人胚胰腺导管上皮细胞的体外长期培养及永生化
摘要
有关人的胰腺导管上皮细胞的生理功能调节、受体分布、基因表达调控、癌变机制等方面的研究，仍是较为薄弱的领域，其中最重要的原因是分离培养以胰腺导管上皮细胞为主的细胞群十分困难，特别是体外长期培养的问题至今没有解决。基于本领域研究工作的迫切需要，我们进行了这方面的工作。
首先，采用胶原酶消化的方法，分离人胚(中期引产的胎儿，20-30周)胰腺上皮细胞为细胞团，将10％FBS完全培基加入到这些细胞团中，接种至50ml塑料培养瓶，24小时后换2％FBS完全培基进行原代培养。经过近20个胎儿的胰腺标本的分离培养，摸索出了适合于胰腺导管上皮细胞原代培养的方法和条件。若原代培养的细胞不传代，其生活状态可维持长达2月之久；也可以传代，传第3代的细胞仍以上皮细胞为主，细胞生长良好。
对原代培养细胞进行了初步的鉴定，结果为培养77、96小时的原代细胞，淀粉酶检测阴性，Cytokeratin免疫细胞化学染色阳性，说明原代培养的细胞为导管上皮细胞而非腺泡细胞，为进行体外转化奠定了基础。
在成功地建立胰腺导管上皮细胞原代和传代培养的基础上，利用SV40 T基因掺入细胞基因组DNA后可以造成细胞转化，使转化细胞能够在体外长期传代培养的特性，将载有SV40 T基因的质粒pSV-T，通过脂质体Lipofectin介导，转染原代和传代(第3代)培养的胰腺导管上皮细胞，经过2-7周的筛选，终于
Little is known about the regulation of physiological function, receptor distribution, control of gene expression, and carcinogenesis in human pancreatic ductal cells.One of the major reasons is that it is difficult to isolate and culture the main pancreatic ductal cells. It is also due to the fact that normal human epithelial cells can only be cultured for very limited number of passages.As a result, it is virtually impossible to carry on any specific long-term research on these cells. Therefore, to establish an immortalized normal human pancreatic ductal cell line is of prime importance, since it can serve as a model for various purposes in research.
Human fetal pancreas (20th-30th week of gestation) was digested with collagenase into cell pellets, which were then plated to flasks in 10% FBS complete medium.Twenty-four hours later, these pellets were cultured in 2% FBS complete medium.When the cells reached 90% confluency, they were subcultured. These subcultures were propagated at least for 2 generations. If primary cultures were not subcultured, they might live for 2 months.
首先，采用胶原酶消化的方法，分离人胚(中期引产的胎儿，20-30周)胰腺上皮细胞为细胞团，将10％FBS完全培基加入到这些细胞团中，接种至50ml塑料培养瓶，24小时后换2％FBS完全培基进行原代培养。经过近20个胎儿的胰腺标本的分离培养，摸索出了适合于胰腺导管上皮细胞原代培养的方法和条件。若原代培养的细胞不传代，其生活状态可维持长达2月之久；也可以传代，传第3代的细胞仍以上皮细胞为主，细胞生长良好。
对原代培养细胞进行了初步的鉴定，结果为培养77、96小时的原代细胞，淀粉酶检测阴性，Cytokeratin免疫细胞化学染色阳性，说明原代培养的细胞为导管上皮细胞而非腺泡细胞，为进行体外转化奠定了基础。
在成功地建立胰腺导管上皮细胞原代和传代培养的基础上，利用SV40 T基因掺入细胞基因组DNA后可以造成细胞转化，使转化细胞能够在体外长期传代培养的特性，将载有SV40 T基因的质粒pSV-T，通过脂质体Lipofectin介导，转染原代和传代(第3代)培养的胰腺导管上皮细胞，经过2-7周的筛选，终于
Little is known about the regulation of physiological function, receptor distribution, control of gene expression, and carcinogenesis in human pancreatic ductal cells.One of the major reasons is that it is difficult to isolate and culture the main pancreatic ductal cells. It is also due to the fact that normal human epithelial cells can only be cultured for very limited number of passages.As a result, it is virtually impossible to carry on any specific long-term research on these cells. Therefore, to establish an immortalized normal human pancreatic ductal cell line is of prime importance, since it can serve as a model for various purposes in research.
Human fetal pancreas (20th-30th week of gestation) was digested with collagenase into cell pellets, which were then plated to flasks in 10% FBS complete medium.Twenty-four hours later, these pellets were cultured in 2% FBS complete medium.When the cells reached 90% confluency, they were subcultured. These subcultures were propagated at least for 2 generations. If primary cultures were not subcultured, they might live for 2 months.
