【作者】 杨华锋；

【导师】 吴爱国；

【作者基本信息】 南方医科大学 ， 外科学， 2010， 博士

【摘要】 背景乳腺癌是全世界女性最常见的恶性肿瘤之一,严重威胁女性的身心健康。全世界乳腺癌的发病率一直呈稳步上升的趋势,20世纪末的统计资料表明世界每年约有115万人诊断为乳腺癌,有41万人死于该病,发病年龄也有年轻化趋势,是导致女性肿瘤患者死亡的第一大原因。然而,乳腺癌的病因尚不十分清楚,其发病机制也非常复杂,影响发病的因素很多。近年来,随着肿瘤分子病理学和分子生物学研究的深入,人们认识到肿瘤的发生和发展是一个多阶段、多基因改变参与的渐进过程。多个癌基因的异常激活和编码蛋白的过度表达以及抑癌基因的缺失、突变失活导致细胞增殖失控而恶性转化,可能是肿瘤发生的分子生物学基础。随着新的治疗理念的提出和综合治疗的实施,乳腺癌的治疗水平不断提高,总生存率也在逐渐提高,但复发和转移仍是乳腺癌治疗失败和死亡的主要原因,因此研究乳腺癌生物学行为,寻找治疗敏感性相关生物学标志物将有助于临床工作。HIF-1α和Her-2是乳腺癌两个独立的预后因子,对乳腺癌的发生、发展和转移有重要影响,在乳腺癌中的生物学作用以及作为提示乳腺癌的预后因素和针对其研制特异性的单克隆抗体来治疗乳腺癌日益成为近年来乳腺癌研究的热点。但以往国内外研究多见于免疫组化对乳腺癌的研究,免疫组化研究操作比较复杂、耗时、费力,而且不能对患者进行实时、动态检测病情变化,目前对HIF-1α和Her-2ECD在乳腺癌血清中的联合定量检测尚无相关报导。甘氨酸、丙氨酸作为肿瘤细胞重要的合成原料和代谢产物,受HIF-1α通道调控,在肿瘤的发生、发展和转归都有举足轻重的作用,但相关研究亦未见报道,故寻找一种新的检测方法,定量分析人体代谢途径中相关的肿瘤标志物,尽可能提高肿瘤标志物的检出率是非常重要的。本课题利用悬浮芯片技术联合高效液相色谱定量检测乳腺浸润性导管癌患者血清中HIF-1α、Her-2ECD、Ala和Gly含量变化规律,分析其内在的相关性及其与乳腺癌临床病理、生物学特性的关系；探讨HIF-1α、Her-2ECD、Ala和Gly在乳腺癌诊断和监测病情的作用,以及评估其对乳腺癌诊断的价值。目的本研究拟从乳腺癌患者机体的代谢途径寻找动态的肿瘤标志物,探讨他们之间内在相关性。研究采用悬浮芯片联合高效液相色谱技术,分析HIF-1α、Her.2ECD、甘氨酸和丙氨酸的含量变化,及其与临床病理因素和预后的关系,并进一步探索HIF-1α、HER-2ECD、Ala和Gly在乳腺癌诊断、预后评估和疗效评价中的价值和意义。方法1.病例来源选自宁波大学医学院附属医院乳腺外科2006-2008年住院病人,其中乳腺原发浸润性导管癌117例,术后复发乳腺浸润导管癌10例,乳腺良性肿癌20例,体检健康拟行乳腺美容手术31例(正常对照)。乳腺原发浸润性导管癌术前均未进行相关放疗、化疗、内分泌治疗及免疫治疗。2.血清标本采集所有入选对象在清晨空腹采肘正中静脉血10ml,静置120分钟,3000r/min离心20分钟取血清,编号,-80℃冰箱保存,待检(根据术后的病理结果筛选保存的乳腺浸润性导管癌和良性肿瘤及乳腺美容患者血清标本冷藏待检)。3.组织标本的采集术中将手术切除的恶性肿瘤或良性肿瘤标本切取无坏死的瘤组织各约1g,以及在行乳腺美容手术时切取正常组织约1g,然后用冰盐水冲洗除掉血污,立即置入液氮中,保存于-80℃低温冰箱。检测前分别取少许肿瘤组织,称重后用质量分数为0.9%的冰冷生理盐水按150mg:1.5ml的比例在冰浴下超声粉碎制成10%的肿瘤组织匀浆,以4000r/min(离心半径8cm)离心10min,取上清液冷藏保存待检测。4.检测方法血清HIF-1α和Her-2ECD含量采用悬浮芯片试剂盒检测(检测范围>1pg),并用酶联免疫吸附(ELISA)法测定做对照(HIF-1α检测范围：10000-156pg;Her-2ECD检测范围：>0.39ng/ml)。血清和组织中甘氨酸和丙氨酸利用高效液相色谱法检测,利用配套软件计算出含量。5.统计学分析采用SPSS13.0软件进行,所有数据以均数±标准差(x±s)表示,多组间比较采用方差分析(F检验),组间两两比较采用t检验,P<0.05为差异,有统计学意义。对液相芯片法与ELISA或HPLC的检测结果采用Spearson积矩相关分析,检验水准,a=0.05。结果1.临床病例资料：本研究四组共178例,均为女性,年龄分布为27-91岁,中位年龄49.2岁。乳腺原发浸润性导管癌117例,年龄27-91岁(平均51.56岁)；术后复发乳腺浸润导管癌10例,年龄35-57岁(平均47.6岁)；乳腺良性肿瘤20例,年龄40-63岁(平均50.38岁)；乳腺美容31例(正常对照),年龄27-65岁(平均48.77岁)。四组入选病人年龄没有统计学差异(P>0.05)。乳腺原发浸润性导管癌术前均未进行相关放疗、化疗、内分泌治疗及免疫治疗,临床病理分期：Ⅰ期25例(21.5%)、Ⅱ期61例(52.1%)、Ⅲ期19例(16.2%)、Ⅳ期12例(10.3%)；病理组织学分级：G1期12例(10.3%)、G2期41例(35.0%)、G3期64例(54.7%)；肿瘤大小：T1组<2cm 41例(35.0%)、T2组>2cm,≤5cm 61例(52.1%)、T3组>5cm15例(12.8%)；淋巴结转移情况：无转移69例(59.0%)、转移48例(41%)。2.悬浮芯片法检测结果2.1原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组和正常对照组血清HIF-1α含量组间有统计学差异(F=200.035,p=0.000)；复发浸润导管癌组血清HIF-1α含量(231.72±38.39)高于原发浸润导管癌(166.18±59.15)(P<0.05)；良性肿瘤组(47.77±15.26)和正常对照组(47.84±11.36)血清中HIF-1α含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清HIF-1α含量均高于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清HIF-1α含量也均高于良性肿瘤组及正常对照组(P<0.01)；原发乳腺浸润性导管组血清HIF-1水平含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=15.030,p=0.000)：Ⅰ期(150.96+38.85)、Ⅱ期(149.52±52.96)之间没有统计学差异(P>0.05)；Ⅲ期(189.03±60.36)、Ⅳ期(249.54±44.84)之间有统计学差异(P<0.01)；Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)；Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)：发乳腺浸润性导管癌组血清HIF-1水平含量在组织学分级中组间有统计学差异(F=18.772,p=0.000)：随低分化(182.42±61.79)、中分化(154.39±52.67)和高分化(119.80±23.28)组织分化程度升高其含量逐渐减低,有统计学差异(P<0.01)；肿瘤直径大于5cm(T3)其血清HIF-1水平含量(215.19±57.11),高于T1(152.42±41.05)、T2(161.82±61.69)两组,有统计学差异(P<0.01),但T1组、T2组没有统计学差异(P>0.05)。