SPC-neu-polyA转基因小鼠的建立以及表型鉴定
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摘要
目的：目前,肺癌已经成为威胁人类健康的第二大癌症,本实验通过构建整合外源基因SPC-neu-polyA的转基因小鼠,对其表型进行鉴定,旨在通过该转基因小鼠模型研究neu基因在肺癌发展中的作用以及肺炎与肺癌的关系。
方法：本实验分为两大部分,第一部分为显微注射得到整合SPC-neu-polyA的转基因小鼠,通过PCR、Southern blot、Western blot、FISH、免疫组化对其进行鉴定,第二部分为转基因小鼠的表型鉴定,病理切片HE染色病理鉴定,Real-Time PCR鉴定炎症因子的表达、免疫组化鉴定炎症因子蛋白的表达。补充实验：免疫组化检测10例肺癌病人组织活检标本的neu蛋白表达和炎症因子的表达。
结果：通过PCR、Southern blot、FISH、检测SPC-neu-polyA完整的整合到转基因小鼠的16号染色体上。Western blot检测到了转基因小鼠中有neu蛋白的表达,IHC实验显示neu蛋白在转基因阳性小鼠的肺泡和支气管上皮表达。
病理切片HE染色发现转基因阳性鼠较正常小鼠有明显的炎症病变,并在年长小鼠中出现上皮细胞增生严重等癌前病变现象。免疫组化检测到10例肺癌病人肺组织活检标本均呈neu阳性或弱阳性,并伴有炎症因子表达。
结论：从以上实验结果,SPC-neu-polyA转基因小鼠已建系成功,表型鉴定为其为炎症到癌前病变的炎症疾病动物模型。该动物模型可用于研究neu在肺癌发展中的作用和研究肺炎与肺癌的关系。
Objective:Lung cancer has currently become the No. 2 killer in cancers threatening human health. Our study was to establish a transgenic mice model integrated with an exogenous SPC-neu-polyA construct, identify their phenotype, so to investigate how the neu gene work in lung cancer development and the relationship between pneumonia and lung cancer.
Methods:Our study included two parts. First, The SPC-neu-polyA sequence was microinjected into mice to generate transgenic mice, which were identified by PCR, Southern blot, Western blot, FISH and immunohistochemistry. Second, the phenotype of transgenic mice was identified by histological HE staining, Real Time-PCR and IHC. Supplement expreiment:10 cases of lung cancer biopsy specimens were detected by IHC to analyze the neu protein expression and inflammation factor expression.
Results:The results of PCR, Southern blot and FISH suggested that SPC-neu-polyA was completely integrated into the genome of transgenic mice, and located in the 16th Chromosome. The results of Western blot suggested that the neu protein was expressed in transgenic mice; IHC experiments suggested that neu protein was expressed in the transgenic mice's alveolar and bronchial epithelium. Significant inflammatory changes were found in the transgenic mice but not in the normal mice by Pathology HE staining. Some transgenic mice showed epithelial cell hyperplasia, one of precancerous lesions. Immunohistochemistry detection of 10 cases of lung biopsy specimens of lung cancer patients showed neu-positive and the expression of inflammatory factors.
Conclusions:From these results, SPC-neu-polyA transgenic mice had been successfully established. Their phenotypes included inflammatory changes and precancerous lesions, indicating they were inflammatory disease models. The animal model can be used to study the role of neu in lung cancer development and the relationship between pneumonia and lung cancer.
