肺癌功能性抗原的分离鉴定及其与肺癌临床相关性研究
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摘要
目的：采用功能性抗体库技术已筛选出的抗肺癌单抗13C9,确定其识别的抗原在肺癌细胞和组织中的表达,测定该单抗抑制肺癌生长的能力,进一步鉴定其识别的抗原蛋白。研究该抗原蛋白的表达与肺癌临床参数的关系,以期为肺癌靶向治疗提供有价值的分子靶位和治疗剂。
方法：采用细胞免疫荧光、免疫组织化学检测13C9单抗识别的抗原蛋白在肺癌细胞及组织中的表达和定位；ELISA测定13C9单抗的产量和亚型；裸鼠体内移植瘤治疗实验和体外肺癌细胞增殖实验,评价13C9单抗对肺癌细胞生长的作用；采用斑点杂交优化、RIPA法提取13C9单抗识别的抗原蛋白,行蛋白印迹(Western blotting)、免疫沉淀、MALDI-TOF-PMF质谱技术,鉴定该抗原蛋白。收集临床肺癌标本,应用免疫组织化学检测M蛋白的表达,分析其与肺癌临床病理参数的相关性。
结果：细胞免疫荧光检测13C9单抗识别的抗原在肺癌12种固定细胞中表达阳性率达90%以上,强度为强阳性(+++)；在活细胞中,其识别的抗原表达于细胞膜上。免疫组织化学检测结果：174例配对组织中,13C9单抗识别的抗原在肺癌组织中阳性表达率87.4%,在配对癌旁组织中阳性表达率16.7%,差异非常显著(t=3.5555E-48,P<0.01)。培养杂交瘤细胞产生13C9单抗,ELISA测定其产量为4.38ug/ml,亚型为小鼠IgM、k型。裸鼠体内移植瘤抑制实验,13C9单抗抑瘤率达63.29%,与对照组比较,差异显著(P=0.01,P<0.05)。斑点杂交优化13C9单抗识别的抗原蛋白主要定位于细胞膜,经RIPA法提取、免疫沉淀、电泳后切胶行MALDI-TOF-PMF质谱技术鉴定,结果13C9单抗识别的抗原蛋白为M蛋白,分子量为226KD。商业化抗体进行质谱结果回证,证明13C9单抗与商业化的抗体识别的抗原蛋白同为M蛋白。对于肺癌临床标本的免疫组织化学检测及其与肺癌临床病理参数的相关性的分析结果显示,在331例肺癌组织中,M蛋白的表达在鳞癌组织高于在腺癌组织(P=0.000,P<0.01),在T3+T4期肺组织高于T1+T2期肺组织(P=0.001,P<0.01)。在161例鳞癌中M蛋白的表达随T分期的增加而增强；在137例腺癌中M蛋白的表达随分化程度的减低而增强。
结论：本实验结果证明：①13C9单抗为功能性单抗,其识别的抗原在肺癌多种细胞和组织中表达,对裸鼠体内移植瘤具有明显的抑制作用。②13C9单抗识别的抗原M蛋白为肺癌功能性抗原蛋白,分子量为226KD,主要定位于肺癌细胞膜。③M蛋白的表达与肺癌的类型及临床病理参数存在相关性：在肺鳞癌中的表达高于肺腺癌；在肺鳞癌中,M蛋白的表达与T分期呈正相关；在肺腺癌中,M蛋白的表达与分化程度呈负相关。本研究有望为研制新的肺癌靶向治疗剂和治疗靶标提供新的实验依据。
Objective A monoclonal antibody 13C9, which had been targeted by screening of functional monoclonal antibody library against lung cancer, was adopted to recognize its specific antigen on multiple cultured-cell-types and tissues of lung cancer. Its function to the growth of lung cancer was detected both in vivo and in vitro. Furthermore the 13C9-specific antigen was identified. And the relationship between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied. This study may provide a clue for exploring valuable molecular targets and treatment agents for targeted therapy of lung cancer.
Methods With immunofluorescence and immunohistochemistry techniques, the expression of 13C9-specific antigen was detected and localized on multiple cultured-cell-types and tissues of lung cancer. ELISA was applied to determine the production of 13C9 monoclonal antibody by culturing hybridoma cells and its subtype. Experiments of tumor treatment in vivo and vitro were carried to evaluate the inhibiting functions of the 13C9 antibody to the growth of lung cancer. Thereafter, the 13C9-specific antigen was optimized by dot blotting techque, extracted by RIPA technique. And with Western blotting, immunoprecipitation, MALDI-TOF-PMF mass spectrometry, the 13C9-specific antigen was identified. On the other hand, clinical specimens of lung cancer were collected and detected with immunohistochemistry. The correlation between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied.