淋巴结转移组血清HIF-1水平含量(197.48±55.28)高于没有淋巴结转移组(144.40±51.80),有统计学差异(P<0.01),孕激素受体阴性表达组(186.16±59.35)高于阳性表达组(153.23±55.69),有统计学差异(P<0.01)；雌激素受体阳性表达组(159.91±56.41)与阴性表达组(179.20±63.27)之间没有统计学差异(P>0.05)。2.2原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组和正常对照组血清Her-2ECD含量组间有统计学差异(F=50.924,p=0.000)；复发浸润导管癌组血清Her-2ECD含量(17.61±3.63)高于原发浸润导管癌(11.20±6.116)(P＜0.05)；良性肿瘤组(4.91±3.40)和正常对照组(4.56±2.95)血清中Her-2ECD含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Her-2ECD含量均高于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Her-2ECD含量也均高于良性肿瘤组及正常对照组(P<0.01)；原发乳腺浸润性导管组血清Her-2ECD水平含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=71.912,p=0.000)；Ⅰ期(7.89±2.94)与Ⅱ期(9.30±4.48)之间没有统计学差异(P>0.05)；Ⅲ期(15.44±6.38)与Ⅳ期(21.67±2.76)之间有统计学差异(P＜0.01)：Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)；Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)；发乳腺浸润性导管癌组血清Her-2ECD水平含量在组织学分级中组间有统计学差异(F=18.086,p=0.000)：肿瘤组织低分化组的Her-2ECD含量(13.04±6.53)高于中分化组(9.33±5.33)和高分化组(7.80±1.12),有统计学差异(P<0.01)中分化组与高分化组之间没有统计学差异(P=0.251)：Her-2ECD水平含量随T1组(7.98±3.35)、T2组(11.17±6.03)、T：组(18.02±5.57)肿瘤直径增大而含量增高(T3>T：>T1),有统计学差异(P<0.01)；淋巴结转移组Her-2ECD的含量(15.75±7.06)高于没有淋巴结转移组(8.04±2.15),有统计学差异(P<0.01)；雌激素受体阴性表达组血清Her-2ECD水平(13.49±6.43)高于雌激素受体阳性表达组(10.10±5.68),有统计学差异(P<0.01)。孕激素受体阴性表达组的Her-2ECD水平(13.05±6.42)高于孕激素受体阳性表达组(10.01±5.64),有统计学差异(P＜0.01)。3.高效液相色谱法检测结果3.1原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组及正常对照组血清中Ala含量组间有统计学差异(F=20.635,p=0.000)；复发浸润导管癌组血清中Ala含量(240.71±74.33)低于原发浸润导管癌(338.57±97.37)(P<0.05)；良性肿瘤组(414.49±64.94)和正常对照组(423.37±80.57)血清中Ala含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Ala含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Ala含量也均低于良性肿瘤组及正常对照组(P<0.01)原发浸润性导管癌、复发浸润性导管癌、良性肿瘤组及正常对照组血清中Gly含量组间有统计学差异(F=150.986,p=0.000)；复发浸润导管癌组血清中Gly含量(135.49±31.34)低于原发浸润导管癌(176.06±46.96)(P＜0.01)良性肿癌组(320.22±42.84)和正常对照组(342.19±48.15)血清中Gly含量之间没有统计学意义(P>0.05)。原发浸润性导管癌血清Gly含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌血清Gly含量也均低于良性肿癌组及正常对照组(P<0.01)原发浸润性导管癌、复发浸润性导管癌、良性肿癌组及正常对照组组织中Ala含量组间有统计学差异(F=161.011,p=0.000)：复发浸润导管癌组织中Ala含量(224.62±44.96)低于原发浸润导管癌(177.14±67.99)(P<0.05)；良性肿瘤组(437.44±58.10)和正常对照组(406.50±66.85)组织中Ala含量之间没有统计学意义(P>0.05)。原发浸润性导管癌组织Ala含量均低于良性肿瘤组及正常对照组(P<0.01)：复发浸润性导管癌组织Ala含量也均低于良性肿瘤组及正常对照组(P<0.01)。原发浸润性导管癌含量、复发浸润性导管癌、良性肿瘤组及正常对照组组织中Gly含量组间有统计学差异(F=141.398,p=0.000)；复发浸润导管癌组组织中Gly含量(204.64±40.94)低于原发浸润导管癌(147.92±56.74)(P＜0.01)；良性肿瘤组(327.32±46.93)和正常对照组(338.06±49.60)组织中Gly含量之间没有统计学意义(P>0.05)。原发浸润性导管癌组织Gly含量均低于良性肿瘤组及正常对照组(P<0.01)；复发浸润性导管癌组织Gly含量也均低于良性肿瘤组及正常对照组(P<0.01)。3.2原发乳腺浸润性导管组血清Ala含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=32.863,p=0.000)；Ⅰ期(433.51±67.69)、Ⅱ(342.90±74.78)、Ⅲ期(272.42±60.76)及Ⅳ期(210.05±82.32)随临床病理分期增高含量逐渐降低,有统计学差异(P<0.01)；原发乳腺浸润性导管癌组血清Ala含量在组织学分级中组间有统计学差异(F=5.511,p=0.005)；低分化(389.91±76.27)、中分化(362.99±92.43)和高分化(313.29±97.36)三组随肿瘤组织分化程度升高而含量水平逐渐下降,均有统计学差异(P<0.01)；原发乳腺浸润性导管组血清Ala含量在不同大小肿瘤组之间有统计学差异(F=51.067,p=0.000)；T1组(421.19±75.45)、T2组(323.10±87.08)、T3组(241.02±43.87)随肿瘤体积增大含量逐渐减少,有统计学差异(P<0.01)；淋巴结转移组血清Ala水平(297.72±96.01)低于没有发生转移组的水平(366.98±88.35),有统计学差异(P<0.01)：雌激素受体的阳性表达组(343.37±90.31)和阴性表达组(328.58±111.27)孕激素受体的阳性表达组(349.52±96.23)与阴性表达组(321.67±97.75)的血清Ala水平之间没有统计学意义(P>0.05)。