癌胚抗原相关结直肠癌的临床及蛋白质组学初步研究
摘要
CRC是世界上最常见的三大恶性肿瘤之一,我国CRC的死亡率在常见肿瘤中排在第四位,发病率在逐渐上升。目前临床上早期诊断率不高,就诊的患者大多数已经属于进展期。CEA是临床上应用最广泛的血清肿瘤学标志物,但其敏感性和特异性不高。与CEA~+CRC相比,CEA~-CRC更缺乏血清学诊断和监测指标。因此,很迫切需要寻找一些更有效的CRC分子标志物。稳定同位素标记联合液相串联质谱技术(iTRAQ-LC-MS/MS)的建立和发展,为寻找有效肿瘤分子标志物提供了可靠的技术手段。本研究以CRC为研究对象,分以下几个部分进行,试图筛选出有价值的CRC候选标志物。
第一部分血清CEA阴性和阳性结直肠癌的临床和流行病特征比较——附1969例分析
本部分的研究目的是通过回顾性调查与CEA相关1969例CRC患者的临床和流行病学特征,了解CEA相关的这两类CRC有何不同以及CEA的血清阳性率情况,为后续基础研究做铺垫。
结果显示, CEA的血清阳性率为38.04%,CEA阴性结直肠癌平均发病年龄比CEA阳性结直肠癌年轻2岁多,中位发病年龄较后者提前3岁,发病人群以60岁以下的青壮年为主,其中A型血患者比例较高,而B型血患者比例较低,并发2型糖尿病和结肠肠梗阻比例较低。
本研究说明,CEA血清阳性率不高,缺乏敏感性,CEA阴性结直肠癌和CEA阳性结直肠癌在临床和流行病学上确实有一定的差异,为后续深入研究奠定了基础。
第二部分CEA阴性和阳性结直肠癌的组织蛋白质组学比较与验证
本部分的研究目的是比较血清CEA~-CRC和CEA~+CRC与正常粘膜组织(normal mucosa, NM)之间差异表达蛋白,验证部分有差异表达的蛋白质,试图寻找潜在的候选标志物。
结果显示,在筛出的57个差异蛋白质中,大多数细胞功能定位在胞内,生物学过程主要体现为调控和代谢,分子功能主要参与结合。许多差异蛋白之间有联系,尤其是差异蛋白TTN、PRDX1、TAGLN、TP11等,处于蛋白相互作用网络的中心枢纽节点上。采用Western Blot (WB)和免疫组织化学染色验证差异蛋白Annexin A5和Trangelin表达,它们趋势与质谱鉴定信息一致。采用荧光定量PCR验证Lumican蛋白对应基因,mRNA表达水平,结果与质谱信息不一致。
本研究说明,iTRAQ定量标记是寻找CRC有效分子标志物的重要方法,但需要验证确认。本实验筛选出一组与CEA相关组织差异蛋白,其中有些可能是CRC潜在的分子标志物。
第三部分CEA阴性和阳性结直肠癌的分泌蛋白质组学比较与验证
本部分的研究目的是比较血清CEA~-CRC、CEA~+CRC与NM微小组织块培养上清之间差异分泌表达蛋白,验证部分有差异表达的蛋白质,试图寻找可能在血清中出现的的潜在候选标志物。
结果显示,在筛出的61个差异蛋白质中,总共有50.82%(31／61)是分泌蛋白,其中有一些是未见报道与肿瘤相关的分泌差异蛋白,Western Blot验证差异蛋白Annexin A5和Trangelin,结果显示它们表达趋势与质谱鉴定信息基本相似,但分泌表达模式与组织表达模式存在分离现象。
本研究说明,分泌蛋白组学是组织蛋白组学的有益补充；本实验筛选出一组与CEA相关的分泌差异蛋白,可能是CRC潜在的血清分子标志物。
第四部分AnnexinA5和Lumican在结直肠癌的表达和意义
本部分的研究目的是扩大样本研究Annexin A5和Lumican在结直肠癌的表达和意义,为下一步进行功能研究进行初步筛选。
结果显示,Annexin A5在CRC阳性率为40%(20／50),明显高于正常组织,其表达与CRC分期、转移有关。Lumican在结直肠癌呈高表达,与病理参数无关。
本研究说明,Annexin A5和Lumican是结直肠癌的重要候选分子标志物,具体的作用机制还有待深入研究。
结论
1.CEA血清阳性率不高,缺乏敏感性,CEA阴性结直肠癌和CEA阳性结直肠癌在临床和流行病学上确实有一定的差异。
2. iTRAQ定量标记是寻找CRC有效分子标志物的重要方法,但需要验证确认。组织蛋白组学筛选出57个与CEA相关差异蛋白,分泌蛋白组学选出61个与CEA相关差异蛋白,两组差异蛋白有较多重复,但差异蛋白的组织表达模式与分泌表达模式可以不一致。本组筛选出的差异蛋白,有些可能CRC潜在分子标志物。
3.改良Alamar blue法是检测组织块活力灵敏有效的新型检测方法之-
4. AnnexinA5和Lumican表达与结直肠癌关系密切,是结直肠癌的重要分子标志物之一。
创新点：
1. iTRAQ定量标记技术应用在结直肠癌分泌蛋白组学研究。
2.以血清CEA表达状态为切入点分组,寻找CRC潜在分子标志物,符合临床实际需求。
3.设计单位质量吸光度评价指标,把Alamar blue法首次应用在组织块活力评价上。
Colorectal carcinoma (CRC) is one of the three common malignant tumors in the world, and ranks fourth in cancer mortality in our country. At present, the incidence rate of CRC is increasing, but early diagnosis level of CRC is poor and majority of CRC belong to the aggressive-phase. carcinoembryonic antigen (CEA) is the most widely used serum marker,but its sensitivity and specificity are far from satisfying. The CEA-negative CRC is more short of serodiagnosis and monitoring marker than the CEA'positive CRC. So, it is urgent to screen for new tumor biomarkers to improve CRC diagnosis. Now, the new tagging technique such as isobaric tag for relative and absolute quantitation (iTRAQ), followed by LC and MS/MS analysis is one of the more powerful methods in the search for disease biomarkers. In this study, CRC is researched in order to screen some worthy tumor biomarkers.
Part One Clinicopathologic and epidemiologic analysis of 1696 cases of the colorectal carcinoma related with preoperative carcinoembryonic antigen
This section aimed to understand the differences between CEA-negative CRC group and CEA-positive group, and the positive expression ratio of CEA by exploring the clinicopathologic and epidemiologic characteristics of 1969 cases CRC.
The result manifested the positive expression ratio of CEA was 38.04 percent. The mean age in the CEA-negative CRC was younger than in the CEA-positive one, and the median age in the CEA-negative CRC was ahead 3 years of that. The negative CEA group patients were younger than 60 years of age. The blood group A ratio was higher in the negative CEA group than in the positive CEA group, and the blood group B one was lower in the negative CEA group than that. The concomitant ratios of type 2 diabetes and colon obstruction in the negative CEA group, apparently lower than the positive CEA group.
This study showed CEA had poor sensitivity for the detection of some CRC, and there really had some differences between the negative CEA group and the positive CEA group, which was the basement of next research.
Part Two The analysis and verification of tissue proteomics between the CEA-negative CRC and CEA-positive CRC.
This section aimed to screen some potential biomarkers of CRC by comparing the differential proteins in the CEA-negative CRC and CEA-positive CRC to the normal mucosa.
Totally 57 proteins were finally identified. Most proteins was intracellular component. The biological process reflected regulation and metabolism. Molecular function mostly participated binding. There had been connected among many differential proteins, especially such as TTN、PRDX1、TAGLN and TP11. These proteins were put in the central key position of network. Annexin A5 and trangelin were chose for identification by western blot and immunohistochemistry stain, lumican was validated by fluorescence quantitative PCR, and majority were proved to be line with the iTRAQ results. Our results proved that iTRAQ results were reliability.
This study showed iTRAQ technique can be as effective manner to screen the differential expressed protein, but it was needed to validate. Some of the differential proteins were probably potential markers of CRC.
Part Three The analysis and verification of secretome between the CEA-negative CRC and CEA-positive CRC.
This section aimed to screen some potential serum biomarkers of CRC by comparing the differential proteins in the CEA-negative CRC supernatant and CEA-positive CRC supernatant to the normal mucosa supernatant.
Totally 61 proteins were finally identified. The secretory proteins ratio were 50.82%by bioinformatics. Some secretory proteins had not been mentioned in tumor in the past records. Annexin A5 and trangelin were chose for identification by western blot, and were proved to be basically similar with the iTRAQ results. But the secretory expression model was probably not line with the tissue expression model.
This study showed the secretome of CRC was a benefical supplement to the tissue proteomics. These differential secretory proteins were probably potential serum biomarkers of CRC.
Part Four The study and significance of annexin A5 and lumican expression in CRC
This section aimed to explore in enlarged sample the annexin A5 and lumican expression in CRC and their significance.