方法：本实验分为两大部分,第一部分为显微注射得到整合SPC-neu-polyA的转基因小鼠,通过PCR、Southern blot、Western blot、FISH、免疫组化对其进行鉴定,第二部分为转基因小鼠的表型鉴定,病理切片HE染色病理鉴定,Real-Time PCR鉴定炎症因子的表达、免疫组化鉴定炎症因子蛋白的表达。补充实验：免疫组化检测10例肺癌病人组织活检标本的neu蛋白表达和炎症因子的表达。
结果：通过PCR、Southern blot、FISH、检测SPC-neu-polyA完整的整合到转基因小鼠的16号染色体上。Western blot检测到了转基因小鼠中有neu蛋白的表达,IHC实验显示neu蛋白在转基因阳性小鼠的肺泡和支气管上皮表达。
病理切片HE染色发现转基因阳性鼠较正常小鼠有明显的炎症病变,并在年长小鼠中出现上皮细胞增生严重等癌前病变现象。免疫组化检测到10例肺癌病人肺组织活检标本均呈neu阳性或弱阳性,并伴有炎症因子表达。
结论：从以上实验结果,SPC-neu-polyA转基因小鼠已建系成功,表型鉴定为其为炎症到癌前病变的炎症疾病动物模型。该动物模型可用于研究neu在肺癌发展中的作用和研究肺炎与肺癌的关系。
Objective:Lung cancer has currently become the No. 2 killer in cancers threatening human health. Our study was to establish a transgenic mice model integrated with an exogenous SPC-neu-polyA construct, identify their phenotype, so to investigate how the neu gene work in lung cancer development and the relationship between pneumonia and lung cancer.
Methods:Our study included two parts. First, The SPC-neu-polyA sequence was microinjected into mice to generate transgenic mice, which were identified by PCR, Southern blot, Western blot, FISH and immunohistochemistry. Second, the phenotype of transgenic mice was identified by histological HE staining, Real Time-PCR and IHC. Supplement expreiment:10 cases of lung cancer biopsy specimens were detected by IHC to analyze the neu protein expression and inflammation factor expression.
Results:The results of PCR, Southern blot and FISH suggested that SPC-neu-polyA was completely integrated into the genome of transgenic mice, and located in the 16th Chromosome. The results of Western blot suggested that the neu protein was expressed in transgenic mice; IHC experiments suggested that neu protein was expressed in the transgenic mice's alveolar and bronchial epithelium. Significant inflammatory changes were found in the transgenic mice but not in the normal mice by Pathology HE staining. Some transgenic mice showed epithelial cell hyperplasia, one of precancerous lesions. Immunohistochemistry detection of 10 cases of lung biopsy specimens of lung cancer patients showed neu-positive and the expression of inflammatory factors.
Conclusions:From these results, SPC-neu-polyA transgenic mice had been successfully established. Their phenotypes included inflammatory changes and precancerous lesions, indicating they were inflammatory disease models. The animal model can be used to study the role of neu in lung cancer development and the relationship between pneumonia and lung cancer.
引文
[1]Waterston, R.H., et al., Initial sequencing and comparative analysis of the mouse genome. Nature,2002.420(6915):p.520-62.
[2]Jemal, A., et al., Cancer statistics,2007. CA Cancer J Clin,2007.57(1):p. 43-66.
[3]Barsky, S.H., et al., Rising incidence of bronchioloalveolar lung carcinoma and its unique clinicopathologic features. Cancer,1994.73(4):p.1163-70.
[4]Parkin, D.M., F.I. Bray and S.S. Devesa, Cancer burden in the year 2000. The global picture. Eur J Cancer,2001.37 Suppl 8:p. S4-66.
[5]Spira, A., et al., Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer. Nat Med,2007.13(3):p.361-6.
[6]Zhang, X., et al., Comparison of smoking-induced gene expression on Affymetrix Exon and 3'-based expression arrays. Genome Inform,2007.18:p. 247-57.
[7]Grinenko, A., V.V. Gryzunov and A.A. Lobzhanidze, [Risk factors in the etiology of lung cancer]. Vestn Khir Im Ⅱ Grek,2000.159(4):p.108-12.
[8]Malkinson, A.M., Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp Lung Res,1998.24(4):p.541-55.
[9]Malkinson, A.M., Primary lung tumors in mice:an experimentally manipulable model of human adenocarcinoma. Cancer Res,1992.52(9 Suppl):p. 2670s-2676s.
[10]Glasser, S.W., et al., Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice. Am J Physiol Lung Cell Mol Physiol,2000.278(5):p. L933-45.
[11]Ehrhardt, A., et al., Development of pulmonary bronchiolo-alveolar adenocarcinomas in transgenic mice overexpressing murine c-myc and epidermal growth factor in alveolar type Ⅱ pneumocytes. Br J Cancer,2001.84(6):p.813-8.