3.3原发乳腺浸润性导管组血清Gly含量在临床病理分期Ⅰ期、Ⅱ期、Ⅲ期及Ⅳ期组间有统计学差异(F=24.621,p=0.000)；Ⅰ期(202.88±37.91)与Ⅱ期(186.18±36.57之间没有统计学差异(P>0.05)；Ⅲ期(152.54±30.75)与Ⅳ期(101.78±45.21)之间有统计学差异(P<0.01)；Ⅰ期与Ⅲ期及Ⅳ期之间均有统计学差异(P<0.01)：Ⅱ期与Ⅲ期及Ⅳ期之间也均有统计学差异(P<0.01)；原发乳腺浸润性导管癌组血清Gly含量在组织学分级中组间有统计学差异(F=23.602,p=0.000)；高分化组(217.50±25.89)与中分化组(199.12±39.35)之间没有统计学意义(P>0.05)；低分化组(153.520±42.089)与高、中分化组之间均有统计学差异(P<0.01)；原发乳腺浸润性导管组血清Gly含量在不同大小肿瘤组之间有统计学差异(F=10.403,p=0.000)；T1组(195.832±37.076)与T2组(177.103±45.486)没有统计学意义(P=0.081)；T1与T3组(130.260±43.177)及T2和T3(p=0.000)均有统计学差异；淋巴结转移组血清Gly水平(152.158±46.436)低于没有发生转移组的水平(192.69±39.85),有统计学差异(P<0.01)；雌激素受体的阳性表达组(181.75±42.12)和阴性表达组(164.22±54.41)、孕激素受体的阳性表达组(182.73±45.163)与阴性表达组(165.767±48.31)的血清Gly水平之间亦没有统计学差异(P>0.05)。4. HIF-1α、Her-2ECD和Ala、Gly之间的相关性分析：Spearson积矩相关分析表明,乳腺癌血清HIF-la与Her-2ECD呈正相关(r=0.533,p=0.000)；乳腺癌血清与组织中Ala(r=-0.429,p=0.000)的含量变化呈负相关：血清与组织中Gly(r=-0.481,p=0.000)的含量变化也呈负相关；Spearman等级相关分析示：在乳腺浸润性导管癌中血清HIF-1α水平与血清Ala(r=-0.403,p=0.000)呈负相关,在血清HIF-1α水平与Gly(r=-0.413,p=0.000)呈负相关；血清Her-2ECD与血清Ala(r=-0.509,p=0.000)水平呈负相关,血清Her-2ECD与血清Gly(r=-0.481,p=0.000)也呈负相关。5. HIF-1α(87.1%)和Gly(96.6%)对乳腺癌诊断率高于Her-2CED (69.7%)和Ala(67.4%),HIF-1α的对乳腺癌早期诊断效果高于Her-2CED(P<0.01);Her-2CED(84.6%)对乳腺癌淋巴结转移的符合率高于HIF-1α(67.5%)、Ala(64.1%)和Gly(65.8%)。Her-2CED对乳腺癌淋巴结的转移诊断效果优越HIF-1α(P<0.01)。6.通过配对t检验及其相关性分析、判别分析及ROC曲线分析,运用悬浮芯片技术同时定量检测血清HIF-1α和Her-2ECD的能力不低于单一比对ELISA试剂盒,悬浮芯片检测结果高于对比试剂盒的检测结果,两种方法检测HIF-1α(r=0.995,p=0.000)和Her-2ECD (r=0.998, p=0.000)结果呈正相关,相关性非常密切。结论1.运用悬浮芯片技术联合检测血清HIF-1α和Her-2ECD两种肿瘤标志物的含量,与比对试剂盒比较的相关性好,实验结果可靠,灵敏度高、操作简单,对乳腺癌的诊断和研究具有较高的辅助价值。可用于临床检测和课题实验研究。2.乳腺浸润性导管癌HIF-1α和Her-2ECD水平过表达,是病人预后不良的指征,其过度表达说明它在肿瘤发生、转化中起重要作用。所以动态检测乳腺浸润性导管癌血清HIF-1α和Her-2ECD水平可提供实时、动态的信息,为制定治疗方案提供帮助。因此检测血清HIF-1α和Her-2ECD水平的动态变化对乳腺浸润性导管癌复发、转移的早期判断及预后评估具有重要意义。3.Ala和Gly参与了乳腺浸润性导管癌细胞的合成和能量代谢,在乳腺浸润性导管癌的发生、发展和转归中有重要作用,可能成为乳腺浸润性导管癌靶向治疗新的靶点。4.血清HIF-1α、Her-2ECD、Ala和Gly可作为乳腺癌的早期诊断、评价乳癌恶性程度和预后比较全面的指标,能准确的反映乳腺浸润性导管癌的生物学特性,随着对血清Her-2ECD、HIF-1α、Ala和Gly基础和临床研究深入以其为靶点的治疗可能成为肿瘤治疗和预防的重要手段之一。更多还原

【Abstract】 BackgroundBreast cancer is one of the most common malignancies among women around the world, seriously threatening women’s physical and mental health. The incidence of breast cancer around the world has shown a steady upward trend, in the late 20th century the world’s annual statistics has show that about 1.15 million people are diagnosed with breast cancer,410 thousand people die of this disease, and the patients are younger and younger. Breast cancer has became the leading cause of death among female patients. However, the etiology of breast cancer is not yet clear, its pathogenesis is very complex and many factors affect the incidence. In recent years, with the deepening of research in tumor molecular pathology and molecular biology, it is recognized that the occurrence and development of cancer is a gradual process involving multi-stage and multi-gene changes. A number of oncogene proteins encoded by the abnormal activation, over-expression and deletion of tumor suppressor genes, and malignant transformation caused by inactivating mutations and uncontrolled cell proliferation may be the molecular biological basis of tumor. New ideas of treatment put forward and the implementation of comprehensive treatment, the treatment of breast cancer has improved continuously. The overall survival rate is gradually increased, but recurrence and metastasis of breast cancer are still the main causes for treatment failure and death. Therefore, the study of biological behavior in breast cancer, and discovering the relevant biological markers of sensitivity