The result showed the expression level of annexin A5 and Lumican in CRC were markedly higher than one in normal mucosa. Annexin A5 expression was related with CRC stage and metastasis, but Lumican expression had no this relationship.
This study manifested annexin A5 and Lumican were one of important candidate biomarkers in CRC.
第一部分血清CEA阴性和阳性结直肠癌的临床和流行病特征比较——附1969例分析
本部分的研究目的是通过回顾性调查与CEA相关1969例CRC患者的临床和流行病学特征,了解CEA相关的这两类CRC有何不同以及CEA的血清阳性率情况,为后续基础研究做铺垫。
结果显示, CEA的血清阳性率为38.04%,CEA阴性结直肠癌平均发病年龄比CEA阳性结直肠癌年轻2岁多,中位发病年龄较后者提前3岁,发病人群以60岁以下的青壮年为主,其中A型血患者比例较高,而B型血患者比例较低,并发2型糖尿病和结肠肠梗阻比例较低。
本研究说明,CEA血清阳性率不高,缺乏敏感性,CEA阴性结直肠癌和CEA阳性结直肠癌在临床和流行病学上确实有一定的差异,为后续深入研究奠定了基础。
第二部分CEA阴性和阳性结直肠癌的组织蛋白质组学比较与验证
本部分的研究目的是比较血清CEA~-CRC和CEA~+CRC与正常粘膜组织(normal mucosa, NM)之间差异表达蛋白,验证部分有差异表达的蛋白质,试图寻找潜在的候选标志物。
结果显示,在筛出的57个差异蛋白质中,大多数细胞功能定位在胞内,生物学过程主要体现为调控和代谢,分子功能主要参与结合。许多差异蛋白之间有联系,尤其是差异蛋白TTN、PRDX1、TAGLN、TP11等,处于蛋白相互作用网络的中心枢纽节点上。采用Western Blot (WB)和免疫组织化学染色验证差异蛋白Annexin A5和Trangelin表达,它们趋势与质谱鉴定信息一致。采用荧光定量PCR验证Lumican蛋白对应基因,mRNA表达水平,结果与质谱信息不一致。
本研究说明,iTRAQ定量标记是寻找CRC有效分子标志物的重要方法,但需要验证确认。本实验筛选出一组与CEA相关组织差异蛋白,其中有些可能是CRC潜在的分子标志物。
第三部分CEA阴性和阳性结直肠癌的分泌蛋白质组学比较与验证
本部分的研究目的是比较血清CEA~-CRC、CEA~+CRC与NM微小组织块培养上清之间差异分泌表达蛋白,验证部分有差异表达的蛋白质,试图寻找可能在血清中出现的的潜在候选标志物。
结果显示,在筛出的61个差异蛋白质中,总共有50.82%(31／61)是分泌蛋白,其中有一些是未见报道与肿瘤相关的分泌差异蛋白,Western Blot验证差异蛋白Annexin A5和Trangelin,结果显示它们表达趋势与质谱鉴定信息基本相似,但分泌表达模式与组织表达模式存在分离现象。
本研究说明,分泌蛋白组学是组织蛋白组学的有益补充；本实验筛选出一组与CEA相关的分泌差异蛋白,可能是CRC潜在的血清分子标志物。
第四部分AnnexinA5和Lumican在结直肠癌的表达和意义
本部分的研究目的是扩大样本研究Annexin A5和Lumican在结直肠癌的表达和意义,为下一步进行功能研究进行初步筛选。
结果显示,Annexin A5在CRC阳性率为40%(20／50),明显高于正常组织,其表达与CRC分期、转移有关。Lumican在结直肠癌呈高表达,与病理参数无关。
本研究说明,Annexin A5和Lumican是结直肠癌的重要候选分子标志物,具体的作用机制还有待深入研究。
结论
1.CEA血清阳性率不高,缺乏敏感性,CEA阴性结直肠癌和CEA阳性结直肠癌在临床和流行病学上确实有一定的差异。
2. iTRAQ定量标记是寻找CRC有效分子标志物的重要方法,但需要验证确认。组织蛋白组学筛选出57个与CEA相关差异蛋白,分泌蛋白组学选出61个与CEA相关差异蛋白,两组差异蛋白有较多重复,但差异蛋白的组织表达模式与分泌表达模式可以不一致。本组筛选出的差异蛋白,有些可能CRC潜在分子标志物。
3.改良Alamar blue法是检测组织块活力灵敏有效的新型检测方法之-
4. AnnexinA5和Lumican表达与结直肠癌关系密切,是结直肠癌的重要分子标志物之一。
创新点：
1. iTRAQ定量标记技术应用在结直肠癌分泌蛋白组学研究。
2.以血清CEA表达状态为切入点分组,寻找CRC潜在分子标志物,符合临床实际需求。
3.设计单位质量吸光度评价指标,把Alamar blue法首次应用在组织块活力评价上。
Colorectal carcinoma (CRC) is one of the three common malignant tumors in the world, and ranks fourth in cancer mortality in our country. At present, the incidence rate of CRC is increasing, but early diagnosis level of CRC is poor and majority of CRC belong to the aggressive-phase. carcinoembryonic antigen (CEA) is the most widely used serum marker,but its sensitivity and specificity are far from satisfying. The CEA-negative CRC is more short of serodiagnosis and monitoring marker than the CEA'positive CRC. So, it is urgent to screen for new tumor biomarkers to improve CRC diagnosis. Now, the new tagging technique such as isobaric tag for relative and absolute quantitation (iTRAQ), followed by LC and MS/MS analysis is one of the more powerful methods in the search for disease biomarkers. In this study, CRC is researched in order to screen some worthy tumor biomarkers.
Part One Clinicopathologic and epidemiologic analysis of 1696 cases of the colorectal carcinoma related with preoperative carcinoembryonic antigen
This section aimed to understand the differences between CEA-negative CRC group and CEA-positive group, and the positive expression ratio of CEA by exploring the clinicopathologic and epidemiologic characteristics of 1969 cases CRC.
The result manifested the positive expression ratio of CEA was 38.04 percent. The mean age in the CEA-negative CRC was younger than in the CEA-positive one, and the median age in the CEA-negative CRC was ahead 3 years of that. The negative CEA group patients were younger than 60 years of age. The blood group A ratio was higher in the negative CEA group than in the positive CEA group, and the blood group B one was lower in the negative CEA group than that. The concomitant ratios of type 2 diabetes and colon obstruction in the negative CEA group, apparently lower than the positive CEA group.
This study showed CEA had poor sensitivity for the detection of some CRC, and there really had some differences between the negative CEA group and the positive CEA group, which was the basement of next research.
Part Two The analysis and verification of tissue proteomics between the CEA-negative CRC and CEA-positive CRC.
This section aimed to screen some potential biomarkers of CRC by comparing the differential proteins in the CEA-negative CRC and CEA-positive CRC to the normal mucosa.
Totally 57 proteins were finally identified. Most proteins was intracellular component. The biological process reflected regulation and metabolism. Molecular function mostly participated binding. There had been connected among many differential proteins, especially such as TTN、PRDX1、TAGLN and TP11. These proteins were put in the central key position of network. Annexin A5 and trangelin were chose for identification by western blot and immunohistochemistry stain, lumican was validated by fluorescence quantitative PCR, and majority were proved to be line with the iTRAQ results. Our results proved that iTRAQ results were reliability.
This study showed iTRAQ technique can be as effective manner to screen the differential expressed protein, but it was needed to validate. Some of the differential proteins were probably potential markers of CRC.
Part Three The analysis and verification of secretome between the CEA-negative CRC and CEA-positive CRC.
This section aimed to screen some potential serum biomarkers of CRC by comparing the differential proteins in the CEA-negative CRC supernatant and CEA-positive CRC supernatant to the normal mucosa supernatant.
Totally 61 proteins were finally identified. The secretory proteins ratio were 50.82%by bioinformatics. Some secretory proteins had not been mentioned in tumor in the past records. Annexin A5 and trangelin were chose for identification by western blot, and were proved to be basically similar with the iTRAQ results. But the secretory expression model was probably not line with the tissue expression model.
This study showed the secretome of CRC was a benefical supplement to the tissue proteomics. These differential secretory proteins were probably potential serum biomarkers of CRC.
Part Four The study and significance of annexin A5 and lumican expression in CRC
This section aimed to explore in enlarged sample the annexin A5 and lumican expression in CRC and their significance.
The result showed the expression level of annexin A5 and Lumican in CRC were markedly higher than one in normal mucosa. Annexin A5 expression was related with CRC stage and metastasis, but Lumican expression had no this relationship.
This study manifested annexin A5 and Lumican were one of important candidate biomarkers in CRC.
引文
[1]Gao Y, Pan X, Su T, et al. Glutathione S-transferase P1 Ile105Val polymer-phism and colorectal cancer risk:a meta-analysis and HuGE review.Eur J Cancer.2009;45(18):3303-3314.
[2]袁庶强,周志伟,万德森,等。 癌胚抗原半衰期在术前癌胚抗原升高的结直肠癌患者中判断预后的作用[J]。癌症,2008,27(6)：612-617.
