1、食管鳞状细胞癌组织中BMP7的表达及其临床意义摘要 背景与目的：骨形成蛋白7(bone morphogenetic protein 7,BMP7)是TGF-家族成员之一，近来研究发现在许多肿瘤中BMP7的表达发生了改变。本研究旨在探讨BMP7在人类食管鳞状细胞癌组织中的表达及其临床意义，研究BMP7与食管鳞癌发生，发展，病理分级，临床分期，浸润程度和淋巴结转移的关系。方法:采用免疫组织化学链霉素抗生物素过氧化酶连接(SP)法和RT-PCR (reverse transcription-polymerase chain reaction) 法检测56例新鲜食管鳞癌及对应癌旁正常组织标本中BMP7
2、及BMP7mRNA的表达, 结合临床病理资料，研究其与食管鳞癌的相关性。结果:免疫组化显示BMP7在食管鳞癌组织细胞膜和细胞质中有表达, 食管鳞癌组织中BMP7的阳性表达率为73.2% (41/56) ，高于正常食管组织的12.5%(7/56)（2=27.225，P0.001）。 RT-PCR显示食管鳞癌组织BMP7mRNA表达明显高于癌旁正常组织（2=28.195，P0.001），食管鳞癌BMP7mRNA的阳性表达与浸润程度，淋巴结转移和TNM分期有关（P0.05），而与性别，年龄和组织学分级无关。结论：BMP7可能在食管鳞癌的发生、发展中起重要作用, BMP7的检测可能有助于判断患者预后。
3、关键词 骨形成蛋白BMP7；食管鳞状细胞癌；免疫组织化学法；临床意义Expressions of BMP7 in Esophageal Squamous Cell Carcinoma and its Clinical Significance YAN Lin1，REN Jing-li2，YANG Kun-peng1 ( 1. Department of Thoracic Surgery，the Second Affiliated Hospital，Zhengzhou University，Zhengzhou 450014 ,China; 2. Department of Pathology，t
4、he Second Affiliated Hospital，Zhengzhou University，Zhengzhou 450014,China )Correspondence to：REN Jing-li E-mail: Abstract Background and Purpose: Bone morphogenetic protein7（BMP7）is a member of the TGF-family, which is demonstrated that the expression pattern is altered in many different tumors in
5、recent studies. To explore the relationship of the BMP7 and cancer occurrence, development, histological grade, clinical stage, invasion and lymph node metastasis by investigating the BMP7 expression and Clinical significance in esophageal squamous cell carcinoma. Methods: Immunohistochemical SP sta
6、ining and RT-PCR were employed to determine the expression of BMP7 and BMP7mRNA in 56 pairs of fresh esophageal carcinoma tissue and the adjacent normal tissues. We study the correlation of BMP7 and esophageal squamous cell carcinoma combination with the clinical pathological data. Results: BMP7 exp
7、ression was found in the cytoplasm and nucleus of cancer cells。The positive expression rate of BMP7 was 73.2%(41/56) in esophageal quamous cell carcinoma significantly higher than normal esophageal tissue 12.5%(7/56) (2=28.195，P0.001). The positive expression of BMP7mRNA correlated with invasion, ly
8、mph node metastasis and TNM stage (P 5cm),立即冻存于-80超低温冰箱中。肿瘤组织经常规病理切片检查证实均为食管鳞状细胞癌,所有患者术前均未行放化疗或其他免疫治疗。56例中,男51例,女5例,年龄4379岁,其中60岁24例；组织学分级G1+G239例，G317例；肿瘤浸润深度T1+T2 26例，T3+T430例；淋巴结转移33例，无淋巴结转移23例；肿瘤分期按国际抗癌联盟（UICC）食管癌TNM分期标准（第7版 2009年），+期21例，+期35例。1.2 主要试剂 免疫组化SP检测试剂盒和鼠抗人BMP7单克隆抗体（生研(上海)生化试剂有限公司），Tr
9、izol试剂（Invitrogen），MMIV 逆转录酶（Promega），引物(北京赛百胜公司)。1.3 免疫组织化学检测BMP7表达 本实验采用鼠抗人BMP7单克隆抗体及免疫组织化学SP配套试剂盒(均购自生研(上海)生化试剂有限公司)。常规制片, 4m厚石蜡切片,操作步骤严格按照SP免疫组化说明书进行，一抗工作浓度为1:200。已知阳性的食管癌组织切片标本为阳性对照, 用磷酸缓冲液(PBS)代替一抗作为空白对照。光学显微镜下, BMP7阳性表达主要位于食管鳞癌细胞的细胞膜和细胞浆内, 为浅棕色至深棕色颗粒（如图1）。判定标准在高倍显微镜下（200）按阳性细胞占肿瘤细胞的百分比20%认为该组