will contribute to clinical work. HIF-1αand Her-2ECD are two independent prognostic factors of breast cancer, which have significant influence on the incidence of, development and metastasis of breast cancer. Due to their biological roles in breast cancer, they are implications of prognostic factors of breast cancer and have important functions in specific monoclonal antibodies to treat breast cancer, HIF-1αand Her-2ECD have increasingly become hot research issues in recent years. In the past domestic and foreign researches prevalent in the immunohistochemical study of breast cancer, immunohistochemical study of operation is relatively more complex, time-consuming, laborious, and can not detect the real-time and dynamic changes in patients, and there is no relevant reports about co-quantitative detection of HIF-1αand Her-2ECD in the serum of breast cancer patients. Glycine and alanine, regulated by HIF-1αchannel, which are important synthetic raw materials of tumor cells and metabolic products of tumor, play very pivotal roles in the occurrence, development and metastasis of the cancer, but no related research has ever been reported. So it is very important to look for a new detection method, quantitatively analyzing the tumor markers of related metabolic pathways, to increase the detection rate to the maximum.This research subject is to apply floating-chip technology combining with high-performance liquid chromatography with quantitative detection of change in content of HIF-1α, Her-2ECD, Ala and Gly content in breast carcinoma patients, analzing its relevance and its relationship with the internal breast cancer pathology, and the relationship between the biological characteristics, and approach the role of HIF-la, Her-2ECD, Ala and Gly in breast cancer diagnosis and monitoring of illness, as well as to assess its diagnostic value of breast cancer.ObjectivesThis study is based on chip technology combined with high-performance liquid chromatography suspended from the body’s metabolic pathways of breast cancer patients to find the dynamic tumor markers to explore the intrinsic correlation between them, to analyze HIF-1α, Her-2ECD, glycine and alanine acid changes and clinical pathological factors and prognosis and study the prognosis of breast cancer. And to further explore the value and significance of her-2ECD, HIF-la, Ala and Gly in the diagnosis of breast cancer.Methods1. Cases from Breast surgery of Ningbo University School of Medicine Affiliated Hospital from 2006 to 2008, Primary breast infiltrating ductal carcinoma:117 cases. Postoperative recurrence of breast infiltrating ductal carcinoma:10 cases. Benign breast tumor:20 cases. breast cosmetic surgery of the health:31 cases(The normal control group). Primary invasive ductal breast cancer were not treated by preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy.2. Serum specimen collection:with the patient’s own consent, each selected object is collected blood 10ml in median cubital vein in the early morning. Put them aside for 120 minutes, then take serum after 3000r/min decentralization for 20 minutes, number, and preserve in -80℃refrigerator, screening serum of the breast invasive ductal carcinoma and benign tumor and breast cosmetic surgery of the health patients for inspection according to surgery pathological findings. 