[3]Hill LB, O'Connell JB, Ko CY. Colorectal cancer:epidemiology and health services research. Surg Oncol ClinN Am[J].2006;15(1):21-37.
[4]Beydoun HA, Beydoun MA. Predictors of colorectal cancer screening behaviors among average-risk older adults in the United States[J]. Cancer Causes Control.2008;19(4):339-359.
[5]Henry KA, Niu X, Boscoe FP. Geographic disparities in colorectal cancer survival[J]. Int J Health Geogr.2009;8:48.
[6]Edwards BK, Ward E, Kohler BA, et al. Annual report to the nation on the status of cancer,1975-2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates[J]. Cancer.2010;116(3):544-573.
[7]Tas F, Keskin S. Age-specific incidence ratios of colorectal cancer (CRC) in Turkey:CRC in older people is increasing[J]. Arch Gerontol Geriatr. 2011;19.
[8]中国抗癌协会大肠癌专业委员会.中国结直肠癌性别、年龄和部位变化趋势—19家医院31246例初步分析.2009欧亚结直肠外科会议论文汇编：14-19.
[9]曹卡加,马国胜,刘奕龙,等.广州市2000—2002年大肠癌的发病率分析[J].癌症,2009,28(4)：441.
[10]李泓澜,高玉堂,郑莹,等.上海市区居民1973—2005年结直肠癌发病趋势 分析[J].中华预防医学杂志,2009,43(10)：875-879.
[11]Risch HA, Yu H, Lu L, et al. ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer:a case-control study [J]. J Natl Cancer Inst.2010;102(7):502-505.
[12]Greer JB, Yazer MH, Raval JS, et al. Significant association between ABO blood group and pancreatic cancer[J]. World J Gastroenterol. 2010;16(44):5588-5591.
[13]Xie J, Qureshi AA, Li Y, et al. ABO blood group and incidence of skin cancer[J]. PLoS One.2010;5(8):e 11972.
[14]Qiu MZ, Zhang DS, Ruan DY, et al.A relationship between ABO blood groups and clinicopathologic characteristics of patients with gastric adeno-carcinoma in China[J]. Med Oncol.2011; 28 Suppl 1:S268-273.
[15]Khalili H, Wolpin BM, Huang ES, et al. ABO blood group and risk of colorectal cancer[J]. Cancer Epidemiol Biomarkers Prev.2011;20(5):1017-1020.
[16]Wirth T, Soeth E, Czubayko F, et al. Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth [J]. Clin Exp Metastasis. 2002; 19(2):155-160.
[17]Li Y, Cao H, Jiao Z, et al. Carcinoembryonic antigen interacts with TGF-｛beta｝ receptor and inhibits TGF-｛beta｝ signaling in colorectal cancers. Cancer Res[J].2010;70(20):8159-8168.
[18]Lee MS, Hsu CC, Wahlqvist ML, et al. Type 2 diabetes increases and metformin reduces total, colorectal, liver and pancreatic cancer incidences in Taiwanese:a representative population prospective cohort study of 800,000 individuals[J]. BMC Cancer.2011;11:20.
[19]田小平,徐宁,惠媛,等。DM2患者HbAlC与肿瘤标志物的相关性分析[J].放射免疫学杂志,2010,23(2)：195-197.
[1]Cao Y, Tan A, Gao F, et al. Alysis of randomized controlled trials comparing chemotherapy plus bevacizumab with chemotherapy alone in metastatic colorectal cancer[J]. It J Colorectal Dis.2009;24(6):677-685.
[2]Winawer S, Fletcher R, Rex D et al., Colorectal cancer screening and surveillance:clinical guidelines and rationale-Update based on new evidence[J]. Gastroenterology,2003.124(2):544-560.
[3]Walsh JM, Terdiman JP. Colorectal cancer screening:scientific review[J]. JAMA.2003;289(10):1288-1296.
[4]Niu D, Sui J, Zhang J, et al. iTRAQ-coupled 2-D LC-MS/MS analysis of protein profile associated with HBV-modulated DNA methylation[J]. Proteomics,2009,9(15):3856-3868.
[5]Feng H, Wang M, Chen W N. iTRAQ-Coupled 2D LC-MS/MS Analysis of Secreted Proteome of HBV-Replicating HepG2 Cells:Potential in Biomarkers for Prognosis of HCC[J].Curr Microbiol,2010.
[6]Ho J, Kong J W, Choong L Y, et al. Novel breast cancer metastasis-associated proteins[J].J Proteome Res,2009,8(2):583-594.
[7]Li SL, Ye F, Cai WJ, et al.Quantitative proteome analysis of multidrug resistance in human ovarian cancer cell line[J].J Cell Biochem.2010; 109(4): 625-633.
[8]Xiao Z, Li G, Chen Y, et al.Quantitative proteomic analysis of formalin-fixed and paraffin-embedded nasopharyngeal carcinoma using iTRAQ labeling, two-dimensional liquid chromatography, and tandem mass spectrometry[J].J Histochem Cytochem.2010;58(6):517-527.
[9]Fan NJ, Gao CF, Wang CS, et al.Discovery and verification of gelsolin as a potential biomarker of colorectal adenocarcinoma in the Chinese population: Examining differential protein expression using an iTRAQ labelling-based proteomics approach. Can J Gastroenterol.2012;26(1):41-47.
[10]Besson D, Pavageau AH, Valo I, et al. A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker.Mol Cell Proteomics. 2011;10(12):M111.009712.
[11]Ghosh D, Yu H, Tan XF, et al.Identification of key players for colorectal cancer metastasis by iTRAQ quantitative proteomics profiling of isogenic SW480 and SW620 cell lines.J Proteome Res.2011;10(10):4373-4387.
[12]Tan HT, Lim TK, Chung MC, et al. iTRAQ~TM labeling coupled with LC-MALDI mass spectrometry for monitoring temporal response of colorectal cancer cells to butyrate treatment.Methods Mol Biol. 2011;716:207-224.
[13]Fan CW, Chan CC, Chen KT, et al. Identification of SEC61βand its autoantibody as biomarkers for colorectal cancer.Clin Chim Acta. 2011;412(11-12):887-893.
[14]Ali NA, McKay MJ, Molloy MP. Proteomics of Smad4 regulated transforming growth factor-beta signalling in colon cancer cells.Mol Biosyst. 2010;6(11):2332-2338.
[15]Chen JS, Chen KT, Fan CW, et al.Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry.FEBS J. 2010;277(14):3028-3038.
[16]Tan HT, Tan S, Lin Q, et al.Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.Mol Cell Proteomics. 2008;7(6):1174-1185.
[17]Chen WT, Chang SC, Ke TW, et al.Identification of biomarkers to improve diagnostic sensitivity of sporadic colorectal cancer in patients with low preoperative serum carcinoembryonic antigen by clinical proteomic analysis. Clin Chim Acta.2011;412(7-8):636-641.
[18]廖存,贺永明,吴留成,等.血清CEA阴性结直肠癌患者血清差异蛋白质的初步筛选。结直肠肛门外科杂志.2011；17(3)：131-136.
[19]Chen WZ, Pang B, Yang B, et al. Differential proteome analysis of conditioned medium of BPH-1 and LNCaP cells. Chin Med J (Engl). 2011;124(22):3806-3809.
[20]Weinkauf M, Zimmermann Y, Hartmann E, et al.2-D PAGE-based comparison of proteasome inhibitor bortezomib in sensitive and resistant mantle cell lymphoma. Electrophoresis.2009;30(6):974-986.
[21]Turashvili G, Bouchal J, Burkadze G, et al.Differentiation of tumours of ductal and lobular origin:II. Genomics of invasive ductal and lobular breast carcinomas. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2005;149(1):63-68.
[22]Hamaguchi T, Iizuka N, Tsunedomi R, et al.Glycolysis module activated by hypoxia-inducible factor 1 alpha is related to the aggressive phenotype of hepatocellular carcinoma. Int J Oncol.2008;33(4):725-731.
[23]黄志勇,熊刚,张军,等。应用二维电泳和质谱技术筛选食管癌及癌旁组织的差异表达蛋白质。南方医科大学学报.2007；27(9)：1406-1409.
[24]Shirahata A, Sakata M, Sakuraba K, et al.Vimentin methylation as a marker for advanced colorectal carcinoma. Anticancer Res.2009;29(1):279-281.
[25]Ngan CY, Yamamoto H, Seshimo I, et al. Quantitative evaluation of vimentin expression in tumour stroma of colorectal cancer. Br J Cancer. 2007;96(6):986-992.
[26]Penuelas S, Noe V, Ciudad CJ. Modulation of IMPDH2, survivin, topoisomerase I and vimentin increases sensitivity to methotrexate in HT29 human colon cancer cells. FEBS J.2005;272(3):696-710.
[27]Ishiwata T, Cho K, Kawahara K, et al. Role of lumican in cancer cells and adjacent stromal tissues in human pancreatic cancer. Oncol Rep.2007; 18(3): 537-543.
