【作者】 郭变琴；

【导师】 涂植光；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2013， 博士

【摘要】 目的：1.研究Wnt2在人乳腺癌细胞株（MDA-MB-231、ZR-75-30和MCF-7）及患者血清中的表达情况，并探讨在乳腺癌患者血清中Wnt2与CA-153的相关性。2.探讨Wnt2及β-catenin在乳腺癌组织及配对癌旁乳腺组织中的表达及临床意义。3.探讨肿瘤相关成纤维细胞（carcinoma-associated fibroblasts，CAFs）及培养上清中Wnt2含量及其培养上清对人乳腺癌细胞MCF-7的增殖、细胞周期及细胞侵袭力的影响；以及对β-catenin在MCF-7中的表达及细胞内分布的影响。方法：1.采用RT-PCR技术检测3种人乳腺癌细胞（MDA-MB-231、ZR-75-30和MCF-7）株Wnt2基因mRNA转录水平，采用蛋白免疫印迹检测3株人乳腺癌细胞中Wnt2蛋白表达水平；此外，采用酶联免疫吸附试验检测30例乳腺癌患者及15名健康成人血清中Wnt2蛋白含量，电化学发光技术检测CA-153含量。2.采用RT-PCR检测7例乳腺癌及配对癌旁组织Wnt2和β-catenin基因mRNA的转录水平；组织芯片及免疫组织化学方法检测40例浸润性乳腺导管癌及其配对癌旁乳腺组织中Wnt2和β-catenin的表达情况，并分析二者与乳腺癌临床病理特征的关系，同时分析Wnt2和β-catenin联合检测与乳腺癌恶性程度及淋巴结转移的关系。3.采用蛋白印迹检测从乳腺癌组织及配对癌旁乳腺组织分离的成纤维细胞（normal fibroblasts,NFs）中Wnt2蛋白表达情况；酶联免疫吸附试验检测成纤维细胞培养上清中Wnt2含量；将实验分为4个组：MCF-7对照组、NFs培养上清+MCF-7组、低表达Wnt2的CAFs培养上清+MCF-7组、高表达Wnt2的CAFs培养上清+MCF-7组；以MTT、流式细胞术、Millicell小室比较各组细胞增殖力、细胞周期的分布及细胞侵袭能力；免疫荧光检测各组细胞中β-catenin的表达及其在细胞内分布。结果:1. RT-PCR结果表明：乳腺癌细胞株MDA-MB-231、ZR-75-30、MCF-7中未检测到Wnt2mRNA；蛋白印迹同样未检测到Wnt2蛋白表达；30例乳腺癌病人血清中Wnt2平均含量[(10.34±4.25) ng/mL]显著高于健康成年女性对照[(1.85±0.98) ng/mL]（P<0.05），且Wnt2在血清中含量与CA-153呈正相关（P<0.05）。2. RT-PCR结果表明：乳腺癌组织中Wnt2和β-catenin基因mRNA的相对转录量显著高于配对癌旁组织（P<0.05）；组织芯片及免疫组化结果表明：Wnt2在部分配对癌旁乳腺组织细胞浆与间质中呈弱表达，在浸润性乳腺导管癌组织细胞浆与间质中呈高表达，Wnt2在浸润性乳腺导管癌阳性表达率77.5%(31/40)显著高于配对癌旁乳腺组织15.0%(6/40)(P<0.05)；配对癌旁乳腺组织中的β-catenin均表达于细胞膜中，而在浸润性乳腺导管癌组织细胞膜、细胞浆及细胞核中呈阳性表达，异常表达率为67.5%（27/40）；Wnt2和β-catenin的表达与浸润性乳腺导管癌患者年龄无关（P>0.05)，但与临床分期、病理组织学分级及淋巴结转移有关(P<0.05)；此外，Wnt2和β-catenin均为阳性表达者的肿瘤恶性程度和淋巴结转移的发生率显著高于两者均为阴性或两者之一为阳性表达者（P<0.05）。3. Western blot结果表明：从4例乳腺癌组织分离的CAFs中有1例检测到Wnt2蛋白高表达（1.33±0.13），其余3例乳腺癌组织分离的CAFs也有表达（0.22±0.10），而4例癌旁乳腺组织中均未检出Wnt2蛋白。ELISA结果表明：4例乳腺癌组织分离的CAFs培养上清中，有1例上清中Wnt2高表达(6.58±1.02) ng/ml，其表达量显著高于配对NFs中Wnt2含量[(0.92±0.34)ng/ml]，以及另外3例乳腺癌组织分离的CAFs培养上清中Wnt2含量[(1.67±0.37) ng/ml](P均<0.05)。此外，其它3例CAFs培养上清中Wnt2含量[(1.67±0.37) ng/ml]也显著高于配对NFs培养上清Wnt2含量[(0.54±0.22)ng/ml]（P<0.05）。MTT、流式细胞术、Millicell小室检测结果表明：与NFs培养上清比较，经CAFs培养上清孵育的MCF-7，其细胞增殖能力明显提高（P<0.05），细胞周期S期细胞比例明显增高（P<0.05）、G2期细胞比例降低（P<0.05），而侵袭穿膜细胞数明显增加(P<0.05);与低表达Wnt2的CAFs培养上清相比，经高表达Wnt2的CAFs上清孵育的MCF-7细胞，其细胞增殖能力明显提高（P<0.05），侵袭穿膜细胞数明显增加(P<0.05)。免疫荧光结果表明：与NFs培养上清比较，经CAFs培养上清孵育的MCF-7，β-catenin的表达明显增强。与低表达Wnt2的CAFs培养上清相比，经高表达Wnt2的CAFs上清孵育的MCF-7细胞，β-catenin在细胞核的表达明显增强。结论:1.血清中Wnt2可协同乳腺癌肿瘤标志物CA-153，作为乳腺癌筛查的血清学辅助指标，二者联合平行检测可提高乳腺癌筛查的灵敏度。2. Wnt2、β-catenin在乳腺癌的发生和侵袭转移中发挥协同作用，联合检测Wnt2和β-catenin可作为乳腺癌诊断和预后判断指标。3. CAFs能通过旁分泌Wnt2促进乳腺癌细胞增殖、分裂及侵袭，有望成为乳腺癌治疗的新靶标。更多还原

【Abstract】 Objective1. To investigate the expressions of Wnt2in the cell lines（MDA-MB-231, ZR-75-30and MCF-7）and tissues of breast cancer，andto observe the relation of Wnt2with CA-153in the serum of patients withbreast cancer.2. To investigate the expressions of Wnt2and β-catenin in the matchedpairs of breast cancer and normal breast tissues, and its possible clinicalsignificance.3. To investigate the expression of Wnt2in the carcinoma-associatedfibroblasts (CAFs) and normal fibroblasts (NFs), as well as in their culturesupernatant. At the same time，to observe the effects of different culturesupernatants on the invasion, proliferation, cell cycle of MCF-7, and theexpression and distribution of β-catenin in MCF-7cells.Methods1. The levels of Wnt2mRNA and protein were measured bypolymerase chain reaction (PCR) and Western blotting in3human breastcancer cell lines（MDA-MB-231, ZR-75-30and MCF-7), respectively.2. Enzyme-linked immunosorbent assay (ELISA) was used to examineWnt2, and electrochemiluminescence technique was applied to detect CA-153in the serum of15normal women subjects (control) and30patients with breast cancer. Furthermore, the relation of Wnt2with CA-153was analyzed. The transcription levels of Wnt2and β-catenin mRNA weredetected by RT-PCR in the seven pairs of breast cancer and normal breasttissues. Expressions of Wnt2and β-catenin were also detected with tissuechip technique by immunohistochemistry in the forty pairs of invasivebreast ductal carcinoma and normal breast tissues, and the relation of thetwo protein expressions with clinical and pathological characteristics wasanalized. At the same time, the relationship between parallel combinationdetection of Wnt2and β-catenin with the degree of malignancy of breastcancer and lymph node metastasis was analized.3. Carcinoma-associated fibroblasts (CAFs) and normal fibroblasts(NFs) were isolated from breast cancer tissues and the correspondingadjacent breast tissues，and their culture supernatants were collected,respectively；Wnt2in CAFs and NFs was examined by Western blot, and inthe culture supernatants by ELISA. The following experiments weredivided into4groups: MCF-7control group, NFs culture supernatant+MCF-7group, CAFs culture supernatant containing low Wnt2+MCF-7group, culture supernatant of CAFs containing high Wnt2+MCF-7group.The cell proliferation was detected by the MTT，cell cycle distribution wasassayed by flow cytometry, and cell invasion ability was investigated withMillicell chamber； And expression and intracellular distribution of β-catenin in the MCF-7were detected by immunofluorescence.Results1. The expressions of Wnt2mRNA and protein were negative in threehuman breast cancer cell lines. The levels of Wnt2in the serum of patientswith breast cancer [(10.34±4.25) ng/mL] were higher than those in normalwomen[(1.85±0.98) ng/mL](P<0.05), and Wnt2levels in the serum of breastcancer patients were positively correlated with serum CA-153levels.2. The relative transcription levels of Wnt2and β-catenin gene mRNAwere obviously higher in the breast cancer tissues than adjacent normalbreast tissues（P<0.05)；Wnt2were weakly expressed in the cytoplasm ofepithelial and interstitial cells of some adjacent normal breast tissues，andstrongly expressed in the cytoplasm of epithelial and interstitial cells ofinvasive breast ductal carcinoma. The positive