3. The collection of tissue samples:with the consent of selected cancer patients, in which surgery would be surgical resection of malignant or benign tumor specimens were cut without necrosis of tumor tissue, each about 1g, and the removal of breast cosmetic surgery at the same time normal tissue of about 1g, and then rinse off bloody with ice saline, immediately placed in liquid nitrogen and stored at -80℃low temperature refrigerators. Before test, take some tumor tissues, respectively, and uonicate to 10% homogenatation under the ice bath after weighing with the ice-cold 0.9% saline by the ratio of 150mg:1.5ml,4000r/min (centrifugal radius 8cm) centrifuge for 10min.The supernatant stored frozen for testing.4. Sample detection:Serum HIF-1αand Her-2ECD content by suspension chip detection kit (detection range>1pg), and enzyme-linked immunosorbent assay (ELISA) is determinated as controls (HIF-la detection range:10000-156pg; Her-2ECD detection range:> 0.39 ng/ml). Serum and tissues of glycine and alanine are tested by high-performance liquid chromatography, using software packages to calculate concentration.5. Statistical analysis:SPSS13.0 data were used, all data are mean±standard deviation (x±s). Use analysis of variance (F test)to compare between the two groups. T test was used to compare among two groups, P<0.05 for the difference, there were statistical significances. On the liquid-phase ELISA or HPLC-chip method and the results of tests were using Spearson rank correlation analysis. Test level,α=0.05.Results1. Clinical Case Data:178 cases in this group were female, age distribution from 27 to 91 years old, with a median age of 49.2 years old. Primary invasive ductal breast carcinoma of 117 patients, aged from 27 to 91 years old (mean 51.56 years); postoperative recurrence of breast invasive ductal carcinoma of 10 cases, from 35 to 57 years old (mean 47.6 years); 20 cases of benign breast tumor, age 40 to 63 years old (mean 50.38 years); breast cosmetic surgery of 31 cases(The normal control group), aged from 27 to 65 years old (mean 48.77 years); Four groups selected for the patient’s age were not statistically significant (P>0.05), All women.Primary invasive ductal breast cancer were not associated preoperative radiotherapy, chemotherapy, endocrine therapy and immunotherapy, clinical pathological stage:Ⅰperiod of 25 cases (21.5%),Ⅱperiod of 61 cases (52.1%),Ⅲperiod of 19 cases (16.2%);Ⅳ,12 cases (10.3%) histopathological grading:G1 phase 12 cases (10.3%), G2 phase 41 cases (35.0%), G3 period of 64 cases (54.7%); tumor size: T1 group≤2cm 41 Li (35.0%), T2 Group> 2cm,≤5cm 61 Li (52.1%), T3 group> 5cm15 cases (12.8%); lymph node metastasis:69 cases without metastasis (59.0%), the transfer of 48 patients (41%).2. Results of suspension micro-array test2.1 Primary invasive ductal carcinoma (166.18±59.15) and recurrent invasive ductal carcinoma (231.72±38.39) of serum levels of HIF-1αwere higher than benign tumor group (47.77±15.26) and normal control group (47.84±11.36) (P<0.01); Recurrent infiltrating ductal carcinoma of HIF-1αserum levels were significantly higher than those in primary invasive ductal carcinoma of (P<0.01和P<0.05); There were not statistically significant between the benign tumor group and