Results Results from immunofluorescence showed that among 12 fixed lung cancer cell lines, positive rate of the recognition of the antibody 13C9 to its antigen was above 90%, intensity was +++; with viable cells of lung cancer cell lines, the antibody 13C9-recognized antigen was demonstrated on the cell membrane. Immunohistochemical results showed that within 174 cases of paired tissues of lung cancer, positive rate of the 13C9-recognized antigen in lung cancer tissues was up to 87.4%, compared with the paired adjacent tissues which was just 16.7%,had a significant difference (t=3.5555E-48, P<0.01). The production of the antibody 13C9 by culturing hybridoma cells was 4.38ug/ml, belong to IgM, Kapa subtype, measured by ELISA. Transplant tumor inhibition experiment showed that the antibody 13C9 inhibit the lung tumor growth in mice by 63.29%, compared with control group (P=0.01, P<0.05). By dot blotting, the 13C9-recognized antigen protein was optimized and localized on the cell membrane. Then with RIPA extraction, immunoprecipitation and electrophoresis, the antigen protein band on the gel-line was excised and identified by MALDI-TOF-PMF. The molecular weight of the 13C9-recognized antigen protein was 226KD, and we named it as M protein. Conformation experiment was taken with commercial antibody, which demonstrated that M protein was recognized by both the antibody 13C9 and the commercial antibody. Investigation to the results from immunohistochemistry showed, that in 331 cases of lung cancer the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma (P=0.000, P<0.01), the expression of M protein in T3+T4 stage was higher than in T1+T2 stage, (P=0.001, P<0.01). The expression of M protein in squamous cell carcinoma of 161 lung cancer patients was more intense with T stage advance, while in adenocarcinoma of 137 lung cancer patients, the expression of M protein was more intense with decreasion of tissue differentiation.
Conclusion Results from this study demonstrated:①13C9 possesses characteristics of a functional monoclonal antibody,13C9-recognized antigen expressed in multiple cultured-cell-types and tissues of lung cancer, inhibitive effect of antibody 13C9 to the growth of the tumor in nude mice was obvious.②The antibody 13C9-recognized antigen which named M protein, may belong as a functional protein in lung cancer; its molecular weight was 226KD, localized on cell membrane.③There are possible correlation between the expression of M protein and the type as well as the clinical parameters of lung cancer:the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma, the expression of M protein shows a positive correlation with T stage advance especially in lung squamous cell carcinoma, while in lung adenocarcinoma it shows a negative correlation with the advance of tissue differentiation. This study may provide new experimental data for further exploring and developing of new targets and new targeting agents in lung cancer treatment.
方法：采用细胞免疫荧光、免疫组织化学检测13C9单抗识别的抗原蛋白在肺癌细胞及组织中的表达和定位；ELISA测定13C9单抗的产量和亚型；裸鼠体内移植瘤治疗实验和体外肺癌细胞增殖实验,评价13C9单抗对肺癌细胞生长的作用；采用斑点杂交优化、RIPA法提取13C9单抗识别的抗原蛋白,行蛋白印迹(Western blotting)、免疫沉淀、MALDI-TOF-PMF质谱技术,鉴定该抗原蛋白。收集临床肺癌标本,应用免疫组织化学检测M蛋白的表达,分析其与肺癌临床病理参数的相关性。
结果：细胞免疫荧光检测13C9单抗识别的抗原在肺癌12种固定细胞中表达阳性率达90%以上,强度为强阳性(+++)；在活细胞中,其识别的抗原表达于细胞膜上。免疫组织化学检测结果：174例配对组织中,13C9单抗识别的抗原在肺癌组织中阳性表达率87.4%,在配对癌旁组织中阳性表达率16.7%,差异非常显著(t=3.5555E-48,P<0.01)。培养杂交瘤细胞产生13C9单抗,ELISA测定其产量为4.38ug/ml,亚型为小鼠IgM、k型。裸鼠体内移植瘤抑制实验,13C9单抗抑瘤率达63.29%,与对照组比较,差异显著(P=0.01,P<0.05)。斑点杂交优化13C9单抗识别的抗原蛋白主要定位于细胞膜,经RIPA法提取、免疫沉淀、电泳后切胶行MALDI-TOF-PMF质谱技术鉴定,结果13C9单抗识别的抗原蛋白为M蛋白,分子量为226KD。商业化抗体进行质谱结果回证,证明13C9单抗与商业化的抗体识别的抗原蛋白同为M蛋白。对于肺癌临床标本的免疫组织化学检测及其与肺癌临床病理参数的相关性的分析结果显示,在331例肺癌组织中,M蛋白的表达在鳞癌组织高于在腺癌组织(P=0.000,P<0.01),在T3+T4期肺组织高于T1+T2期肺组织(P=0.001,P<0.01)。在161例鳞癌中M蛋白的表达随T分期的增加而增强；在137例腺癌中M蛋白的表达随分化程度的减低而增强。
结论：本实验结果证明：①13C9单抗为功能性单抗,其识别的抗原在肺癌多种细胞和组织中表达,对裸鼠体内移植瘤具有明显的抑制作用。②13C9单抗识别的抗原M蛋白为肺癌功能性抗原蛋白,分子量为226KD,主要定位于肺癌细胞膜。③M蛋白的表达与肺癌的类型及临床病理参数存在相关性：在肺鳞癌中的表达高于肺腺癌；在肺鳞癌中,M蛋白的表达与T分期呈正相关；在肺腺癌中,M蛋白的表达与分化程度呈负相关。本研究有望为研制新的肺癌靶向治疗剂和治疗靶标提供新的实验依据。
Objective A monoclonal antibody 13C9, which had been targeted by screening of functional monoclonal antibody library against lung cancer, was adopted to recognize its specific antigen on multiple cultured-cell-types and tissues of lung cancer. Its function to the growth of lung cancer was detected both in vivo and in vitro. Furthermore the 13C9-specific antigen was identified. And the relationship between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied. This study may provide a clue for exploring valuable molecular targets and treatment agents for targeted therapy of lung cancer.