rate of Wnt2expressionswas15.0%(6/40) in the cancer adjacent normal tissues and77.5%(31/40)in the invasive breast ductal carcinoma (P<0.05). β-catenin was onlyexpressed in the cytomembrane of adjacent normal breast tissues，incontrast, it was strongly expressed in the cytomembrane, cytoplasm andnucleus of invasive breast ductal carcinoma. The abnormal expression rateof β-catenin was67.5%（27/40）in invasive breast ductal carcinomain. theexpression levels of Wnt2and β-catenin were correlated with clinical stage,histological granding and lymph nodes metastases (P<0.05)，while nosignificant association was found with patient age (P>0.05). However, the malignant degree and the rate of lymph nodes metastases were higher inpatients with both of Wnt2and β-catenin positive expression than thosewith Wnt2or β-catenin alone expressed.3. CAFs and NFs were separated from four breast cancer tissues andadjacent normal breast tissues, Wnt2was strongly expressed in1of4CAFs (1.33±0.13) and low expressed in other3CAFs (0.22±0.10),however, there was no Wnt2expression in the NFs by Western blotting.Culture supernatant Wnt2test with ELISA showed: Wnt2was high lever[(6.58±1.02) ng/ml] in one culture supernatan of4CAFs, which wasobviously higher than paired NFs [(0.92±0.34)ng/ml] and other3CAFs[(1.67±0.37) ng/ml](all P<0.05); And Wnt2in the culture supernatan ofother3CAFs [(1.67±0.37) ng/ml] was also significantly higher than pairedNFs [(0.54±0.22) ng/ml]（P<0.05).In addition，MTT, FCM and Millicell chamber tests revealed thatcompared with the culture supernatant of NFs, MCF-7hatched with theculture supernatant of CAFs showed significantly higher proliferationability, percentage of S phase cells and invasion ability (all P<0.05);compared with the culture supernatant of CAFs with low Wnt2, MCF-7hatched with the culture supernatant of CAFs with high Wnt2showedsignificantly higher proliferation ability and invasion ability (all P<0.05).Furthermore, compared with the culture supernatant of NFs, β-cateninexpression was increased in the MCF-7hatched with the culture supernatant of CAFs; in addition, compared with the culture supernatant ofCAFs with low Wnt2, the nuclear expression of β-catenin was increased inthe MCF-7hatched with the culture supernatant of CAFs with high Wnt2.Conclusions1. Wnt2cooperating with CA-153can act as markers for breast cancerscreening，parallel combined detection of both may help to improve thesensitivity of breast cancer screening.2. Overexpression of Wnt2and β-catenin in breast carcinoma mayplay some role in the oncogenesis and metastasis of breast cancer. Thecombined detection of Wnt2and β-catenin may be useful makers in thediagnosis and prognosis of breast cancer patients.3. CAFs could promote the proliferation, meiosis and invasion ofbreast cancer cells through Wnt2paracrine, and it may be a novel target ofbreast cancer treatment in the future.更多还原