[12]Ohashi, K., et al., Chemopreventive effects of gefitinib on nonsmoking-related lung tumorigenesis in activating epidermal growth factor receptor transgenic mice. Cancer Res,2009.69(17):p.7088-95.
[13]Gamier, N., et al., Influence of a mutation in the transmembrane domain of the p185c-erbB2 oncogene-encoded protein studied by molecular dynamics simulations. J Biomol Struct Dyn,1994.11(5):p.983-1002.
[14]Tsai, C.M., et al., Correlation of intrinsic chemoresistance of non-small-cell lung cancer cell lines with HER-2/neu gene expression but not with ras gene mutations. J Natl Cancer Inst,1993.85(11):p.897-901.
[15]Tsai, C.M., et al., Interrelationships between cellular nucleotide excision repair, cisplatin cytotoxicity, HER-2/neu gene expression, and epidermal growth factor receptor level in non-small cell lung cancer cells. Jpn J Cancer Res,2000.91(2): p.213-22.
[16]Ehrhardt, A., et al., Increased susceptibility to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in transgenic mice overexpressing c-myc and epidermal growth factor in alveolar type Ⅱ cells. J Cancer Res Clin Oncol,2003.129(2):p.71-5.
[17]Tokumo, M., et al., The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin Cancer Res,2005.11(3):p.1167-73.
[18]Lynch, T.J., et al., Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med,2004.350(21):p.2129-39.
[19]Yun, C.H., et al., Structures of lung cancer-derived EGFR mutants and inhibitor complexes:mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell,2007.11(3):p.217-27.
[20]Houghton, J., et al., Gastric cancer originating from bone marrow-derived cells. Science,2004.306(5701):p.1568-71.
[21]Prescott, S.M. and F.A. Fitzpatrick, Cyclooxygenase-2 and carcinogenesis. Biochim Biophys Acta,2000.1470(2):p. M69-78.
[22]Chuang, M.J., et al., Tumor-derived tumor necrosis factor-alpha promotes progression and epithelial-mesenchymal transition in renal cell carcinoma cells. Cancer Sci,2008.99(5):p.905-13.
[23]Yoshida, N., et al., Interleukin-6, tumour necrosis factor alpha and interleukin-lbeta in patients with renal cell carcinoma. Br J Cancer,2002.86(9): p.1396-400.
[24]Littman, A.J., L.A. Jackson and T.L. Vaughan, Chlamydia pneumoniae and lung cancer:epidemiologic evidence. Cancer Epidemiol Biomarkers Prev,2005.14(4): p.773-8.
[25]Santillan, A. A., C.J. Camargo and G.A. Colditz, A meta-analysis of asthma and risk of lung cancer (United States). Cancer Causes Control,2003.14(4):p. 327-34.
[26]Malkinson, A.M., Role of inflammation in mouse lung tumorigenesis:a review. Exp Lung Res,2005.31(1):p.57-82.
[27]Parimon, T., et al., Inhaled corticosteroids and risk of lung cancer among patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med,2007. 175(7):p.712-9.
[28]Adjei, A.A., Ras signaling pathway proteins as therapeutic targets. Curr Pharm Des,2001.7(16):p.1581-94.
[29]Vojtek, A.B. and Der CJ, Increasing complexity of the Ras signaling pathway. J Biol Chem,1998.273(32):p.19925-8.
[30]Ji, H., et al., K-ras activation generates an inflammatory response in lung tumors. Oncogene,2006.25(14):p.2105-12.
[31]Campa, D., et al., Association of a common polymorphism in the cyclooxygenase 2 gene with risk of non-small cell lung cancer. Carcinogenesis, 2004.25(2):p.229-35.
[32]Chen, D., et al., Genetic variants in peroxisome proliferator-activated receptor-gamma gene are associated with risk of lung cancer in a Chinese population. Carcinogenesis,2008.29(2):p.342-50.
[33]Engels, E.A., et al., Systematic evaluation of genetic variants in the inflammation pathway and risk of lung cancer. Cancer Res,2007.67(13):p.6520-7.