Methods With immunofluorescence and immunohistochemistry techniques, the expression of 13C9-specific antigen was detected and localized on multiple cultured-cell-types and tissues of lung cancer. ELISA was applied to determine the production of 13C9 monoclonal antibody by culturing hybridoma cells and its subtype. Experiments of tumor treatment in vivo and vitro were carried to evaluate the inhibiting functions of the 13C9 antibody to the growth of lung cancer. Thereafter, the 13C9-specific antigen was optimized by dot blotting techque, extracted by RIPA technique. And with Western blotting, immunoprecipitation, MALDI-TOF-PMF mass spectrometry, the 13C9-specific antigen was identified. On the other hand, clinical specimens of lung cancer were collected and detected with immunohistochemistry. The correlation between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied.
Results Results from immunofluorescence showed that among 12 fixed lung cancer cell lines, positive rate of the recognition of the antibody 13C9 to its antigen was above 90%, intensity was +++; with viable cells of lung cancer cell lines, the antibody 13C9-recognized antigen was demonstrated on the cell membrane. Immunohistochemical results showed that within 174 cases of paired tissues of lung cancer, positive rate of the 13C9-recognized antigen in lung cancer tissues was up to 87.4%, compared with the paired adjacent tissues which was just 16.7%,had a significant difference (t=3.5555E-48, P<0.01). The production of the antibody 13C9 by culturing hybridoma cells was 4.38ug/ml, belong to IgM, Kapa subtype, measured by ELISA. Transplant tumor inhibition experiment showed that the antibody 13C9 inhibit the lung tumor growth in mice by 63.29%, compared with control group (P=0.01, P<0.05). By dot blotting, the 13C9-recognized antigen protein was optimized and localized on the cell membrane. Then with RIPA extraction, immunoprecipitation and electrophoresis, the antigen protein band on the gel-line was excised and identified by MALDI-TOF-PMF. The molecular weight of the 13C9-recognized antigen protein was 226KD, and we named it as M protein. Conformation experiment was taken with commercial antibody, which demonstrated that M protein was recognized by both the antibody 13C9 and the commercial antibody. Investigation to the results from immunohistochemistry showed, that in 331 cases of lung cancer the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma (P=0.000, P<0.01), the expression of M protein in T3+T4 stage was higher than in T1+T2 stage, (P=0.001, P<0.01). The expression of M protein in squamous cell carcinoma of 161 lung cancer patients was more intense with T stage advance, while in adenocarcinoma of 137 lung cancer patients, the expression of M protein was more intense with decreasion of tissue differentiation.
Conclusion Results from this study demonstrated:①13C9 possesses characteristics of a functional monoclonal antibody,13C9-recognized antigen expressed in multiple cultured-cell-types and tissues of lung cancer, inhibitive effect of antibody 13C9 to the growth of the tumor in nude mice was obvious.②The antibody 13C9-recognized antigen which named M protein, may belong as a functional protein in lung cancer; its molecular weight was 226KD, localized on cell membrane.③There are possible correlation between the expression of M protein and the type as well as the clinical parameters of lung cancer:the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma, the expression of M protein shows a positive correlation with T stage advance especially in lung squamous cell carcinoma, while in lung adenocarcinoma it shows a negative correlation with the advance of tissue differentiation. This study may provide new experimental data for further exploring and developing of new targets and new targeting agents in lung cancer treatment.
引文
[1]Garcia M, Jemal A, Ward EM, et al. Global Cancer Facts and Figures 2007. American Cancer Society,2007
[2]Peto R, Darby S, Deo H,等.1950年以来英国的吸烟、戒烟和肺癌状况：全国统计和两项病例对照研究的综合报告.英国医学杂志中文版,2000,3：169
[3]Kris MG, Natale RB, Herbst RS. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase.in symptomatic patients with non-small cell lung cancer:a randomized trial. JAMA,2003,290(16):2149-2158
[4]McLeod C, Bagust A, Boland A, et al. Erlotinib for the Treatment of Relapsed Non-Small Cell Lung Cancer. Health Technol Assess.2009,13 Suppl 1:41-47
[5]Nassar D, Soutou B, Aractingi S. Cutaneous side effects of EGF receptor inhibitors. Bull Cancer, 2009,96 Suppl:S85-91
[6]卢燕来,冉宇靓,遇珑等.功能差异细胞模型筛选抗肺癌功能性单克隆抗体及其抗原.中国肿瘤生物治疗杂志,2008,15(6)：501-507
[7]周晓彬,纪新强,徐莉.医用统计学软件PPMS1.5的组成和应用特点[J].齐鲁医学杂志,2009,24(1)：29-32.
