【作者】 刘小丰；

【导师】 邵志敏；

【作者基本信息】 复旦大学 ， 肿瘤学， 2008， 博士

【摘要】 [目的]DARC是Duffy抗原/趋化因子受体(Duffy antigen/Receptor 0fChemokine)的简称。Duffy抗原和DARC是有同样结构和功能的蛋白质分子,它们的区别仅仅在于Duffy抗原位于红细胞上,而DARC位于乳腺癌细胞等其它非红细胞上。Duffy抗原决定了Duffy血型。DARC是乳腺癌的负性调控因子,它和乳腺癌关系密切;另外,已知炎症性疾病时DARC上调。因此,我们提出TNF-α等致炎性细胞因子调节乳腺癌细胞DARC表达的假设并加以验证。在患者Duffy血型与临床病理关系的研究方面,目的是验证Duffy血型(Duffy Blood Group,DBG)的表型是否与乳腺癌的发生和恶性程度有关。相关的研究背景包括:流行病学的研究表明,不同民族有不同的DBG表型分布,同时,不同民族在乳腺癌的发病率和死亡率方面也有所不同。【方法】选择常见的四种致炎性细胞因子TNF-α、IL-1β、IFN-γ和IL-8,以乳腺癌MDA-MB-231细胞系为研究对象,应用Real-Time PCR和Western-Blot方法,检测这些致炎性细胞因子能否调节MDA-MB-231的DARC表达。在初步验证TNF-α可以上调DARC的表达κ之后,进一步检测TNF-α抗体和NF-κB(nuclear factor-kappaB,核转录因子κB)抑制剂AEA(anadmide,NF-κB阻滞剂)对于DARC表达的影响。本实验还采用ELISA定量内源性TNF-α的浓度,确定内源性TNF-α对DARC表达的影响。最后应用Transwell观察TNF-α上调乳腺癌细胞系DARC表达后,细胞对于趋化因子CXCL8、CCL2的清除能力、细胞的趋化运动、侵袭能力是否改变以确定DARC的这种上调是否具有功能。在Duffy血型与临床病理关系的研究方面,我们采用了非直接抗人球蛋白法检验了复旦大学附属上海肿瘤医院乳腺科的连续253例女性病人的DBG表型,根据凝血情况分为Fya+Fyb-、Fya-Fvb+、Fya+Fyb+和Fya-Fyb-四种表型。在此基础上分析DBG表型与病理之间的关系。【结果】0.5～1ng/ml的TNF-α上调mDARC4～8倍。4～8ng/ml的IL-1β可以上调DARC 7倍左右。IFN-γ和IL-8对DARC表达的影响较小。应用TNF-α抗体后,TNF-α上调DARC的作用减弱。DARC的上调或下调时,其mRNA和蛋白的变化是同步的。AEA25μM作用后,TNF-α上调DARC的作用降低。内源性TNF-α对DARC表达没有显著影响。TNF-α上调乳腺癌细胞系的DARC表达后,细胞对于趋化因子CXCL8、CCL2的清除能力提高了,同时提高了细胞对于CXCL8、CCL2的趋化运动,并且降低了细胞的侵袭运动能力。在Duffy血型与病理关系的研究方面,我们发现有乳腺疾病或有乳腺癌的汉族女性患者和普通汉族人群在DBG表型分布方面的差异无显著性。在乳腺癌的发病方面,Fya-Fyb-和Fya-Fyb+有更高的乳腺癌的发病倾向性,但没有统计学的差异;Fya-表型的患者群和Fya+表型的患者群相比有更高的恶性率,但没有统计学的差异(两者分别为57.14%和39.02%,P=0.28)。各表型之间在ER、PR、Her-2、P53、PCNA、PS2、nm23和P450状态方面没有差异;在乳腺癌的病理分级方面也没有统计学差异。表型Fya-的患者比Fya+的患者有更高的腋淋巴结转移率(分别是100%和39.13%,P＜0.05,FisHer精确检验)。[结论]TYF-α和IL-1β对于DARC的表达起重要作用。TNF-α对DARC的调节可能通过NF-κB起作用。乳腺癌细胞系MDA-MB-231自身分泌的TNF-α对于DARC的调节功能并不重要。在Dufry血型与病理关系的研究方面,本研究表明,对于乳腺癌患者的腋淋巴结转移,Dufry血型表型体系中的Fya抗原比Fyb抗原更加重要。Fya-表型对于发现腋淋巴结转移的倾向的乳腺癌患者有帮助更多还原