[1]Bonnet D and JE Dick Human acute myeloid leukemiais organized as a hierarchy that originates from a primitivehematopoietic cell[J]. Nat Med,1997 Jul; 3 (7):730-7.
[2]Sun ZG, Huang SD, Zhang BR, Xu ZY, Liu XH, Gong DJ, Yuan Y Isolation and identification of cancer stem cells from human esoPHageal carcinoma[J] cell,2009, Feb 10; 89 (5):291-5
[3]Prince ME, R Sivanandan, A Kaczorowski, GT Wolf, MJ Kaplan, P Dalerba, IL Weissman, MF Clarke and LE Ailles. Identifi cation of a subpopulation of cells with cancer stem cellproperties in head and neck squamous cell carcinoma. [J] Proc Natl Acad Sci U S A,2007, Jan 16; 104 (3):973-8. Epub 2007 Jan 8.
[4]Al-Hajj M, MS Wicha, A Benito-Hernandez, SJ Morrison and MFClarke. Prospective identifi cation of tumorigenic breastcancer cells.[J] Proc Natl Acad Sci U S A,2003, May 27; 100 (11):6890.
[5]Li C, DG Heidt, BA Daler, CF Burant, L Zhang, VAdsay, MWicha, MF Clarke and DM Simeone. (2007). Identifi cation of pancreatic cancer stem cells.[J] Cancer Res,2007, Feb 1; 67 (3):1030-7.
[6]Qi Wang, Zhi-Guo Chen, Chang-Zheng Du, Hong-Wei Wang, Li Yanl & Jin Gu Cancer stem cell marker CD133+ tumour cells and clinical outcome in rectal cancer; [J] Histopathology,2009, Sep; 55 (3):284-93.
[7]Cheshier SH, Kalani MY, Lim M, Ailles L, Huhn SL, Weissman IL; A neurosurgeon's guide to stem cells, cancer stem cells, and brain tumor stem cells[J]; Neurosurgery,2009 Aug; 65 (2):237-49; discussion 249-50; quiz N6
[8]Singh SK, ID Clarke, M Terasaki, VE Bonn, C Hawkins, J Squireand PB Dirks. Identifi cation of a cancer stem cell inhuman brain turnors[J] Cancer
Res,2003, Sep 15; 63 (18):5821-8.
[9]O'Brien CA, A Pollett, S Gallinger and JE Dick. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J] Nature,2007, Jan 4; 445 (7123):106-10. Epub 2006 Nov 19.
[10]Ricci-Vitiani L, DG Lombardi, E Pilozzi, M Biffoni, M Todaro, C Peschle and R De Maria. Identifi cation and expansion of human colon-cancer-initiating cells.[J] Nature,2007, Jan 4; 445 (7123):111-5. Epub 2006 Nov 19
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[13]Goodell MA, Brose K, Paradis G, et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo[J] J Exp Med, 1996, Apr 1; 183 (4):1797-806.
[14]Dingzhi Huang, Quanli Gao, Liping Guo, Chunpeng Zhang, Wei Jiang, Hongxia Li, Jing Wang, Xiaohong Han. Identification of Cancer Stem-Like Cells in EsoPHagealCarcinoma Cells and Its Tumor Stemness[J] Ultrastruct Pathol, 2009, Jul-Aug; 33 (4):175-81
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[18]Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells[J]. Cancer Res,2005, Dec 1; 65 (23):10946-51.
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[2]Jemal, A., et al., Cancer statistics,2007. CA Cancer J Clin,2007.57(1):p. 43-66.
[3]Barsky, S.H., et al., Rising incidence of bronchioloalveolar lung carcinoma and its unique clinicopathologic features. Cancer,1994.73(4):p.1163-70.
[4]Parkin, D.M., F.I. Bray and S.S. Devesa, Cancer burden in the year 2000. The global picture. Eur J Cancer,2001.37 Suppl 8:p. S4-66.
[5]Spira, A., et al., Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer. Nat Med,2007.13(3):p.361-6.