[8]尹光浩,刘伟,刘国津等.非小细胞肺癌的EGFR表达与EGFR基因突变的比较.中国实验诊断学,2009,13(2)：200－202
[9]Cersosimo RJ.Monoclonal antibodies in the treatment of cancer(Part2). Am J Health System Pharmacy,2003,60(16):1631-1641
[10]Nguyen DT, Amess JA, Doughty H, et al. IDEC-C2B8 anti-CD20 (rituximab) immunotherapy in patients with low-grade non-Hodgkin's lymphoma and lymphoproliferative disorders:evaluation of response on 48 patients. [J]. Eur J Hacmatol,1999.62(2):76-82.
[11]WitzigTE, GordonLI. CabanillasF, et al. Randomized controlled trial of ytthrium-90-labeled ibfitumomab tiuxetan radioimmtmotherapy versus rituximab immunotherapy for panents with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin's lympharna[J]. JClin Oncol, 2002,20(10):2453-2463
[12]Pegram MD, LDpezA, KonecnyG, et al. Trastuzumab and chemotherapeutic drug interactions andsynergies[J]. Selllin Oncol,2000,27(6Suppl 11):21-25
[13]Arteaga CL. Trastuzumab, an appropriate first line single agent therapy for HER2 over-expressing metastatic breast cancer. Breast Cancer Res,2003,5(2):96-100.
[14]Hainsworth JD, Sosman JA, Spigel DR, et al. Phase II trial of heracizunmb and edotinib in patients with metastatiic renal carcinoma(RCC). Pro Am Soc CIinOncol,2004,22:382
[15]PankinDM, Bray F, Ferlay J, et al. Global cancer statistics 2002[J]. CA Cancer J Clin.2005, 55:74
[16]Ko EC, Wang X, Ferrone S.Immunotherapy of malignant diseases. Challenges and strategies.Int Arch Allergy Immunol,2003,132(4):294-309
[17]Simon R. Targets for treatment succes. Nat Clin Plaet Oncol,2006,3(1):1-5
[18]Ciardiello F, Caputo R, Bianco R, et al. Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839(Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor [J]. Clin Cancer Res,2001,7(5):1459-1465.
[19]Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefinitib for previously treated patients with advanced non-small cell lung cancer(The IDEAL 1 Trial)(corrected)[J].J Clin Oncol,2003,21(12):2237-2246
[20]Kris MG, Natale RB, Herbst RS, et al. Efficacy of Gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer 1-J]. JAMA, 2003,290(16):2149-2158
[21]Perez SR, Chachoua A, Hammond LA, et al. Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer[J]. J Clin Oncol,2004,22(16):3238-3247
[22]Kim ES. Cetuximab as a single agent or incombination with chemotherapy in lung cancer[J]. Clin Lung Cancer,2004,6(Suppl 2):80-84
[23]Sandier AB, Gray R, BrahmerJ, et al. Paclitaxel carboplatin alone or with bevacizumab for non-small-cell lung cancer[J].N Engl J Med,2006,355(24):2542—2550
[24]Sjoblom T, Jones S, Wood LD, et al. The Consensus Coding Sequences of Human Breast and Colorectal Cancers[J]. Science,2006,314:268-274.