[28]Wisniewski JR, Ostasiewicz P, Mann M. High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers. J Proteome Res. 2011;10(7):3040-3049.
[29]Xu BJ, Li J, Beauchamp RD, et al.Identification of early intestinal neoplasia protein biomarkers using laser capture microdissection and MALDI MS. Mol Cell Proteomics.2009;8(5):936-945..
[30]Han CL, Chen JS, Chan EC, et al. An informatics-assisted label-free approach for personalized tissue membrane proteomics:case study on colorectal cancer. Mol Cell Proteomics.2011;10(4):M110.003087.
[31]Bates RC, Mercurio AM.The epithelial-mesenchymal transition (EMT) and colorectal cancer progression. Cancer Biol Ther.2005;4(4):365-370.
[32]Oshiro R, Yamamoto H, Takahashi H, et al.C4.4A is associated with tumor budding and EMT of colorectal cancer. Cancer Sci.2012.doi:10.1111/ j.1349-7006.2012.02263.x.
[33]Matsuo N, Tanaka S, Yoshioka H, et al.Collagen XXIV (Co124al) gene expression is a specific marker of osteoblast differentiation and bone formation. Connect Tissue Res.2008;49(2):68-75.
[34]Takahira T, Oda Y, Tamiya S, et al. Detection of COLlAl-PDGFB fusion transcripts and PDGFB/PDGFRB mRNA expression in dermatofibro- sarcoma protuberans. Mod Pathol.2007;20(6):668-675.
[35]Chakravarti S, Petroll WM, et al. Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma. Invest Ophthalmol Vis Sci.2000;41(11):3365-3373.
[36]Jepsen KJ, Wu F, Peragallo JH, et al. A syndrome of joint laxity and impaired tendon integrity in lumican-and fibromodulin-deficient mice. J Biol Chem.2002;277(38):35532-35540.
[37]Nikitovic D, Berdiaki A, Zafiropoulos A, et al. Lumican expression is positively correlated with the differentiation and negatively with the growth of human osteosarcoma cells FEBS J.2008 Jan;275(2):350-361.
[38]Matsuda Y, Yamamoto T, Kudo M, et al. Expression and roles of lumican in lung adenocarcinoma and squamous cell carcinoma. Int J Oncol.2008; 33(6):1177-1185.
[39]Brezillon S, Radwanska A, Zeltz C, et al. Lumican core protein inhibits melanoma cell migration via alterations of focal adhesion complexes. Cancer Lett.2009;283(1):92-100.
[1]陈功,万德森,侯景辉,等.结肠直肠癌组织CEA表达及其临床生物学意义[J].癌症.2001；20(6)：628-630.
[2]邱春华,欧阳钦.环氧合酶-2选择性抑制剂对大肠腺瘤细胞的作用机制[J].中华内科杂志.2001；40(6)：377-380.
[3]蔡慧云,于波,白雪等.简便人大肠癌原代细胞培养方法的探讨[J].解放军医杂志.2011；39(4)：611-612.
[4]R.弗雷纳,R.工.弗雷谢尼主编.人肿瘤细胞培养[M].北京：化学工业出版社.2006：54-55.
[5]Smoot EC 3rd, Kucan JO, Roth A, et al. In vitro toxicity testing for antibacterials against human keratinocytes[J]. Plast Reconstr Surg.1991; 87(5):917-924.
[6]朱德妹,汪复,胡付品,等.2010年中国CHINET细菌耐药性监测[J].中国感染与化疗杂志.2010；11(5)：321-329.
[7]周曾同,黄吉燕,周晓健.人口腔黏膜异常增生上皮细胞体外培养体系的研究[J].临床口腔医学杂志.2003；19(9)：520-523.
[8]Muller G, Kramer A. Comparative study of in vitro cytotoxicity of povidone-iodine in solution, in ointment or in a liposomal formulation (Repithel) and selected antiseptics[J]. Dermatology.2006;212 Suppl 1: 91-93.
[9]钱毅春,樊树理,尹幸念,等.碘伏消毒液的毒性研究[J].中国消毒学杂志.2004；21(2)：128-129.
[10]Cao J, Shen C, Zhang J, et al. Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells[J]. Sci China Life Sci.2011;54(1):34-38.
[11]Schwarz K, Fiedler T, Fischer RJ, et al. Standard Operating Procedure (SOP) for the preparation of intra-and extracellular proteins of Clostridium acetobutylicum for proteome analysis[J]. J Microbiol Methods.2007;68(2): 396-402.
[12]Klein-Scory S, Kiibler S, Diehl H, et al. Immunoscreening of the extracellular proteome of colorectal cancer cells[J]. BMC Cancer.2010; 10:70.
[13]Xue H, Lii B, Zhang J, et al.Identification of serum biomarkers for colorectal cancer metastasis using a differential secretome approach[J]. J Proteome Res.2010;9(1):545-555.
[14]Stoddart MJ. WST-8 analysis of cell viability during osteogenesis of human[J]. Methods Mol Biol.2011;740:21-25.
[15]Han SB, Shin YJ, Hyon JY,et al. Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells[J]. Antimicrob Agents Chemother.2011; 55(10):4519-4523.
[16]Kilani-Jaziri S, Frachet V, Bhouri W, et al. Flavones inhibit the proliferation of human tumor cancer cell lines by inducing apoptosis[J]. Drug Chem Toxicol.2012;35(1):1-10.
[17]贺旭,伍津津,杨桂红.复方壳多糖组织工程皮肤活力测定的研究[J].重庆医学.2008,37(9)：914-917.
[18]贾晓明,纪晓峰,Yang Hongyou.双重核酸染色法测定皮肤活力的研究[J].中华外科杂志.2002,40(2)：139-141.
[19]Chapkin RS, Hong MY, Fan YY, et al. Dietary n-3 PUFA alter colonocyte mitochondrial membrane composition and function[J]. Lipids.2002;37(2): 193-199.
[20]王通,曾耀英,何贤辉,等.阻断p38途径对地塞米松诱导的胸腺细胞凋亡的影响[J].第三军医大学学报.2006；28(16)：1667-1670.
[21]Hathout Y. Approaches to the study of the cell secretome[J]. Expert Rev Proteomics.2007;4(2):239-248.
[22]Celis JE, Gromov P, Cabezon T, et al. Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment:a novel resource for biomarker and therapeutic target discovery[J]. Mol Cell Proteomics.2004;3(4):327-344.
[23]Wu CC, Chien KY, Tsang NM, et al. Cancer cell-secreted proteomes as a basis for searching potential tumor markers:nasopharyngeal carcinoma as a model[J]. Proteomics.2005;5(12):3173-3182.
[24]Gronborg M, Kristiansen TZ, Iwahori A, et al. Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach[J]. Mol Cell Proteomics.2006;5(1):157-171.
[25]Sardana G, Marshall J, Diamandis EP. Discovery of candidate tumor markers for prostate cancer via proteomic analysis of cell culture-conditioned medium[J]. Clin Chem.2007; 53(3):429-437.
[26]Volmer MW, Stuhler K, Zapatka M, et al. Differential proteome analysis of conditioned media to detect Smad4 regulated secreted biomarkers in colon cance[J]r. Proteomics.2005;5(10):2587-2601.
[27]Rangiah K, Tippornwong M, Sangar V, et al. Differential secreted proteome approach in murine model for candidate biomarker discovery in colon cancer[J]. J Proteome Res.2009;8(11):5153-5164.
[28]Xue H, Lii B, Zhang J, et al. Identification of serum biomarkers for colorectal cancer metastasis using a differential secretome approach[J]. J Proteome Res.2010;9(1):545-555.
[29]Zwickl H, Traxler E, Staettner S, et al. A novel technique to specifically analyze the secretome of cells and tissues[J]. Electrophoresis.2005; 26(14):2779-2785.
[30]Mbeunkui F, Fodstad O, Pannell LK. Secretory protein enrichment and analysis:an optimized approach applied on cancer cell lines using 2D LC-MS/MS[J]. J Proteome Res.2006;5(4):899-906.
[31]Huang CM, Ananthaswamy HN, Barnes S, et al. Mass spectrometric proteomics profiles of in vivo tumor secretomes:capillary ultrafiltration sampling of regressive tumor masses[J]. Proteomics.2006;6(22): 6107-6116.
[32]Cao J, Hu Y, Shen C, et al. Nanozeolite-driven approach for enrichment of secretory proteins in human hepatocellular carcinoma cells[J]. Proteomics. 2009;9(21):4881-4888.
[33]Fan NJ, Gao CF, Wang CS, et al. Discovery and verification of gelsolin as a potential biomarker of colorectal adenocarcinoma in the Chinese population:Examining differential protein expression using an iTRAQ labelling-based proteomics approach[J]. Can J Gastroenterol.2012;26(1): 41-47.
[34]Tsai MH, Wu CC, Peng PH, et al. Identification of secretory gelsolin as a plasma biomarker associated with distant organ metastasis of colorectal cancer[J]. J Mol Med (Berl).2012; 90(2):187-200.
[35]Pike SE, Yao L, Jones KD, et al. Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth[J]. J Exp Med.1998;188(12): 2349-2356.