[6]Zhang, X., et al., Comparison of smoking-induced gene expression on Affymetrix Exon and 3'-based expression arrays. Genome Inform,2007.18:p. 247-57.
[7]Grinenko, A., V.V. Gryzunov and A.A. Lobzhanidze, [Risk factors in the etiology of lung cancer]. Vestn Khir Im Ⅱ Grek,2000.159(4):p.108-12.
[8]Malkinson, A.M., Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp Lung Res,1998.24(4):p.541-55.
[9]Malkinson, A.M., Primary lung tumors in mice:an experimentally manipulable model of human adenocarcinoma. Cancer Res,1992.52(9 Suppl):p. 2670s-2676s.
[10]Glasser, S.W., et al., Human SP-C gene sequences that confer lung epithelium-specific expression in transgenic mice. Am J Physiol Lung Cell Mol Physiol,2000.278(5):p. L933-45.
[11]Ehrhardt, A., et al., Development of pulmonary bronchiolo-alveolar adenocarcinomas in transgenic mice overexpressing murine c-myc and epidermal growth factor in alveolar type Ⅱ pneumocytes. Br J Cancer,2001.84(6):p.813-8.
[12]Ohashi, K., et al., Chemopreventive effects of gefitinib on nonsmoking-related lung tumorigenesis in activating epidermal growth factor receptor transgenic mice. Cancer Res,2009.69(17):p.7088-95.
[13]Gamier, N., et al., Influence of a mutation in the transmembrane domain of the p185c-erbB2 oncogene-encoded protein studied by molecular dynamics simulations. J Biomol Struct Dyn,1994.11(5):p.983-1002.
[14]Tsai, C.M., et al., Correlation of intrinsic chemoresistance of non-small-cell lung cancer cell lines with HER-2/neu gene expression but not with ras gene mutations. J Natl Cancer Inst,1993.85(11):p.897-901.
[15]Tsai, C.M., et al., Interrelationships between cellular nucleotide excision repair, cisplatin cytotoxicity, HER-2/neu gene expression, and epidermal growth factor receptor level in non-small cell lung cancer cells. Jpn J Cancer Res,2000.91(2): p.213-22.
[16]Ehrhardt, A., et al., Increased susceptibility to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in transgenic mice overexpressing c-myc and epidermal growth factor in alveolar type Ⅱ cells. J Cancer Res Clin Oncol,2003.129(2):p.71-5.
[17]Tokumo, M., et al., The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin Cancer Res,2005.11(3):p.1167-73.
[18]Lynch, T.J., et al., Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med,2004.350(21):p.2129-39.
[19]Yun, C.H., et al., Structures of lung cancer-derived EGFR mutants and inhibitor complexes:mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell,2007.11(3):p.217-27.
[20]Houghton, J., et al., Gastric cancer originating from bone marrow-derived cells. Science,2004.306(5701):p.1568-71.
[21]Prescott, S.M. and F.A. Fitzpatrick, Cyclooxygenase-2 and carcinogenesis. Biochim Biophys Acta,2000.1470(2):p. M69-78.
[22]Chuang, M.J., et al., Tumor-derived tumor necrosis factor-alpha promotes progression and epithelial-mesenchymal transition in renal cell carcinoma cells. Cancer Sci,2008.99(5):p.905-13.
[23]Yoshida, N., et al., Interleukin-6, tumour necrosis factor alpha and interleukin-lbeta in patients with renal cell carcinoma. Br J Cancer,2002.86(9): p.1396-400.
[24]Littman, A.J., L.A. Jackson and T.L. Vaughan, Chlamydia pneumoniae and lung cancer:epidemiologic evidence. Cancer Epidemiol Biomarkers Prev,2005.14(4): p.773-8.
[25]Santillan, A. A., C.J. Camargo and G.A. Colditz, A meta-analysis of asthma and risk of lung cancer (United States). Cancer Causes Control,2003.14(4):p. 327-34.
[26]Malkinson, A.M., Role of inflammation in mouse lung tumorigenesis:a review. Exp Lung Res,2005.31(1):p.57-82.
[27]Parimon, T., et al., Inhaled corticosteroids and risk of lung cancer among patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med,2007. 175(7):p.712-9.