[1]侯雪梅,崔黎丽,李国栋,等.抗肿瘤药物靶向制剂研究进展[J].药学实践杂志,2007,25(5)：273－275
[2]Marcove RC. A 17-year review of cryosurgery in the treatment of bone tumors [J]. Clin Orthop Relat Res,1982,163:231-234
[3]Coppres. Cryogenic surgery:a new method of destruction or extirpation of benign or malignant tissues[J]. N Engl J Med,1963,268:743-749
[4]Southam CM, Immunotherapeutic trials in sarcoma patients using autogenous tumorvaccine[G]. Proceedings of the 7th animal San Francisco cancer sym.1972,199-204
[5]Seifert J K, Morris D L. World survey on the complications of hepatic and prostate cryotherapy[J]. World J Surg,1999,23 (2):109-113
[6]Ivarsson K, Myllymaki L, Jansner K,et al. Heat shock protein 70 (HSP70) after laser thermotherapy of an adenocarcinoma transplanted into rat liver[J]. Anticancer Res,2003,23(5A):3703-0712
[7]Kramer G, Steiner GE Grobl M, et al. Response to sublethal heat treatment of prostatic tumor cells and of prostatic tumor infilt rating T-cells[J]. Prostate.2004,58(2):109-120
[8]Wang SZ, Wang L, Gao XD, et al. Influence of the expression of heatshock protein 70 in maxillofacial squamous cell carcinoma by thermochemo-therapy[J]. Hua Xi Kou Qiang Yi Xue Za Zhi, 2005,23(4):277-279
[9]Schell SR, Wessels FJ, Abouhamze A, et al. Pro-and antiinflammatory cyto-kine production after radiofrequency ablation of unresectable hepatic tumors[J]. Jam Coll Surg,2002,195(6):774-782
[10]Falk MH. Issels RD. Hypexthexmia in oncology[J]. Int J Hypexthermia.2001,17(1):1-18
[11]Zolzer F, Strefer C. G2-phase delays after irradiation and/or heat treatment assessed by two-parameter flow cytometry[J]. Radiat Res,2001,155(1):50-56
[12]Mohamed F, Marchettini P, Stuart OA et Al. Thermal enhancement of new the motherapeutic agents at moderate hyper-thermia[J]. Ann Surg Oncol,2003,10(4):463-468
[13]Ling CC, Humm J,Larson S, et al. Towards multidimensional radiotherapy(MD-CRT):biological imaging and biological conforreality[J]. Int J Radiat Oneol Biol Phys,2000,47,551-560
[14]Dwamena BA, Sonnad SS, Angobaldo JO, et al. Metastases from non-small cell lung cancer:mediastinal staging in the 1990s-meta-analytic comparison of PET and CT[J]. Radiology,1999,213:530-536
[15]Eschmann SM, Friedel G, Paulsen F, et al. Impact of staging with 18F-FDG-PET on outcome of patients with stage III no-small cell lung Cancer:PET identifies potential survivors[J]. Eur J Nu-cl Med Mol, Imaging,2007,34:54-59
[16]Moureau-Zabotto L, Touboul E, Lorouge D, et al. Impact of CT and 18F-deoxyglucose positron emission tomography image fusion for conformal radiotherapy in esophageal carcinoma [J]. Int J Radiat Oncol Biol Phys,2005,63(2):340-345
[17]Vrieze O, Hanstermans K, De Wever W, et al. Is there a role for FGD-PET in radiotherapy planning in esophageal carcinom [J]. Radiother Oncol,2004,73(3),269-275
[18]Hutchings M, Loft A, Hansen M, et al. Clinical impact of FDG-PET/CT in the planning of radiotherapy for early-stage Hodgkin lymphoma[J]. Eur J Haematol,2007,78(3):206-212
[19]Daisne JF, Duprez T, Weynand B, et al. Tumor volume in pharyngolaryngeal squamous cell carcinoma:comparison at CT, MR imagin, and FDG-PET and validation with surgieal specimen [J]. Radiology,2004,233(1):93-100
[20]Schwartz DL, Ford EC, Rajendran J, et al. FDG-PET/CT-guided intensity modulated head and neck radiotherapy:a pilot investigation [J]. Head Neck,2005,27(6):478-487
[21]Chang JY, Dong L, Liu H, et al. Image-guided radiation therapy for non-small cell lung cancer[J]. J Thorae Oneol,2008; 3(2):177-186
[22]李曾.恶性肿瘤的电化学治疗[J].临床肿瘤学杂志.2004,9(3)：302-304
[23]王秀丽5-氨基酮戊酸光动力疗法在皮肤科的应用[J].上海医学,2007,30(1)5-7
[24]常金环,郭海霞.31例光动力疗法治疗皮肤肿瘤的护理[J].天津护理,2009,17(5)：268－269
[25]O'Brien CA, Pollett S, Gallinger, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J]. Nature,2007,445:106-110
[26]Al-Hajj MM, W Becker, M Wicha, et al. Therapeutic implications of cancer stem cells[J].Curr Opin Genet,2004,14:43-47
[27]Wicha MS. Cancer stem cells and metastasis:lethal seeds[J]. Clin Cancer Res,2006, 12:5606-5607
[28]Bonnet D, J.E. Dick. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[J]. Nat Med,1997.3:730-737
[29]Patrawala, L, T. Calhoun, R. Schneider-Broussard, et al. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic[J]. Cancer research 2005.65:6207-6219
[30]Perez-Losada J, A Balmain. Stem-cell hierarchy in skin cancer[J]. Nat Rev Cancer,2003. 3:434-443
[31]Houghton JC, Stoicov S, Nomura A, et al. Gastric cancer originating from bone marrow-derived cells[J]. Science,2004,306:1568-1571
[32]Kim CF, EL Jackson, AEWoolfenden,et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer[J]. Cell,2005,121:823-835
[33]Collins AT, PA Berry, C Hyde,et al. Prospective identification of tumorigenic prostate cancer stem cells[J]. Cancer Res,2005,65:10946-10951.
[34]Tu SM, SH Lin, CJ Logothetis. Stem-cell origin of metastasis and heterogeneity in solid tumours[J]. Lancet Oncol,2002,3:508-513.
[35]Hixson DC, L Chapman, A McBride, et al. Antigenic phenotypes common to rat oval cells, primary hepatocellular carcinomas and developing bile ducts[J]. Carcinogenesis,1997,18:1169-1175.