[36]Toquet C, Jarry A, Bou-Hanna C, et al. Altered Calreticulin expression in human colon cancer:maintenance of Calreticulin expression is associated with mucinous differentiation [J]. Oncol Rep.2007; 17(5):1101-1107.
[37]Liu M, Imam H, Oberg K, et al. Gene transfer of vasostatin, a calreticulin fragment, into neuroendocrine tumor cells results in enhanced malignant behavior[J]. Neuroendocrinology.2005;82(1):1-10.
[38]李里,彭佳远,马延磊,等.Transgelin蛋白在人结直肠癌中的表达及其临床意义[J].肿瘤.2009；29(8)：765-771.
[1]Vangestel C, Van de Wiele C, Van Damme N, et al. (99)mTc-(CO)(3) His-annexin A5 micro-SPECT demonstrates increased cell death by irinotecan during the vascular normalization window caused by bevacizumab[J]. JNucl Med.2011;52(11):1786-1794.
[2]Mattes MJ, Michel RB, Goldenberg DM, et al. Induction of apoptosis by cross-linking antibodies bound to human B-lymphoma cells:expression of Annexin Ⅴ binding sites on the antibody cap[J]. Cancer Biother Radiopharm. 2009;24(2):185-193.
[3]Rand JH, Wu XX, Quinn AS, et al. Resistance to annexin A5 anticoagulant activity:a thrombogenic mechanism for the antiphospholipid syndrome[J]. Lupus.2008;17(10):922-930.
[4]Rand JH. Antiphospholipid antibody-mediated disruption of the annexin-Ⅴ antithrombotic shield:a thrombogenic mechanism for the antiphospholipid syndrome[J].J Autoimmun.2000; 15(2):107-111.
[5]Rand JH, Wu XX, Quinn AS,et al. Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug[J]. Blood. 2010;115(11):2292-2299.
[6]辛虹,王惠兰.膜联蛋白V在孕妇外周血及胎盘组织中的表达及其与子痫前期发病的关系[J].中华妇产科杂志.2011；46(2)：88-93.
[7]Wahezi DM, Ilowite NT, Rajpathak S, et al. Prevalence of annexin A5 resistance in children and adolescents with rheumatic disease[J]s. J Rheumatol.2012;39(2):382-388.
[8]TP, Reddy SP, Wilborn TW, et al. Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR[J]. Biochem Biophys Res Commun.2003;306(4):1026-1036.
[9]Wehder L, Arndt S, Murzik U, et al. Annexin A5 is involved in migration and invasion of oral carcinoma[J]. Cell Cycle.2009;8(10):1552-1558.
[10]李岭,庄英帜.Annexin A5表达与鼻咽癌分化转移的关系[J].肿瘤防治研究.2011；38(4)：389-393.
[11]李欣,王立平,梁秀军,等.宫颈鳞癌患者血清膜联蛋白A5含量的检测及其临床意义[J].解剖学报.2012；43(1)：68-71.
[12]Xue G, Hao LQ, Ding FX, et al. Expression of annexin a5 is associated with higher tumor stage and poor prognosis in colorectal adenocarcinomas[J]. J Clin Gastroenterol.2009;43(9):831-837.
[13]Horisberger K, Erben P, Strobel P, et al.Annexin and survivin in locally advanced rectal cancer:indicators of resistance to preoperative chemoradiotherapy?[J] Onkologie.2010;33(8-9):439-444.
[14]Brezillon S, Radwanska A, Zeltz C, et al.Lumican core protein inhibits melanoma cell migration via alterations of focal adhesion complexes[J]. Cancer Lett.2009;283(1):92-100.
[15]Ishiwata T, Cho K, Kawahara K, et al.Role of lumican in cancer cells and adjacent stromal tissues in human pancreatic cancer[J]. Oncol Rep. 2007;18(3):537-543.
[16]Matsuda Y, Yamamoto T, KUDO M, et al. Expression and roles of lumican in lung adenocarcinoma and squamous cell carcinoma[J]. Int J Oncol.2008; 33(6):1177-1185.
[17]宋洪江,盂祥宁,佟静,等.Lumican基因在结直肠癌中表达的相关研究[J].国际遗传学杂志.2007；30(6)：416-418.
[18]宋洪江,蓝秀文,魏玉哲,等.Lumican基因在大肠癌中的表达及其与预后的关系[J].国际遗传学杂志.2010；33(5)：268-271.
[1]焦丽燕,应万涛,蔡耘,等.肿瘤研究中的蛋白质组学研究策略[J].肿瘤防治研究.2005,32(4)：245-247.
[2]Chaerkady R, Harsha H C, Nalli A, et al. A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma[J]. J Proteome Res,2008,7(10):4289-4298.
[3]Zhang J, Niu D, Sui J, et al. Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ-coupled 2-D LC-MS/MS analysis:Insights on liver angiogenesis[J]. Proteomics,2009,9(10):2836-2845.
[4]Niu D, Sui J, Zhang J, et al. iTRAQ-coupled 2-D LC-MS/MS analysis of protein profile associated with HBV-modulated DNA methylation[J]. Proteomics,2009,9(15):3856-3868.
[5]Feng H, Wang M, Chen W N. iTRAQ-Coupled 2D LC-MS/MS Analysis of Secreted Proteome of HBV-Replicating HepG2 Cells:Potential in Biomark-ers for Prognosis of HCC[J].Curr Microbiol,2010,61(4):280-284.
[6]Lee H J, Na K, Choi E Y, et al. Simple method for quantitative analysis of N-linked glycoproteins in hepatocellular carcinoma specimens[J].J Proteome Res,2010,9(1):308-318.
[7]Tan H T, Tan S, Lin Q, et al. Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells[J].Mol Cell Proteomics.2008,7(6): 1174-1185.
[8]Deng L, Su T, Leng A, et al. Upregulation of soluble resistance-related calcium-binding protein (sorcin) in gastric cancer[J].Med Oncol,2010; 27(4):1102-1108.
[9]Chen Y, Choong L Y, Lin Q, et al. Differential expression of novel tyrosine kinase substrates during breast cancer development[J].Mol Cell Proteomics, 2007,6(12):2072-2087.
[10]Lim S, Choong L Y, Kuan C P, et al. Regulation of macrophage inhibitory factor (MIF) by epidermal growth factor receptor (EGFR) in the MCF10AT model of breast cancer progression[J]J Proteome Res,2009,8(8):4062-4076.
[11]朱镭,倪国新,张政希,等.用于相对和绝对定量的等量异位标签-二维液相色谱-串联质谱法分析雌二醇和血清剥夺对乳腺癌细胞内蛋白质含量的影响[J].色谱.2009,27(3)：270-278.
[12]倪国新,郭立海,朱镭,等.基于四重iTRAQ标记结合LC-MALDI-TOF-TOF方法分析雌二醇和它莫西芬对MCF-7乳腺癌细胞内蛋白质含量影响的研究[J].分析测试学报.2008,27(12)：1283-1287.
[13]Armenta J M, Hoeschele I, Lazar I M. Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology [J]. J Am Soc Mass Spectrom,2009,20(7):1287-1302.
[14]Ho J, Kong J W, Choong L Y, et al. Novel breast cancer metastasis-associated proteins[J].J Proteome Res,2009,8(2):583-594.
[15]Bouchal P, Roumeliotis T, Hrstka R, et al. Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis[J].J Proteome Res,2009,8(1):362-373.
[16]Smith L, Qutob O, Watson M B, et al. Proteomic identification of putative biomarkers of radiotherapy resistance:a possible role for the 26S proteaso-me?[J].Neoplasia,2009,11(11):1194-1207.
[17]Streckfus C F, Mayorga-Wark O, Arreola D, et al. Breast cancer related proteins are present in saliva and are modulated secondary to ductal carcino-ma in situ of the breast[J].Cancer Invest,2008,26(2):159-167.
[18]Streckfus C F, Storthz K A, Bigler L, et al. A Comparison of the Proteomic Expression in Pooled Saliva Specimens from Individuals Diagnos ed with Ductal Carcinoma of the Breast with and without Lymph Node Involvement [J].J Oncol,2009:737619.
[19]Wu W W, Wang G, Baek S J, et al. Comparative study of three proteom-ic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF[J].J Proteome Res,2006,5(3):651-658.
[20]Goshe M B, Smith R D. Stable isotope-coded proteomic mass spectrometry [J].Curr Opin Biotechnol,2003,14(1):101-109.
[21]Tao W A, Aebersold R. Advances in quantitative proteomics via stable isotope tagging and mass spectrometry[J].Curr Opin Biotechnol,2003,14(1): 110-118.
[2]袁庶强,周志伟,万德森,等。 癌胚抗原半衰期在术前癌胚抗原升高的结直肠癌患者中判断预后的作用[J]。癌症,2008,27(6)：612-617.
[3]Hill LB, O'Connell JB, Ko CY. Colorectal cancer:epidemiology and health services research. Surg Oncol ClinN Am[J].2006;15(1):21-37.