[28]Adjei, A.A., Ras signaling pathway proteins as therapeutic targets. Curr Pharm Des,2001.7(16):p.1581-94.
[29]Vojtek, A.B. and Der CJ, Increasing complexity of the Ras signaling pathway. J Biol Chem,1998.273(32):p.19925-8.
[30]Ji, H., et al., K-ras activation generates an inflammatory response in lung tumors. Oncogene,2006.25(14):p.2105-12.
[31]Campa, D., et al., Association of a common polymorphism in the cyclooxygenase 2 gene with risk of non-small cell lung cancer. Carcinogenesis, 2004.25(2):p.229-35.
[32]Chen, D., et al., Genetic variants in peroxisome proliferator-activated receptor-gamma gene are associated with risk of lung cancer in a Chinese population. Carcinogenesis,2008.29(2):p.342-50.
[33]Engels, E.A., et al., Systematic evaluation of genetic variants in the inflammation pathway and risk of lung cancer. Cancer Res,2007.67(13):p.6520-7.
[1]Bonnet D and JE Dick Human acute myeloid leukemiais organized as a hierarchy that originates from a primitivehematopoietic cell[J]. Nat Med,1997 Jul; 3 (7):730-7.
[2]Sun ZG, Huang SD, Zhang BR, Xu ZY, Liu XH, Gong DJ, Yuan Y Isolation and identification of cancer stem cells from human esoPHageal carcinoma[J] cell,2009, Feb 10; 89 (5):291-5
[3]Prince ME, R Sivanandan, A Kaczorowski, GT Wolf, MJ Kaplan, P Dalerba, IL Weissman, MF Clarke and LE Ailles. Identifi cation of a subpopulation of cells with cancer stem cellproperties in head and neck squamous cell carcinoma. [J] Proc Natl Acad Sci U S A,2007, Jan 16; 104 (3):973-8. Epub 2007 Jan 8.
[4]Al-Hajj M, MS Wicha, A Benito-Hernandez, SJ Morrison and MFClarke. Prospective identifi cation of tumorigenic breastcancer cells.[J] Proc Natl Acad Sci U S A,2003, May 27; 100 (11):6890.
[5]Li C, DG Heidt, BA Daler, CF Burant, L Zhang, VAdsay, MWicha, MF Clarke and DM Simeone. (2007). Identifi cation of pancreatic cancer stem cells.[J] Cancer Res,2007, Feb 1; 67 (3):1030-7.
[6]Qi Wang, Zhi-Guo Chen, Chang-Zheng Du, Hong-Wei Wang, Li Yanl & Jin Gu Cancer stem cell marker CD133+ tumour cells and clinical outcome in rectal cancer; [J] Histopathology,2009, Sep; 55 (3):284-93.
[7]Cheshier SH, Kalani MY, Lim M, Ailles L, Huhn SL, Weissman IL; A neurosurgeon's guide to stem cells, cancer stem cells, and brain tumor stem cells[J]; Neurosurgery,2009 Aug; 65 (2):237-49; discussion 249-50; quiz N6
[8]Singh SK, ID Clarke, M Terasaki, VE Bonn, C Hawkins, J Squireand PB Dirks. Identifi cation of a cancer stem cell inhuman brain turnors[J] Cancer
Res,2003, Sep 15; 63 (18):5821-8.
[9]O'Brien CA, A Pollett, S Gallinger and JE Dick. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[J] Nature,2007, Jan 4; 445 (7123):106-10. Epub 2006 Nov 19.
[10]Ricci-Vitiani L, DG Lombardi, E Pilozzi, M Biffoni, M Todaro, C Peschle and R De Maria. Identifi cation and expansion of human colon-cancer-initiating cells.[J] Nature,2007, Jan 4; 445 (7123):111-5. Epub 2006 Nov 19
[11]Collins AT, PA Berry, C Hyde, MJ Stower and NJ Maitland. (2005) Prospective identifi cation of tumorigenic prostate cancer stem cells.[J] Cancer Res,2005, Dec 1; 65 (23):10946-51
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