[36]FDA. A catalog of FDA approved drug products[DB]. http://www.accessdata. fad. gov/scripts/cder/drugsatfda,2005
[37]FDA. FDA approves eribitux for colorectal cancer[EB/OL]. http://www.fda.gov/bbs/topics/NEWS/2O04/NEWO1024.html,2004,2,12.
[38]FDA. FDA approves first angiogenesis inhibitor to treat colorectal cancer[EB/OL]. http://www. fda. Gov/bbs/topics/NEWS/2O04/NEW01027.html,2004,2,26.
[39]Czuczman MS, Grillo Lopez AJ, White CA, et al. Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy[J]. J Clin Oncol,1999,17(1):268.
[40]Baselga J, Tripathy D, Mendelsohn J, et al. Phase Ⅱ study of weekly intravenous trastuzumab(herceptin)in patients with HER2/neu-overexpressing metastatic breast cancer[J]. Semin Oncology,1999,26(4 Suppl 12):78
[41]Morishima Y, Ogura M, Nishimura M, et al. Efficacy and safety of imatinib mesylate for patients in the first chronic phase of chronic myeloid leukemia:results of a Japanese phase 11 clinical study [J].Int J Hematof,2004,80(3):261
[42]Codess CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors[J].J Clin Oncol,2004,22(18):3813
[43]Cunningham D, Humblet Y, Siena S,et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan refractory metastatic colorectal cancer[J].N Engl J Med,2004,351(4): 337
[44]Cersosimo RJ. Monoclonal antibodies in the treatment of cancer(Part2). Am J Health System Pharmacy,2003,60(16):1631-1643
[45]桑国卫.我国创新药物和中药现代化研究近况[J].中国药事,2007,21(1)：3-7
[46]Thome SH, Negrin RS, Contag CH. Synergistic antitumor effects of immune cell-viral biotherapy[J]. Science,2006,311(5768):1780-1784
[47]徐辉碧.纳米医药[M].北京：清华大学出版社,2004：54
[2]Peto R, Darby S, Deo H,等.1950年以来英国的吸烟、戒烟和肺癌状况：全国统计和两项病例对照研究的综合报告.英国医学杂志中文版,2000,3：169
[3]Kris MG, Natale RB, Herbst RS. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase.in symptomatic patients with non-small cell lung cancer:a randomized trial. JAMA,2003,290(16):2149-2158
[4]McLeod C, Bagust A, Boland A, et al. Erlotinib for the Treatment of Relapsed Non-Small Cell Lung Cancer. Health Technol Assess.2009,13 Suppl 1:41-47
[5]Nassar D, Soutou B, Aractingi S. Cutaneous side effects of EGF receptor inhibitors. Bull Cancer, 2009,96 Suppl:S85-91
[6]卢燕来,冉宇靓,遇珑等.功能差异细胞模型筛选抗肺癌功能性单克隆抗体及其抗原.中国肿瘤生物治疗杂志,2008,15(6)：501-507
[7]周晓彬,纪新强,徐莉.医用统计学软件PPMS1.5的组成和应用特点[J].齐鲁医学杂志,2009,24(1)：29-32.
[8]尹光浩,刘伟,刘国津等.非小细胞肺癌的EGFR表达与EGFR基因突变的比较.中国实验诊断学,2009,13(2)：200－202
[9]Cersosimo RJ.Monoclonal antibodies in the treatment of cancer(Part2). Am J Health System Pharmacy,2003,60(16):1631-1641
[10]Nguyen DT, Amess JA, Doughty H, et al. IDEC-C2B8 anti-CD20 (rituximab) immunotherapy in patients with low-grade non-Hodgkin's lymphoma and lymphoproliferative disorders:evaluation of response on 48 patients. [J]. Eur J Hacmatol,1999.62(2):76-82.
[11]WitzigTE, GordonLI. CabanillasF, et al. Randomized controlled trial of ytthrium-90-labeled ibfitumomab tiuxetan radioimmtmotherapy versus rituximab immunotherapy for panents with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin's lympharna[J]. JClin Oncol, 2002,20(10):2453-2463
[12]Pegram MD, LDpezA, KonecnyG, et al. Trastuzumab and chemotherapeutic drug interactions andsynergies[J]. Selllin Oncol,2000,27(6Suppl 11):21-25
[13]Arteaga CL. Trastuzumab, an appropriate first line single agent therapy for HER2 over-expressing metastatic breast cancer. Breast Cancer Res,2003,5(2):96-100.
[14]Hainsworth JD, Sosman JA, Spigel DR, et al. Phase II trial of heracizunmb and edotinib in patients with metastatiic renal carcinoma(RCC). Pro Am Soc CIinOncol,2004,22:382
[15]PankinDM, Bray F, Ferlay J, et al. Global cancer statistics 2002[J]. CA Cancer J Clin.2005, 55:74
[16]Ko EC, Wang X, Ferrone S.Immunotherapy of malignant diseases. Challenges and strategies.Int Arch Allergy Immunol,2003,132(4):294-309
[17]Simon R. Targets for treatment succes. Nat Clin Plaet Oncol,2006,3(1):1-5
[18]Ciardiello F, Caputo R, Bianco R, et al. Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839(Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor [J]. Clin Cancer Res,2001,7(5):1459-1465.