[4]Beydoun HA, Beydoun MA. Predictors of colorectal cancer screening behaviors among average-risk older adults in the United States[J]. Cancer Causes Control.2008;19(4):339-359.
[5]Henry KA, Niu X, Boscoe FP. Geographic disparities in colorectal cancer survival[J]. Int J Health Geogr.2009;8:48.
[6]Edwards BK, Ward E, Kohler BA, et al. Annual report to the nation on the status of cancer,1975-2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates[J]. Cancer.2010;116(3):544-573.
[7]Tas F, Keskin S. Age-specific incidence ratios of colorectal cancer (CRC) in Turkey:CRC in older people is increasing[J]. Arch Gerontol Geriatr. 2011;19.
[8]中国抗癌协会大肠癌专业委员会.中国结直肠癌性别、年龄和部位变化趋势—19家医院31246例初步分析.2009欧亚结直肠外科会议论文汇编：14-19.
[9]曹卡加,马国胜,刘奕龙,等.广州市2000—2002年大肠癌的发病率分析[J].癌症,2009,28(4)：441.
[10]李泓澜,高玉堂,郑莹,等.上海市区居民1973—2005年结直肠癌发病趋势 分析[J].中华预防医学杂志,2009,43(10)：875-879.
[11]Risch HA, Yu H, Lu L, et al. ABO blood group, Helicobacter pylori seropositivity, and risk of pancreatic cancer:a case-control study [J]. J Natl Cancer Inst.2010;102(7):502-505.
[12]Greer JB, Yazer MH, Raval JS, et al. Significant association between ABO blood group and pancreatic cancer[J]. World J Gastroenterol. 2010;16(44):5588-5591.
[13]Xie J, Qureshi AA, Li Y, et al. ABO blood group and incidence of skin cancer[J]. PLoS One.2010;5(8):e 11972.
[14]Qiu MZ, Zhang DS, Ruan DY, et al.A relationship between ABO blood groups and clinicopathologic characteristics of patients with gastric adeno-carcinoma in China[J]. Med Oncol.2011; 28 Suppl 1:S268-273.
[15]Khalili H, Wolpin BM, Huang ES, et al. ABO blood group and risk of colorectal cancer[J]. Cancer Epidemiol Biomarkers Prev.2011;20(5):1017-1020.
[16]Wirth T, Soeth E, Czubayko F, et al. Inhibition of endogenous carcinoembryonic antigen (CEA) increases the apoptotic rate of colon cancer cells and inhibits metastatic tumor growth [J]. Clin Exp Metastasis. 2002; 19(2):155-160.
[17]Li Y, Cao H, Jiao Z, et al. Carcinoembryonic antigen interacts with TGF-｛beta｝ receptor and inhibits TGF-｛beta｝ signaling in colorectal cancers. Cancer Res[J].2010;70(20):8159-8168.
[18]Lee MS, Hsu CC, Wahlqvist ML, et al. Type 2 diabetes increases and metformin reduces total, colorectal, liver and pancreatic cancer incidences in Taiwanese:a representative population prospective cohort study of 800,000 individuals[J]. BMC Cancer.2011;11:20.
[19]田小平,徐宁,惠媛,等。DM2患者HbAlC与肿瘤标志物的相关性分析[J].放射免疫学杂志,2010,23(2)：195-197.
[1]Cao Y, Tan A, Gao F, et al. Alysis of randomized controlled trials comparing chemotherapy plus bevacizumab with chemotherapy alone in metastatic colorectal cancer[J]. It J Colorectal Dis.2009;24(6):677-685.
[2]Winawer S, Fletcher R, Rex D et al., Colorectal cancer screening and surveillance:clinical guidelines and rationale-Update based on new evidence[J]. Gastroenterology,2003.124(2):544-560.
[3]Walsh JM, Terdiman JP. Colorectal cancer screening:scientific review[J]. JAMA.2003;289(10):1288-1296.
[4]Niu D, Sui J, Zhang J, et al. iTRAQ-coupled 2-D LC-MS/MS analysis of protein profile associated with HBV-modulated DNA methylation[J]. Proteomics,2009,9(15):3856-3868.
[5]Feng H, Wang M, Chen W N. iTRAQ-Coupled 2D LC-MS/MS Analysis of Secreted Proteome of HBV-Replicating HepG2 Cells:Potential in Biomarkers for Prognosis of HCC[J].Curr Microbiol,2010.
[6]Ho J, Kong J W, Choong L Y, et al. Novel breast cancer metastasis-associated proteins[J].J Proteome Res,2009,8(2):583-594.
[7]Li SL, Ye F, Cai WJ, et al.Quantitative proteome analysis of multidrug resistance in human ovarian cancer cell line[J].J Cell Biochem.2010; 109(4): 625-633.
[8]Xiao Z, Li G, Chen Y, et al.Quantitative proteomic analysis of formalin-fixed and paraffin-embedded nasopharyngeal carcinoma using iTRAQ labeling, two-dimensional liquid chromatography, and tandem mass spectrometry[J].J Histochem Cytochem.2010;58(6):517-527.
[9]Fan NJ, Gao CF, Wang CS, et al.Discovery and verification of gelsolin as a potential biomarker of colorectal adenocarcinoma in the Chinese population: Examining differential protein expression using an iTRAQ labelling-based proteomics approach. Can J Gastroenterol.2012;26(1):41-47.
[10]Besson D, Pavageau AH, Valo I, et al. A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker.Mol Cell Proteomics. 2011;10(12):M111.009712.
[11]Ghosh D, Yu H, Tan XF, et al.Identification of key players for colorectal cancer metastasis by iTRAQ quantitative proteomics profiling of isogenic SW480 and SW620 cell lines.J Proteome Res.2011;10(10):4373-4387.
[12]Tan HT, Lim TK, Chung MC, et al. iTRAQ~TM labeling coupled with LC-MALDI mass spectrometry for monitoring temporal response of colorectal cancer cells to butyrate treatment.Methods Mol Biol. 2011;716:207-224.
[13]Fan CW, Chan CC, Chen KT, et al. Identification of SEC61βand its autoantibody as biomarkers for colorectal cancer.Clin Chim Acta. 2011;412(11-12):887-893.
[14]Ali NA, McKay MJ, Molloy MP. Proteomics of Smad4 regulated transforming growth factor-beta signalling in colon cancer cells.Mol Biosyst. 2010;6(11):2332-2338.
[15]Chen JS, Chen KT, Fan CW, et al.Comparison of membrane fraction proteomic profiles of normal and cancerous human colorectal tissues with gel-assisted digestion and iTRAQ labeling mass spectrometry.FEBS J. 2010;277(14):3028-3038.
[16]Tan HT, Tan S, Lin Q, et al.Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.Mol Cell Proteomics. 2008;7(6):1174-1185.
[17]Chen WT, Chang SC, Ke TW, et al.Identification of biomarkers to improve diagnostic sensitivity of sporadic colorectal cancer in patients with low preoperative serum carcinoembryonic antigen by clinical proteomic analysis. Clin Chim Acta.2011;412(7-8):636-641.
[18]廖存,贺永明,吴留成,等.血清CEA阴性结直肠癌患者血清差异蛋白质的初步筛选。结直肠肛门外科杂志.2011；17(3)：131-136.
[19]Chen WZ, Pang B, Yang B, et al. Differential proteome analysis of conditioned medium of BPH-1 and LNCaP cells. Chin Med J (Engl). 2011;124(22):3806-3809.
[20]Weinkauf M, Zimmermann Y, Hartmann E, et al.2-D PAGE-based comparison of proteasome inhibitor bortezomib in sensitive and resistant mantle cell lymphoma. Electrophoresis.2009;30(6):974-986.
[21]Turashvili G, Bouchal J, Burkadze G, et al.Differentiation of tumours of ductal and lobular origin:II. Genomics of invasive ductal and lobular breast carcinomas. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2005;149(1):63-68.
[22]Hamaguchi T, Iizuka N, Tsunedomi R, et al.Glycolysis module activated by hypoxia-inducible factor 1 alpha is related to the aggressive phenotype of hepatocellular carcinoma. Int J Oncol.2008;33(4):725-731.
[23]黄志勇,熊刚,张军,等。应用二维电泳和质谱技术筛选食管癌及癌旁组织的差异表达蛋白质。南方医科大学学报.2007；27(9)：1406-1409.
[24]Shirahata A, Sakata M, Sakuraba K, et al.Vimentin methylation as a marker for advanced colorectal carcinoma. Anticancer Res.2009;29(1):279-281.
[25]Ngan CY, Yamamoto H, Seshimo I, et al. Quantitative evaluation of vimentin expression in tumour stroma of colorectal cancer. Br J Cancer. 2007;96(6):986-992.
[26]Penuelas S, Noe V, Ciudad CJ. Modulation of IMPDH2, survivin, topoisomerase I and vimentin increases sensitivity to methotrexate in HT29 human colon cancer cells. FEBS J.2005;272(3):696-710.