10、织BMP7表达阳性6。1.4 RT-PCR法检测BMP7mRNA表达 首先提取标本总RNA，所得RNA经1%的琼脂糖凝胶电泳分析,符合试验要求者在-70冰箱中存放备用。进一步参与反转录，制备食管癌及癌旁正常组织的cDNA。PCR扩增BMP7，同时扩增-actin 作为内参照。BMP7引物序列，正义：5-CCAACGTGGCAGAGAACAG-3；反义5- GGTGGCGTTCATGTAGGAGT-3。-actin引物序列，正义：5-TGACGGGGTCACCCACACTGTGCC-3；反义：5-CTGCATCCTGTCGGCAATGCCAG-3。反应条件如下：预变性94 5 min，变性94
11、45 s，退火54 45 s，延伸72 1 min，后延伸72 5 min，共30个循环。判定标准RT-PCR产物经2%琼脂糖凝胶电泳分析,出现目标条带者为阳性，见图2。1.5统计学处理 所有资料均经SPSS16.0软件包进行数据处理,采用配对2检验比较癌旁组织和食管鳞癌组织中BMP7阳性表达率的差异，采用行列表资料的2检验比较食管癌组织中BMP7mRNA阳性表达与临床病理特征之间的关系。检验水准P=0.05。2 结果2.1 食管鳞癌及正常组织中BMP7的表达 食管鳞癌组织中BMP7的阳性表达率为73.2% (41/56) ，高于正常食管组织的12.5%(7/56)，见表1。表1 食管正常和癌
12、组织中BMP7的表达Tab.1:BMP7 expression in Esophageal Squamous Cell Carcinoma and their adjacent normal tissues.Normal tissueSquamous Cell Carcinoma P0.001 4 3 37 12McNemar test:2=27.225，P0.001.图1 食管鳞癌中BMP7的阳性表达 Fig1: Positive expression of BMP7 in Esophageal Squamous Cell CarcinomaA:SP, 100; B:SP,4002.2 食管
13、鳞癌BMP7 mRNA表达与临床病理因素的关系 56例食管鳞癌组织中,BMP7mRNA呈阳性的为43例, 占76.8%；而56例相应的正常癌旁组织中BMP7mRNA呈阳性的为8例, 占14.3%,显著低于癌组织组(=28.195，P0.001) , 见表2。食管鳞癌BMP7mRNA的阳性表达与浸润程度，淋巴结转移和TNM分期有关（P0.05），而与性别，年龄和组织学分级无关。见表3。图2 BMP7 mRNA RT-PCR扩增结果 Fig2: Expression of BMP7 mRNA by RT-PCR.M: DNA Marker DL2000; 1:BMP7 DNA; 2: -actin
14、表2 食管鳞癌BMP7mRNA的表达Tab.2: BMP7mRNA expression in Esophageal Squamous Cell Carcinoma and their adjacent normal tissues.Normal tissueSquamous Cell Carcinoma P0.001 5 3 38 10McNemar test:2=28.195，P0.001.表3 BMP7mRNA的表达与食管鳞癌患者临床病理特征之间的关系Tab.3 The relationship between BMP7mRNA expression and clinical patho
15、logical characteristics of Esophageal Squamous Cell Carcinoma patientsParameter Case BMP7mRNA positive(n%) 2 PGenderMale 51 40 (78.4) 0.142* 0.706Female 5 3 (60.0)Age/year60 32 25 (78.1) 0.075 0.78460 24 18 (75.0)Histological gradeG1 + G2 39 27 (69.2) 0.478* 0.489G3 17 14 (82.4)Invasion depthT1+T2 2
16、6 16 (61.5) 6.330 0.012T3+T4 30 27 (90.0)Lymphatic metastasis 33 29 (87.9) 5.547 0.019 23 14 (60.9)Clinical stage+ 21 11 (52.4) 9.143* 0.002+ 35 32 (91.4)*Continuity Correction Chi-Square Tests.3 讨论BMP7是TGF-家族成员之一, 人的BMP7cDNA全长1293bp，含有431个氨基酸，BMP7在细胞内以前体形式合成，并在C末端在蛋白水解酶的作用下剪切加工成含有139个氨基酸的成熟BMP7蛋白2。
17、BMP7存在于人体多种组织细胞中, 除了在骨和软骨的发生发育及诱导异位骨和软骨发生过程中具有重要作用外,还参与了胚胎的发生和发育以及调控组织细胞增殖、分化、凋亡等多种生物学作用3。近年来大量研究报道BMP7表达与多种肿瘤细胞的发生发展相关，如骨髓瘤、骨肉瘤、恶性黑色素瘤、前列腺癌、乳腺癌、肾细胞癌、结直肠癌和胃癌411，这些文献报道BMP7具有促进或抑制肿瘤细胞增殖作用，但该蛋白在有关食管鳞癌方面的报道国内外极为罕见。本研究通过免疫组织化学和RT-PCR技术分析了食管鳞癌组织与相应癌旁正常组织之间BMP7表达的差异，结果显示食管鳞癌患者BMP7在鳞癌组织中的表达显著高于癌旁组织，且RT-PCR
18、结果显示BMP7mRNA的阳性表达与浸润程度，淋巴结转移和TNM分期有关（P0.05），而与性别，年龄和组织学分级无关。Motoyama等研究证实BMP7mRNA在结直肠癌的表达增加与肿瘤浸润深度和肝转移显著有关10；Aoki等报道在胃癌中BMP7的表达和肿瘤的深度，淋巴结转移，淋巴的浸润，和静脉的浸润有关11；这些研究结果都支持本次的实验结果。与此相反，Kwak等报道在肾细胞癌中BMP7的表达与更好的外科预后有关9；在正常的前列腺腺体组织中BMP7表达水平最高,随着前列腺癌的发生进展BMP7表达有越来越低的趋势7；还有相关文献报道在骨髓瘤、乳腺癌和前列腺癌中BMP7具有抑制细胞增殖作用4，1