[19]Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefinitib for previously treated patients with advanced non-small cell lung cancer(The IDEAL 1 Trial)(corrected)[J].J Clin Oncol,2003,21(12):2237-2246
[20]Kris MG, Natale RB, Herbst RS, et al. Efficacy of Gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer 1-J]. JAMA, 2003,290(16):2149-2158
[21]Perez SR, Chachoua A, Hammond LA, et al. Determinants of tumor response and survival with erlotinib in patients with non-small-cell lung cancer[J]. J Clin Oncol,2004,22(16):3238-3247
[22]Kim ES. Cetuximab as a single agent or incombination with chemotherapy in lung cancer[J]. Clin Lung Cancer,2004,6(Suppl 2):80-84
[23]Sandier AB, Gray R, BrahmerJ, et al. Paclitaxel carboplatin alone or with bevacizumab for non-small-cell lung cancer[J].N Engl J Med,2006,355(24):2542—2550
[24]Sjoblom T, Jones S, Wood LD, et al. The Consensus Coding Sequences of Human Breast and Colorectal Cancers[J]. Science,2006,314:268-274.
[1]侯雪梅,崔黎丽,李国栋,等.抗肿瘤药物靶向制剂研究进展[J].药学实践杂志,2007,25(5)：273－275
[2]Marcove RC. A 17-year review of cryosurgery in the treatment of bone tumors [J]. Clin Orthop Relat Res,1982,163:231-234
[3]Coppres. Cryogenic surgery:a new method of destruction or extirpation of benign or malignant tissues[J]. N Engl J Med,1963,268:743-749
[4]Southam CM, Immunotherapeutic trials in sarcoma patients using autogenous tumorvaccine[G]. Proceedings of the 7th animal San Francisco cancer sym.1972,199-204
[5]Seifert J K, Morris D L. World survey on the complications of hepatic and prostate cryotherapy[J]. World J Surg,1999,23 (2):109-113
[6]Ivarsson K, Myllymaki L, Jansner K,et al. Heat shock protein 70 (HSP70) after laser thermotherapy of an adenocarcinoma transplanted into rat liver[J]. Anticancer Res,2003,23(5A):3703-0712
[7]Kramer G, Steiner GE Grobl M, et al. Response to sublethal heat treatment of prostatic tumor cells and of prostatic tumor infilt rating T-cells[J]. Prostate.2004,58(2):109-120
[8]Wang SZ, Wang L, Gao XD, et al. Influence of the expression of heatshock protein 70 in maxillofacial squamous cell carcinoma by thermochemo-therapy[J]. Hua Xi Kou Qiang Yi Xue Za Zhi, 2005,23(4):277-279
[9]Schell SR, Wessels FJ, Abouhamze A, et al. Pro-and antiinflammatory cyto-kine production after radiofrequency ablation of unresectable hepatic tumors[J]. Jam Coll Surg,2002,195(6):774-782
[10]Falk MH. Issels RD. Hypexthexmia in oncology[J]. Int J Hypexthermia.2001,17(1):1-18
[11]Zolzer F, Strefer C. G2-phase delays after irradiation and/or heat treatment assessed by two-parameter flow cytometry[J]. Radiat Res,2001,155(1):50-56
[12]Mohamed F, Marchettini P, Stuart OA et Al. Thermal enhancement of new the motherapeutic agents at moderate hyper-thermia[J]. Ann Surg Oncol,2003,10(4):463-468
[13]Ling CC, Humm J,Larson S, et al. Towards multidimensional radiotherapy(MD-CRT):biological imaging and biological conforreality[J]. Int J Radiat Oneol Biol Phys,2000,47,551-560
[14]Dwamena BA, Sonnad SS, Angobaldo JO, et al. Metastases from non-small cell lung cancer:mediastinal staging in the 1990s-meta-analytic comparison of PET and CT[J]. Radiology,1999,213:530-536
[15]Eschmann SM, Friedel G, Paulsen F, et al. Impact of staging with 18F-FDG-PET on outcome of patients with stage III no-small cell lung Cancer:PET identifies potential survivors[J]. Eur J Nu-cl Med Mol, Imaging,2007,34:54-59
[16]Moureau-Zabotto L, Touboul E, Lorouge D, et al. Impact of CT and 18F-deoxyglucose positron emission tomography image fusion for conformal radiotherapy in esophageal carcinoma [J]. Int J Radiat Oncol Biol Phys,2005,63(2):340-345
[17]Vrieze O, Hanstermans K, De Wever W, et al. Is there a role for FGD-PET in radiotherapy planning in esophageal carcinom [J]. Radiother Oncol,2004,73(3),269-275