[27]Ishiwata T, Cho K, Kawahara K, et al. Role of lumican in cancer cells and adjacent stromal tissues in human pancreatic cancer. Oncol Rep.2007; 18(3): 537-543.
[28]Wisniewski JR, Ostasiewicz P, Mann M. High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers. J Proteome Res. 2011;10(7):3040-3049.
[29]Xu BJ, Li J, Beauchamp RD, et al.Identification of early intestinal neoplasia protein biomarkers using laser capture microdissection and MALDI MS. Mol Cell Proteomics.2009;8(5):936-945..
[30]Han CL, Chen JS, Chan EC, et al. An informatics-assisted label-free approach for personalized tissue membrane proteomics:case study on colorectal cancer. Mol Cell Proteomics.2011;10(4):M110.003087.
[31]Bates RC, Mercurio AM.The epithelial-mesenchymal transition (EMT) and colorectal cancer progression. Cancer Biol Ther.2005;4(4):365-370.
[32]Oshiro R, Yamamoto H, Takahashi H, et al.C4.4A is associated with tumor budding and EMT of colorectal cancer. Cancer Sci.2012.doi:10.1111/ j.1349-7006.2012.02263.x.
[33]Matsuo N, Tanaka S, Yoshioka H, et al.Collagen XXIV (Co124al) gene expression is a specific marker of osteoblast differentiation and bone formation. Connect Tissue Res.2008;49(2):68-75.
[34]Takahira T, Oda Y, Tamiya S, et al. Detection of COLlAl-PDGFB fusion transcripts and PDGFB/PDGFRB mRNA expression in dermatofibro- sarcoma protuberans. Mod Pathol.2007;20(6):668-675.
[35]Chakravarti S, Petroll WM, et al. Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma. Invest Ophthalmol Vis Sci.2000;41(11):3365-3373.
[36]Jepsen KJ, Wu F, Peragallo JH, et al. A syndrome of joint laxity and impaired tendon integrity in lumican-and fibromodulin-deficient mice. J Biol Chem.2002;277(38):35532-35540.
[37]Nikitovic D, Berdiaki A, Zafiropoulos A, et al. Lumican expression is positively correlated with the differentiation and negatively with the growth of human osteosarcoma cells FEBS J.2008 Jan;275(2):350-361.
[38]Matsuda Y, Yamamoto T, Kudo M, et al. Expression and roles of lumican in lung adenocarcinoma and squamous cell carcinoma. Int J Oncol.2008; 33(6):1177-1185.
[39]Brezillon S, Radwanska A, Zeltz C, et al. Lumican core protein inhibits melanoma cell migration via alterations of focal adhesion complexes. Cancer Lett.2009;283(1):92-100.
[1]陈功,万德森,侯景辉,等.结肠直肠癌组织CEA表达及其临床生物学意义[J].癌症.2001；20(6)：628-630.
[2]邱春华,欧阳钦.环氧合酶-2选择性抑制剂对大肠腺瘤细胞的作用机制[J].中华内科杂志.2001；40(6)：377-380.
[3]蔡慧云,于波,白雪等.简便人大肠癌原代细胞培养方法的探讨[J].解放军医杂志.2011；39(4)：611-612.
[4]R.弗雷纳,R.工.弗雷谢尼主编.人肿瘤细胞培养[M].北京：化学工业出版社.2006：54-55.
[5]Smoot EC 3rd, Kucan JO, Roth A, et al. In vitro toxicity testing for antibacterials against human keratinocytes[J]. Plast Reconstr Surg.1991; 87(5):917-924.
[6]朱德妹,汪复,胡付品,等.2010年中国CHINET细菌耐药性监测[J].中国感染与化疗杂志.2010；11(5)：321-329.
[7]周曾同,黄吉燕,周晓健.人口腔黏膜异常增生上皮细胞体外培养体系的研究[J].临床口腔医学杂志.2003；19(9)：520-523.
[8]Muller G, Kramer A. Comparative study of in vitro cytotoxicity of povidone-iodine in solution, in ointment or in a liposomal formulation (Repithel) and selected antiseptics[J]. Dermatology.2006;212 Suppl 1: 91-93.
[9]钱毅春,樊树理,尹幸念,等.碘伏消毒液的毒性研究[J].中国消毒学杂志.2004；21(2)：128-129.
[10]Cao J, Shen C, Zhang J, et al. Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells[J]. Sci China Life Sci.2011;54(1):34-38.
[11]Schwarz K, Fiedler T, Fischer RJ, et al. Standard Operating Procedure (SOP) for the preparation of intra-and extracellular proteins of Clostridium acetobutylicum for proteome analysis[J]. J Microbiol Methods.2007;68(2): 396-402.
[12]Klein-Scory S, Kiibler S, Diehl H, et al. Immunoscreening of the extracellular proteome of colorectal cancer cells[J]. BMC Cancer.2010; 10:70.
[13]Xue H, Lii B, Zhang J, et al.Identification of serum biomarkers for colorectal cancer metastasis using a differential secretome approach[J]. J Proteome Res.2010;9(1):545-555.
[14]Stoddart MJ. WST-8 analysis of cell viability during osteogenesis of human[J]. Methods Mol Biol.2011;740:21-25.
[15]Han SB, Shin YJ, Hyon JY,et al. Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells[J]. Antimicrob Agents Chemother.2011; 55(10):4519-4523.
[16]Kilani-Jaziri S, Frachet V, Bhouri W, et al. Flavones inhibit the proliferation of human tumor cancer cell lines by inducing apoptosis[J]. Drug Chem Toxicol.2012;35(1):1-10.
[17]贺旭,伍津津,杨桂红.复方壳多糖组织工程皮肤活力测定的研究[J].重庆医学.2008,37(9)：914-917.
[18]贾晓明,纪晓峰,Yang Hongyou.双重核酸染色法测定皮肤活力的研究[J].中华外科杂志.2002,40(2)：139-141.
[19]Chapkin RS, Hong MY, Fan YY, et al. Dietary n-3 PUFA alter colonocyte mitochondrial membrane composition and function[J]. Lipids.2002;37(2): 193-199.
[20]王通,曾耀英,何贤辉,等.阻断p38途径对地塞米松诱导的胸腺细胞凋亡的影响[J].第三军医大学学报.2006；28(16)：1667-1670.
[21]Hathout Y. Approaches to the study of the cell secretome[J]. Expert Rev Proteomics.2007;4(2):239-248.
[22]Celis JE, Gromov P, Cabezon T, et al. Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment:a novel resource for biomarker and therapeutic target discovery[J]. Mol Cell Proteomics.2004;3(4):327-344.
[23]Wu CC, Chien KY, Tsang NM, et al. Cancer cell-secreted proteomes as a basis for searching potential tumor markers:nasopharyngeal carcinoma as a model[J]. Proteomics.2005;5(12):3173-3182.
[24]Gronborg M, Kristiansen TZ, Iwahori A, et al. Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach[J]. Mol Cell Proteomics.2006;5(1):157-171.
[25]Sardana G, Marshall J, Diamandis EP. Discovery of candidate tumor markers for prostate cancer via proteomic analysis of cell culture-conditioned medium[J]. Clin Chem.2007; 53(3):429-437.
[26]Volmer MW, Stuhler K, Zapatka M, et al. Differential proteome analysis of conditioned media to detect Smad4 regulated secreted biomarkers in colon cance[J]r. Proteomics.2005;5(10):2587-2601.
[27]Rangiah K, Tippornwong M, Sangar V, et al. Differential secreted proteome approach in murine model for candidate biomarker discovery in colon cancer[J]. J Proteome Res.2009;8(11):5153-5164.
[28]Xue H, Lii B, Zhang J, et al. Identification of serum biomarkers for colorectal cancer metastasis using a differential secretome approach[J]. J Proteome Res.2010;9(1):545-555.
[29]Zwickl H, Traxler E, Staettner S, et al. A novel technique to specifically analyze the secretome of cells and tissues[J]. Electrophoresis.2005; 26(14):2779-2785.
[30]Mbeunkui F, Fodstad O, Pannell LK. Secretory protein enrichment and analysis:an optimized approach applied on cancer cell lines using 2D LC-MS/MS[J]. J Proteome Res.2006;5(4):899-906.
[31]Huang CM, Ananthaswamy HN, Barnes S, et al. Mass spectrometric proteomics profiles of in vivo tumor secretomes:capillary ultrafiltration sampling of regressive tumor masses[J]. Proteomics.2006;6(22): 6107-6116.
[32]Cao J, Hu Y, Shen C, et al. Nanozeolite-driven approach for enrichment of secretory proteins in human hepatocellular carcinoma cells[J]. Proteomics. 2009;9(21):4881-4888.
[33]Fan NJ, Gao CF, Wang CS, et al. Discovery and verification of gelsolin as a potential biomarker of colorectal adenocarcinoma in the Chinese population:Examining differential protein expression using an iTRAQ labelling-based proteomics approach[J]. Can J Gastroenterol.2012;26(1): 41-47.
[34]Tsai MH, Wu CC, Peng PH, et al. Identification of secretory gelsolin as a plasma biomarker associated with distant organ metastasis of colorectal cancer[J]. J Mol Med (Berl).2012; 90(2):187-200.