19、214；这可能与肿瘤的特异性有关。由于本实验未对入选对象进行术后随访，尚不能确定患者的中位生存期、远期生存预后与BMP7基因表达之间的联系，但至少证实了BMP7基因在人类食管鳞癌中表达增强，这使我们意识到对其进行进一步研究的必要性和前景，如进一步探讨BMP7在食管鳞状细胞癌组织中的作用机制和发现BMP7参与癌细胞功能的新的信号途径以及在食管鳞癌的诊断治疗提供新的思路和药物靶点等。【参考文献】1 Lee SJ, Lee KS, Yim YJ, et al. Recurrence of squamous cell carcinoma of the oesophagus after curative
20、 surgery: rates and patterns on imaging studies correlated with tumour location and pathological stageJ . Clin Radiol,2005,60(5):547-554. 2 Griffth DL , Keck PC , Sampath TK, et al . Three dimensional structure of recombinant human osteogenic ptotein-1:structural paradigm for the transforming growth
21、 factor beta superfamilyJ. Proc Acad Sci USA,1996 ,93(2):878-883.3 Parmiani G, Rodolfo M, Melani C. Immunological gene therapy with ex vivo gene-modified tumor cells a critique and a reappraisalJ.Hum Gene Ther,2000,11(9):1269-1275.4 Ro TB，Holt RU，Brenne AT，et a1Bone morphogenetic protein-5，-6 and-7
22、inhibit growth and induce apoptosis in human myeloma cellsJ.Oncogene,2004,23(17)：3024-30325 Sulzbacher I, Birner P, Lang S, et al. The expression of bone morphogenetic proteins in osteosarcoma and its relevance as a prognostic parameterJ. J Clin Pathol,2002,55(5):381385.6 Rothhammer T, Wild PJ, Boss
23、erhoff AK, et al. Bone morphogenetic protein 7 (BMP7) expression is a potential novel prognostic marker for recurrence in patients with primary melanomaJ.Cancer biomarkers : section A of Disease markers,2007,3(2):111-117.7 Masuda H, Fukabori Y, Yamanaka H, et al. Expression of bone morphogenetic pro
24、tein-7 (BMp-7) in human prostateJ. Prostate,2004,59(1):101106.8 Alamo EL, Rauta J, Kallioniemi A, et al. Bone morphogenetic protein 7 is widely overexpressed in primary breast cancerJ. Genes Chromosomes & Cancer, 2006,45(4):411419.9 Kwak C, Park YH, Kim IY, et al. Expression of bone morphogenetic pr
25、oteins, the subfamily of the transforming growth factorsuperfamily,in renal cell carcinomaJ. J Urol,2007,178(3):10621067.10Motoyama K, Tanaka F, Kosaka Y, et al. Clinical significance of BMP7 in human colorectal cancerJ. Ann Surg Oncol,2008,15(5):15301537.11 Aoki M, Ishigami S, Natugoe S, et al. Exp
26、ression of BMP-7 in human gastric cancer and its clinical significanceJ. Br J Cancer,2011,104(4):714718.12 Buijs JT，Henriquez NV，van Overveld PG，et a1Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancerJCancer Res,2007,67(18)：8742-875113 Miyazaki H，Wata
27、be T，Kitamura T，et a1BMP signals inhibit proliferation and in vivo tumor growth of androgeninsensitive prostate carcinoma cellsJ.Oncogene,2004,23(58):9326933514 Buijs JT,Rentsch CA，van der Horst G，et a1BMP7，a putative regulator of epithelial homeostasis in the human prostate，is a potent inhibitor of prostate cancer bone metastasis in vivoJAm J Pathol,2007,171(3):1047-1057