[18]Hutchings M, Loft A, Hansen M, et al. Clinical impact of FDG-PET/CT in the planning of radiotherapy for early-stage Hodgkin lymphoma[J]. Eur J Haematol,2007,78(3):206-212
[19]Daisne JF, Duprez T, Weynand B, et al. Tumor volume in pharyngolaryngeal squamous cell carcinoma:comparison at CT, MR imagin, and FDG-PET and validation with surgieal specimen [J]. Radiology,2004,233(1):93-100
[20]Schwartz DL, Ford EC, Rajendran J, et al. FDG-PET/CT-guided intensity modulated head and neck radiotherapy:a pilot investigation [J]. Head Neck,2005,27(6):478-487
[21]Chang JY, Dong L, Liu H, et al. Image-guided radiation therapy for non-small cell lung cancer[J]. J Thorae Oneol,2008; 3(2):177-186
[22]李曾.恶性肿瘤的电化学治疗[J].临床肿瘤学杂志.2004,9(3)：302-304
[23]王秀丽5-氨基酮戊酸光动力疗法在皮肤科的应用[J].上海医学,2007,30(1)5-7
[24]常金环,郭海霞.31例光动力疗法治疗皮肤肿瘤的护理[J].天津护理,2009,17(5)：268－269
[25]O'Brien CA, Pollett S, Gallinger, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J]. Nature,2007,445:106-110
[26]Al-Hajj MM, W Becker, M Wicha, et al. Therapeutic implications of cancer stem cells[J].Curr Opin Genet,2004,14:43-47
[27]Wicha MS. Cancer stem cells and metastasis:lethal seeds[J]. Clin Cancer Res,2006, 12:5606-5607
[28]Bonnet D, J.E. Dick. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[J]. Nat Med,1997.3:730-737
[29]Patrawala, L, T. Calhoun, R. Schneider-Broussard, et al. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic[J]. Cancer research 2005.65:6207-6219
[30]Perez-Losada J, A Balmain. Stem-cell hierarchy in skin cancer[J]. Nat Rev Cancer,2003. 3:434-443
[31]Houghton JC, Stoicov S, Nomura A, et al. Gastric cancer originating from bone marrow-derived cells[J]. Science,2004,306:1568-1571
[32]Kim CF, EL Jackson, AEWoolfenden,et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer[J]. Cell,2005,121:823-835
[33]Collins AT, PA Berry, C Hyde,et al. Prospective identification of tumorigenic prostate cancer stem cells[J]. Cancer Res,2005,65:10946-10951.
[34]Tu SM, SH Lin, CJ Logothetis. Stem-cell origin of metastasis and heterogeneity in solid tumours[J]. Lancet Oncol,2002,3:508-513.
[35]Hixson DC, L Chapman, A McBride, et al. Antigenic phenotypes common to rat oval cells, primary hepatocellular carcinomas and developing bile ducts[J]. Carcinogenesis,1997,18:1169-1175.
[36]FDA. A catalog of FDA approved drug products[DB]. http://www.accessdata. fad. gov/scripts/cder/drugsatfda,2005
[37]FDA. FDA approves eribitux for colorectal cancer[EB/OL]. http://www.fda.gov/bbs/topics/NEWS/2O04/NEWO1024.html,2004,2,12.
[38]FDA. FDA approves first angiogenesis inhibitor to treat colorectal cancer[EB/OL]. http://www. fda. Gov/bbs/topics/NEWS/2O04/NEW01027.html,2004,2,26.
[39]Czuczman MS, Grillo Lopez AJ, White CA, et al. Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy[J]. J Clin Oncol,1999,17(1):268.
[40]Baselga J, Tripathy D, Mendelsohn J, et al. Phase Ⅱ study of weekly intravenous trastuzumab(herceptin)in patients with HER2/neu-overexpressing metastatic breast cancer[J]. Semin Oncology,1999,26(4 Suppl 12):78
[41]Morishima Y, Ogura M, Nishimura M, et al. Efficacy and safety of imatinib mesylate for patients in the first chronic phase of chronic myeloid leukemia:results of a Japanese phase 11 clinical study [J].Int J Hematof,2004,80(3):261
[42]Codess CL, Fletcher JA, Heinrich MC. Biology of gastrointestinal stromal tumors[J].J Clin Oncol,2004,22(18):3813
[43]Cunningham D, Humblet Y, Siena S,et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan refractory metastatic colorectal cancer[J].N Engl J Med,2004,351(4): 337
[44]Cersosimo RJ. Monoclonal antibodies in the treatment of cancer(Part2). Am J Health System Pharmacy,2003,60(16):1631-1643
[45]桑国卫.我国创新药物和中药现代化研究近况[J].中国药事,2007,21(1)：3-7
[46]Thome SH, Negrin RS, Contag CH. Synergistic antitumor effects of immune cell-viral biotherapy[J]. Science,2006,311(5768):1780-1784
[47]徐辉碧.纳米医药[M].北京：清华大学出版社,2004：54