normal control group serum and tissue HIF-1αlevels. Primary invasive ductal carcinoma of serum levels of HIF-1 levels in clinical and pathological staging of one (150.96±38.85), between the two groups (149.52±52.96) there were no statistical significance(P>0.05), but there were a significant difference between three (189.03±60.36), four stages (249.54±44.84) and one, two stages (P<0.01); and with reduce histological grade (well-differentiated, moderately differentiated and poorly differentiated), its content gradually elevated (119.80±23.28 154.39±52.67,182.42±61.79), there were a significant difference (P<0.01); tumor when the diameter of T3 (diameter>5cm), the serum levels of HIF-1 were significantly higher (215.19±57.11) than the other two groups (T1,T2) (152.42±41.05,161.82±61.69), there were a significant difference (P<0.01); Tumor diameter, T1, T2 were not statistically significant (P>0.05). Lymph node metastasis in patients with serum levels of HIF-1 were significantly higher (197.48±55.28) than that in patients with lymph node metastasis did not occur (144.40±51.80), there were a significant difference(P<0.01), progesterone receptor-negative expression (186.16±59.35) was significantly higher than positive expression(153.23±55.69), there were a significant difference (P<0.01); expression of estrogen receptor-positive (159.91±56.41) and negative expression (179.20±63.27) had no significant difference (P>0.05).2.2 Primary invasive ductal carcinoma (11.20±6.116) and invasive ductal carcinoma of recurrence (17.61±3.63) of serum Her-2ECD levels were significantly higher than benign (4.91±3.40) and normal control group (4.56±2.95) (P<0.01), Recurrent infiltrating ductal carcinoma higher than primary invasive ductal carcinoma (P<0.01); There were no statistical significance between the benign tumor group and normal control serum Her-2ECD content(P>0.05).Primary invasive ductal breast cancer patients with Her-2ECD level of content and serum levels of HIF-1 were similar, to the clinical pathological staging one(7.89±2.94), two (9.30±4.48), there were no statistically significant (P>0.05), but there is a significant difference between three (15.44±6.38), four stages (21.67±2.76) and one, two stages (P<0.01); tumor tissue with low (13.04±6.53) credit-based were higher than those higher degree of differentiation (in the differentiation and well-differentiated) (9.33±5.33,7.80±1.12), there were a significant difference (P<0.01); Her-2ECD level of concentration with the increase of tumor diameter for increased levels (T3>T2>T1) (18.02±5.57,11.17±6.03,7.98±3.35), there is a significant difference (P<0.01); Lymph node metastasis (15.75±7.06) than those without metastasis (8.04±2.15) in patients with serum Her-2ECD levels were significantly increased, there were a significant difference (P<0.01); Estrogen and progesterone receptor positive and negative expression of Her-2ECD between the serum levels were different, and negative expression was significantly higher (13.49±6.43,13.05±6.42) than positive expression (10.10±5.68,10.01±5.64), there is a significant difference (P<0.01).3. Results of high-performance liquid chromatography3.1 Analysis found that serum Ala (338.57±97.37), Gly (176.06±46.9) content in the primary, recurrent invasive ductal carcinoma (240.71±74.33,135.49±31.34) were lower than the normal benign tumor group (414.49±64.94,320.22±42.84) and control group (423.37±80.57,342.19±48.15) (P<0.01); That tissues Ala (177.14±67.99), Gly (147.92±56.74) content in the primary, recurrent invasive ductal carcinoma (224.62±44.96,204.64±40.94) were lower than the normal benign tumor group (437.44±58.10,327.32±46.93) and control group too (406.50±66.85,338.06±49.60) (P<0.01);Ala, Gly in recurrent infiltrating ductal carcinoma group were significantly higher than those in primary invasive ductal carcinoma (P<0.01和P<0.05); While the Ala, Gly in the serum of recurrent infiltrating ductal carcinoma were significantly lower than that of primary invasive ductal carcinoma (P<0.01和P<0.05); Ala, Gly levels in serum and tissues were no statistical significance between the benign tumor group and normal control group.3.2 Primary invasive ductal carcinoma of the level of serum Ala levels reduce with the clinical and pathological stage gradually increase (433.51±67.69, 342.90±74.78,272.42±60.76,210.05±82.32), there were a significant difference between the levels of different stages (P<0.01); With the histological grade (pathology classification) increased, the level of serum Ala levels reduce (389.91±76.27,362.99±92.43,313.29±97.36), there were a significant difference between different groups (P<0.01); Serum Ala levels and tumor volume is also closely related to tumor volume increased with decreasing concentration(421.19±75.45,323.10±87.08,241.02±43.87), there are significant differences. And lymph node metastases were also inter-related occurred in patients, with lymph node metastasis (297.72±96.01) in Ala serum levels were significantly lower than the transfer did not occur (366.98±88.35) in patients with the level, there were a significant difference (P<0.01); Estrogen (343.37±90.31,328.58±111.27) and progesterone (349.52±96.23 321.67±97.75) receptors and serum in Ala levels were not statistically significant (P>0.05).3.3 As to primary invasive ductal carcinoma of Gly serum levels in clinical and pathological staging, there were no significant difference betweenⅠ(202.88±37.91),Ⅱperiod (186.18±36.57) (P>0.05), there were a significant difference betweenⅠ,Ⅱperiod andⅢ(152.54±30.75),Ⅳperiod (101.78±45.21) (P<0.01), its content reduced gradually with the classification increased; In tumor cell differentiation, there were no statistical significance between the well-differentiated group (217.50±25.89) and moderately differentiation group (199.12±39.35) (P>0.05), there were statistically significant between the well-differentiated group, moderately differentiation group and poorly differentiation group (153.520±42.089) (P<0.01), with the reduction in the degree of differentiation Gly levels were gradually reduced; Gly serum levels and tumor volume were also closely related to tumor volume increased with decreasing concentration (195.832±37.076, 177.103±45.486,130.260±43.177), there are significant differences(P<0.01). And lymph node metastases were also inter-related occurred in patients with lymph node metastasis, in the Gly serum levels (152.158±46.436) were significantly lower than the transfer did not occur in patients with the level (192.69±39.85), there were a significant difference (P<0.01); Estrogen (181.75±42.12,164.22±54.41) and progesterone receptors (182.73±45.163, 165.767±48.31) and serum of Gly levels were not statistically significant (P> 0.05).4. Correlation between HIF-1α, Her-2ECD and Ala, Gly:Spearson rank correlation analysis showed that HIF-1αwere positive correlation with Her-2ECD in serum (r=0.533,p=0.000);Ala, (r=-0.429, p=0.000) Gly (r=-0.481, p=0.000) content was negatively correlated with changes in breast cancer serum and tissue. It was closely negatively correlated between HIF-1α, Her-2ECD and Ala (r=-0.403, r=-0.413, p=0.000), Gly (r=-0.509, r=-0.481, p=0.000) in breast invasive ductal carcinoma serum, and showed a closely positive correlation with Ala (r=0.973, r=0.969, p=0.000), Gly (r=0.534, r=0.533, p=0.000) in organization. There were no statistically significance between the normal control group and benign tumors(P>0.05).5. HIF-1α(87.1%) and Gly(96.6%) is higher than Her-2CED(69.7%) and Ala(67.4%). in diagnosis on breast cancer. Early diagnosis on breast of HIF-1αis higher than Her-2CED(P<0.01); Her-2CED(84.6%) is higher than HIF-1α(67.5%), Ala(64.1%) and Gly(65.8%) in lymph node metastasis of breast cancer. Her-2CED is superior to HIF-1α(P<0.01) in lymph node metastasis of breast cancer.6. Through the correlation analysis, discriminant analysis and ROC curve analysis, suspended chip technology the ability to detect a single target goal of not less than ELISA kit, test results and contrast the results of tests kits was no significant difference, especially for the determination of the value of tumor samples, suspended Chip kit and the control kit relatively good correlation (r=0.995, r= 0.998, p=0.000)。Conclusion1. In serum, HIF-1αand Her-2ECD can be detected with high sensitivity, high-throughput and reliability by using Suspension Array Technology which had a better auxiliary value than ELISA kit in the diagnosis of breast cancer. Therefore, this theme had a viable method and a reliable result, that can be used for research project.2. The lever of HIF-1αand Her-2ECD in serum of breast invasive ductal carcinoma were overexpression, it were an indication of poor prognosis of the patients, its overexpression indicates that it played an important role in tumorigenesis and tumor induction. So dynamic detection of HIF-1αand Her-2ECD can be used as a real-time, dynamic information, to provide assistance for the development of making treatment program. Therefore, it was important for us to detect the level of HIF-1αand Her-2ECD in serum in the recurrence, early assessment of metastasis and prognosis of breast invasive ductal carcinoma.3. Ala and Gly involved in anabolic and energy metabolism of the tumor cells of the breast invasive ductal carcinoma, played an important role in the occurrence, development and progression of the breast invasive ductal carcinoma, and may become a new target of the targeted therapy.4. In serum, HIF-1α、Her-2ECD、Ala and Gly can be used as the early diagnosis of breast cancer, some more comprehensive indicators to evaluate the degree of malignancy, can reflect the biological characteristics of the breast invasive ductal carcinoma. With the basic and clinical research of HIF-1α、Her-2ECD、Ala and Gly in serum going further, it could be one of means in cancer treatment and prevention for targeted treatment.更多还原