[35]Pike SE, Yao L, Jones KD, et al. Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth[J]. J Exp Med.1998;188(12): 2349-2356.
[36]Toquet C, Jarry A, Bou-Hanna C, et al. Altered Calreticulin expression in human colon cancer:maintenance of Calreticulin expression is associated with mucinous differentiation [J]. Oncol Rep.2007; 17(5):1101-1107.
[37]Liu M, Imam H, Oberg K, et al. Gene transfer of vasostatin, a calreticulin fragment, into neuroendocrine tumor cells results in enhanced malignant behavior[J]. Neuroendocrinology.2005;82(1):1-10.
[38]李里,彭佳远,马延磊,等.Transgelin蛋白在人结直肠癌中的表达及其临床意义[J].肿瘤.2009；29(8)：765-771.
[1]Vangestel C, Van de Wiele C, Van Damme N, et al. (99)mTc-(CO)(3) His-annexin A5 micro-SPECT demonstrates increased cell death by irinotecan during the vascular normalization window caused by bevacizumab[J]. JNucl Med.2011;52(11):1786-1794.
[2]Mattes MJ, Michel RB, Goldenberg DM, et al. Induction of apoptosis by cross-linking antibodies bound to human B-lymphoma cells:expression of Annexin Ⅴ binding sites on the antibody cap[J]. Cancer Biother Radiopharm. 2009;24(2):185-193.
[3]Rand JH, Wu XX, Quinn AS, et al. Resistance to annexin A5 anticoagulant activity:a thrombogenic mechanism for the antiphospholipid syndrome[J]. Lupus.2008;17(10):922-930.
[4]Rand JH. Antiphospholipid antibody-mediated disruption of the annexin-Ⅴ antithrombotic shield:a thrombogenic mechanism for the antiphospholipid syndrome[J].J Autoimmun.2000; 15(2):107-111.
[5]Rand JH, Wu XX, Quinn AS,et al. Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug[J]. Blood. 2010;115(11):2292-2299.
[6]辛虹,王惠兰.膜联蛋白V在孕妇外周血及胎盘组织中的表达及其与子痫前期发病的关系[J].中华妇产科杂志.2011；46(2)：88-93.
[7]Wahezi DM, Ilowite NT, Rajpathak S, et al. Prevalence of annexin A5 resistance in children and adolescents with rheumatic disease[J]s. J Rheumatol.2012;39(2):382-388.
[8]TP, Reddy SP, Wilborn TW, et al. Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR[J]. Biochem Biophys Res Commun.2003;306(4):1026-1036.
[9]Wehder L, Arndt S, Murzik U, et al. Annexin A5 is involved in migration and invasion of oral carcinoma[J]. Cell Cycle.2009;8(10):1552-1558.
[10]李岭,庄英帜.Annexin A5表达与鼻咽癌分化转移的关系[J].肿瘤防治研究.2011；38(4)：389-393.
[11]李欣,王立平,梁秀军,等.宫颈鳞癌患者血清膜联蛋白A5含量的检测及其临床意义[J].解剖学报.2012；43(1)：68-71.
[12]Xue G, Hao LQ, Ding FX, et al. Expression of annexin a5 is associated with higher tumor stage and poor prognosis in colorectal adenocarcinomas[J]. J Clin Gastroenterol.2009;43(9):831-837.
[13]Horisberger K, Erben P, Strobel P, et al.Annexin and survivin in locally advanced rectal cancer:indicators of resistance to preoperative chemoradiotherapy?[J] Onkologie.2010;33(8-9):439-444.
[14]Brezillon S, Radwanska A, Zeltz C, et al.Lumican core protein inhibits melanoma cell migration via alterations of focal adhesion complexes[J]. Cancer Lett.2009;283(1):92-100.
[15]Ishiwata T, Cho K, Kawahara K, et al.Role of lumican in cancer cells and adjacent stromal tissues in human pancreatic cancer[J]. Oncol Rep. 2007;18(3):537-543.
[16]Matsuda Y, Yamamoto T, KUDO M, et al. Expression and roles of lumican in lung adenocarcinoma and squamous cell carcinoma[J]. Int J Oncol.2008; 33(6):1177-1185.
[17]宋洪江,盂祥宁,佟静,等.Lumican基因在结直肠癌中表达的相关研究[J].国际遗传学杂志.2007；30(6)：416-418.
[18]宋洪江,蓝秀文,魏玉哲,等.Lumican基因在大肠癌中的表达及其与预后的关系[J].国际遗传学杂志.2010；33(5)：268-271.
[1]焦丽燕,应万涛,蔡耘,等.肿瘤研究中的蛋白质组学研究策略[J].肿瘤防治研究.2005,32(4)：245-247.
[2]Chaerkady R, Harsha H C, Nalli A, et al. A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma[J]. J Proteome Res,2008,7(10):4289-4298.
[3]Zhang J, Niu D, Sui J, et al. Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ-coupled 2-D LC-MS/MS analysis:Insights on liver angiogenesis[J]. Proteomics,2009,9(10):2836-2845.
[4]Niu D, Sui J, Zhang J, et al. iTRAQ-coupled 2-D LC-MS/MS analysis of protein profile associated with HBV-modulated DNA methylation[J]. Proteomics,2009,9(15):3856-3868.
[5]Feng H, Wang M, Chen W N. iTRAQ-Coupled 2D LC-MS/MS Analysis of Secreted Proteome of HBV-Replicating HepG2 Cells:Potential in Biomark-ers for Prognosis of HCC[J].Curr Microbiol,2010,61(4):280-284.
[6]Lee H J, Na K, Choi E Y, et al. Simple method for quantitative analysis of N-linked glycoproteins in hepatocellular carcinoma specimens[J].J Proteome Res,2010,9(1):308-318.
[7]Tan H T, Tan S, Lin Q, et al. Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells[J].Mol Cell Proteomics.2008,7(6): 1174-1185.
[8]Deng L, Su T, Leng A, et al. Upregulation of soluble resistance-related calcium-binding protein (sorcin) in gastric cancer[J].Med Oncol,2010; 27(4):1102-1108.
[9]Chen Y, Choong L Y, Lin Q, et al. Differential expression of novel tyrosine kinase substrates during breast cancer development[J].Mol Cell Proteomics, 2007,6(12):2072-2087.
[10]Lim S, Choong L Y, Kuan C P, et al. Regulation of macrophage inhibitory factor (MIF) by epidermal growth factor receptor (EGFR) in the MCF10AT model of breast cancer progression[J]J Proteome Res,2009,8(8):4062-4076.
[11]朱镭,倪国新,张政希,等.用于相对和绝对定量的等量异位标签-二维液相色谱-串联质谱法分析雌二醇和血清剥夺对乳腺癌细胞内蛋白质含量的影响[J].色谱.2009,27(3)：270-278.
[12]倪国新,郭立海,朱镭,等.基于四重iTRAQ标记结合LC-MALDI-TOF-TOF方法分析雌二醇和它莫西芬对MCF-7乳腺癌细胞内蛋白质含量影响的研究[J].分析测试学报.2008,27(12)：1283-1287.
[13]Armenta J M, Hoeschele I, Lazar I M. Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology [J]. J Am Soc Mass Spectrom,2009,20(7):1287-1302.
[14]Ho J, Kong J W, Choong L Y, et al. Novel breast cancer metastasis-associated proteins[J].J Proteome Res,2009,8(2):583-594.
[15]Bouchal P, Roumeliotis T, Hrstka R, et al. Biomarker discovery in low-grade breast cancer using isobaric stable isotope tags and two-dimensional liquid chromatography-tandem mass spectrometry (iTRAQ-2DLC-MS/MS) based quantitative proteomic analysis[J].J Proteome Res,2009,8(1):362-373.
[16]Smith L, Qutob O, Watson M B, et al. Proteomic identification of putative biomarkers of radiotherapy resistance:a possible role for the 26S proteaso-me?[J].Neoplasia,2009,11(11):1194-1207.
[17]Streckfus C F, Mayorga-Wark O, Arreola D, et al. Breast cancer related proteins are present in saliva and are modulated secondary to ductal carcino-ma in situ of the breast[J].Cancer Invest,2008,26(2):159-167.
[18]Streckfus C F, Storthz K A, Bigler L, et al. A Comparison of the Proteomic Expression in Pooled Saliva Specimens from Individuals Diagnos ed with Ductal Carcinoma of the Breast with and without Lymph Node Involvement [J].J Oncol,2009:737619.
[19]Wu W W, Wang G, Baek S J, et al. Comparative study of three proteom-ic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF[J].J Proteome Res,2006,5(3):651-658.
[20]Goshe M B, Smith R D. Stable isotope-coded proteomic mass spectrometry [J].Curr Opin Biotechnol,2003,14(1):101-109.
[21]Tao W A, Aebersold R. Advances in quantitative proteomics via stable isotope tagging and mass spectrometry[J].Curr Opin Biotechnol,2003,14(1): 110-118.