overexpression in many tumors. Many researches report that CXCR4 plays very important effects in the tumor-genesis, infiltration, metastasis, myxo-adnexal and externalization. But contraries exists in the present researches, without final conclusions to some questions. During the process of tumor metastasis, the first step is breaking through the barriers of basement membrance and extra cellular matrix. The main ingredients of BM and ECM included two components, one is structural proteins, which composed with collagen, laminin, fiber light beam and beam with vitrectomy et al. The corresponding enzyme is MMPs, which play a important role in the process of tumor invasion and metastasis. MMPs is a big family, MMP-2 and MMP-9 are more closely with tumor invasion and metastasis among the members. Studies have shown that MMP-9 play a key role in the process of the cell-mediated tumor cell extracellular matrix degradation.Presently there are only a few studies on the expression of CXCR4 in ESCC at abroad and there are no related reports at home. The relation between CXCR4 and ESCC is unclear. In order to discuss the correlation of genes of CXCR4 and MMP-9 with growth and trans-infiltration of esophageal squamous cell carcinoma (ESCC), find the targets of early detecting ESCC growth and metabasis, and explore the effetive way which could prevent its growth and metabasis, we detect mRNA and protein expression levels of CXCR4 and MMP-9 in ESCC by in situ hybridization and immunohistochemical staining, analysis the relationship between CXCR4 and MMP-9 and between them and invasion and metastasis of ESCC.Material and methods1. The immunohistochemical staining was used to measure the protein expression of CXCR4 and MMP9 in 62 cases of ESCC,62 cases of normal esophageal mucosa and 31 cases of adjacent atypical hyperplasia tissues.2. In situ hybridization was used to detect the mRNA expression of CXCR4 and MMP9 in 62 cases of ESCC,62 cases of normal esophageal mucosa and 31 cases of adjacent atypical hyperplasia tissues.3. Statistical analysis was carried out using Statistical Package for Social Sciences(SPSS)P, version 13.0 for windows. Chi-square test, one-way anaysis of variances and two indepndent-samples t test were used. P-values of<0.05 were considered to be statistically significant..Result1. The positive rates of CXCR4 protein(59.7%) and mRNA(74.2%) in ESCC were significantly higher than in adjacent atypical hyperplasia(35.5%,40.9%) and in normal esophageal mucosa(25.8%,24.0%). There were significant differences in the expressions of CXCR4 among the three groups(P<0.05).2. The positive rates of CXCR4 protein(68.2%) and mRNA(85.4) with tumor invaded into fiber membrane were significantly higher than those with tumor invaded into deep muscle layer(57.1%,64.3%)and tumor invaded into shallow muscle layer(14.3%,28.6%). The differences among the three groups were statistically significant (P< 0.05).3. With the increase of histological grade, CXCR4 mRNA(77.3%,72.0%and 73.3%) and protein(63.6%,56.0%and 60.0%) expression was no significant change. There were no significant differences among different groups(P>0.05).4. The positive rate of CXCR4 protein(85.0%) and mRNA(95.0%) with lymph node metastasis were significantly higher than those without lymph node metastasis(47.6%,64.3%). The difference between the two groups was statistically significant (P<0.05).5. The positive rates of MMP-9 mRNA(72.6%) and protein(80.6%) in ESCC were significantly higher than in adjacent atypical hyperplasia(51.6%,58.1%) and in normal esophageal mucosa(41.9%,27.4%). There were significant differences in the expressions of MMP-9 among the three groups(P<0.05).6. The positive rates of MMP-9 mRNA(80.5%) and protein(92.7%) with tumor invaded into fiber membrane were significantly higher than those with tumor invaded into deep muscle layer(71.4%,71.4%) and tumor invaded into shallow muscle layer(28.6%,28.6%). The differences among the three groups were statistically significant (P<0.05).7. With the increase of histological grade, MMP-9 mRNA(53.5%,68.0%and 90.9%) and protein(46.7%,88.0%and 95.5%) expression was significant changed. There were significant differences among different groups(P>0.05).8. The positive rate of MMP-9 mRNA(95.0%) and protein(100.0%) with lymph node metastasis were significantly higher than those without lymph node metastasis(61.9%and 71.4%). The difference between the two groups was statistically significant (P<0.05). 9. There was a positive correlation between the mRNA and protein expressions of CXCR4 and MMP-9 in ESCC. (P<0.05)Conclusion1. CXCR4 and MMP-9 may play an important role in the process of invasion and metastasis of ESCC.2. CXCR4 and MMP-9 protein expressions were positively correlated, suggesting that they has a certain synergy in the development of ESCC.3. The mRNA expression and protein expression of CXCR4 and MMP-9 were consistent.更多还原