【Abstract】 Purpose:DARC is the abbreviation of Duffy antigen/Receptor of Chemokine, which is a negative regulator to breast cancer.Duffy Blood Group(DBG) is determined by Duffy antigen.Duffy antigen and DARC is the identical protein which share the same structure and function.The only difference between Duffy antigen and DARC is the former is located in the membrane of red bolld cell and the latter is located in the membrane of breast cancer cells and other cells such as endothelial cells.Our lab previous study suggested that DARC correlated to breast cancer malignancy.Besides,DARC is up-regulated in several inflammatory diseases. In this study we test the hypothesis that whether pro-inflammatory cytokines can up-regulate DARC in breast cancer cell lines based on such background.Another hypothesis we test is that clinical patients’ DBG phenotypes affect on breast cancer occurrence and correlate to malignancy based on the epidemiological facts that different races has definitely different DBG phenotypes distribution and to them, there is a various breast cancer mobidity and mortality.Methods:Real-Time PCR and Western-Blot analysis was used to test four pro-inflammatory cytokines TNF-α, IL-1β,IFN-γand IL-8 effects on MDA-MB-231 cell lines DARC mRNA and protein expression.After we founded that TNF-αincrease DARC expression at both mRNA and protein level,we tested TNF-αantibody and NF-κB inhibitor AEA(Anadmide) effects on DARC by Real-Time PCR and Western-Blot analysis.Concentration of endogenous TNF-αwas determined by Enzyme Linked Immunosorbent Assay (ELISA).Finally,ELISA measured the concentration of CXCL8(CXC chemokine ligand 8,interleukin-8,IL-8),CCL2(CC chemokine ligand 2,two major DARC ligands,in conditioned media were done to determine DARC chemotactic clearance, and Transwell migration assays towards CCL2 as well as in vitro invasion assay were done to determine whether the TNF-α-induced increase in DARC expression was functional.In the study about correlation between DBG and pathological diagnosie,we investigated DBG phenotypes of 253 cases consecutive hospitalized female patients suffering breast disease in Shanghai Cancer Hospital and analysized its relationship with clinical pathological diagnosis.DBG phenotypes were examined by indirect antiglobulin-test with anti-Fya and anti-Fyb reagents and were classified into Fya+Fyb-,Fya-Fyb+,Fya+Fyb+,Fya-Fyb- according agglutination.Results: TNF-α(0.5～1ng/ml) and IL-1β(4～8ng/ml) obviously increased DARC expression by 4 to 8 folds and 7 folds respectly.TNF-αantibody and NF-κB inhibitor AEA(25μM) attenuated TNF-αup-regulation.IFN-γand IL-8 effects on DARC expression were weak.No correlation between concentration of endogenous TNF-αand DARC expression.DARC expression level in mRNA and protein was paralleled.TNF-αinduced DARC expression enhanced clearance of chemokines,moreover,mobility of MDA-MD-231 cell line to CCL2 was increased and invasion capacity of MDA-MD-231 cell line was decreased.DARC increased by TNF-αwas functional. In the study about correlation between DBG and pathological diagnosie,we found neither DBG phenotypes distribution difference existed in breast disease patients nor DBG phenotypes distribution difference existed in breast cancer patients compared to general Chinese Han population.To breast cancer occurring possibility,Fya-Fyb-and Fya-Fyb+ demonstrated more susceptibility to breast cancer than Fya+Fyb- and Fya+Fyb+,but no statistical significant difference.Fya- group(57.14%) had more malignant incidence than that of Fya+ group(39.02%),but no statistical significance (P=0.28).Neither significant difference had been observed in ER,PR,Her-2 status and P53,PCNA,PS2,nm23,P450 status between every DBG phenotypes,nor statistical difference was found to tumor grades in various DBG phenotypes.More patients were involved in auxiliary lymph nodes metastasis in Fya- than that of Fya+ group and reached a statistical significance(100%and 39.13%respectively,P＜0.05, Fisher exact test).Conclusion:Both TNF-αand IL-1βare two important regulators to breast cancer cell lines MDA-MB-231 DARC expression and the function may via NF-κB pathway.Endogenous TNF-αwas not an important redulator to DARC expression.Fya antigen weights more than Fyb antigen in breast cancer lymphnodes metastasis.Fya negative maybe a clinically useful factor to identify the potential axillary lymph nodes metastasis in breast cancer patients.更多还原