引文
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2. Warshaw AL and Fernandez-Del Castillo C Pancreatic carcinomas New Eng J Med 1992;32b:455-465
3. Scarpelle DG, Rao MS, and Reddy JR Studies of pancreatic carcinogenesis in different animal models Enveron Health Perspect 1984;56:219-227
4. Pour PM Hamster model for carcinogenesis of the pancreas Int J Pancreatoi 1994;16:288-289
5．柯杨 DNA肿瘤病毒SV40的细胞转化特性 生物化学与生物物理进展1991：18：186-188
6．柯杨，宁涛，王冰等 人胃粘膜上皮细胞系GES-1的建立及其生物学特性 中华肿瘤杂志 1994；16：7-10
7．吴根生，陈秋燕，孙宗棠，CC．Harris SV40 T抗原介导的人胚肝细胞的体外永生化及其机制探讨 中华肿瘤杂志1993；(6)：415-418
8．徐新来 鼠尾胶原的制备 组织培养技术 修订本，鄂征主编，人民卫生出版社，北京，1988，pp63-64
9. Sambrook JJ, Fritsch EF,Maniatis J eds Molecular Cloning-A Laboratory Manual, 2nd edition, 金冬雁, 黎孟枫等译 科学出版社, 北京 1992, p908, p91010. Molecular Cloning, pp19-21, pp26-27
11. Molecular Cloning, pp55-56
12. Molecular Cloning, pp28-32
13. Hansen MB, Nielsen SE, and Berg K Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell dill J Immunol Methods 1989;119: 203-210
14．鄂征 培养细胞染色体显示法 组织培养和分子细胞学技术，鄂征主编，北京出版社，北京，1995，pp164-167
15．鄂征 细胞转化及恶性程度检查--软琼脂培养 组织培养技术 修订本，鄂征主编，人民卫生出版社，北京，1989，pp156-158
16. Kahn SM, Jiang W, Culbertson TA, et al Rapid and sensitive nonradioactive detection of mutant K-ras genes via "enriched" PCR amplification Oncogene 1991;6:1079-1983
17. Urban T, Ricci S, Grange JD, et al Detection of c-K-ras mutation by PCR/RFLP analysis and diagnosis of pancreatic adenocarcinommnas J Natl Cancer Inst 1993;85:2008-2012
18．王志永，刘彤华，崔全才等 胰腺癌的基因诊断 中华病理学杂志 1994；23：270-273
19. Molecular Cloning pp474-488, pp503
20. Githens S Pancreatic duct cell cultrures Annu Rev Physio 1994;56:419-44321. Trautmann B, Schlitt HJ, Hahn EG, and Lohhr M Isolation, culture, and characterization of human pancreatic duct cells Pancreas 1993; 8:248-254
22. Hall PA and Lemoine NR Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas J Pathol 1992;166:97-103
23. Harris A and Coleman L Establishment of a tissue culture system for epithelial cells derived from human pancreas: a model for the study of cystic fibrosis J cell Sci 1987;87:695-703
24. Vila ME, Lloreta J, Real FX Normal human pancreas cultures display functional ductal characteristics Lab Invest 1994; 71: 423-431
25. Masue T,Wakefield LM, Lechner JF, et al Type β transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells Proc Natl Acad Sci 1986;83:2438
26. Bisgaard HC and Thorgeirsson SS Exiddence For a commnon cell of origin for primitive epithetial cells isolated from rat liver and pancrease J Cell Physiol 1991;147:333-43
27. Leclmer JF and La Veck MA A serum-free method for culturing mormal mormal human bronchial epithelial cells at clonal density J Tissue Culture Methods 1985;9:43-48
28．柯杨，宁涛，高燕等 人胃粘膜上皮细胞原代培养及其生物??学鉴定 解剖学报 1992, 23:384-390
29.Hayflick L The limited in vitro lifetime of human diploid cell strains Exp Cell Res 1965;37:615-636
30.Fausto N and Mead JE Regulation of liver growth: protooncogenes and transforming growth factors Lab Invest 1989;60:4-13
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32. Chang SE In vitro transformation of human epithelial cells Bioch Biophys Acta 1986;823:161-194
33. Harris CC Human tissues and cells in carcinogenesis research Cancer Res 1987;47:1-10
34.Almoguera C, Shibata D, Forresteer K, et al Most carecinomas of the exocrine pancreas contain mutant c-K-ras genes Cell 1988;53:549-559
35. Hrubuan RH,Van Mansfeld AOM, Offerhaus GJA, et al K-ras oncogene activation in adenocarcinomas of the human pancreas: a study of 82 carcinoma using a comnination of mutant-rich PCR analysis and allele oligonucleotide hybridization Am J Pathol 1993;143:545-554
36.Berthelemy P, Bouisson M, Escorror J, et al Identification of K- ras mutations in pancreatec juice in the early diagnosis of??pancreatic cancer Am Intern Med 1995;123:188-191
37. Iguchi H, Sugano K, Fukayama N, et al Analysis of the Ki-ras codon 12 mutations in the duodenal juice of patients with pancreatic cancer Gastroenterology 1996;110:221-116
38. Urrutia R and Di D Magno EP Genetic markers: The Key to early diagnosis and improved survival in pancreatic cancer Gastroenterology 1996;110:306-316
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45．柯杨 宁涛 王冰等 人胃粘膜上皮细胞系GEs-1的建立及其生物学特性中华肿瘤杂志 19994，16：7-10
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13. Kaighn ME,Reddel RR,Lechner JF,et al Transformation of human neonatal prostate epithelial cells by strontium phosphate transfection with a plasmid containing SV40 early region genes Cancer Res 1989;49:3050-3056
14. Lemoine N.R., Mayall E.S., Jones T., et al Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection Br. J. Cancer 1989;60:897-903
15. Wyllie FS, Bond JA, Dawson T, et al A phenotypically and karyotypically stable human thyroid epithelial line conditionally Immortalized by SV40 large T antigen Cancer Res 1992;52: 2938-2945
16. Stoner G,Kaighn ME,Reddel RR,et al Establishment and charaterization of SV40 T-antigen immortalized human esophageal epithelial cells Cancer Res 1991 ;51:365-371
17. Pfeifer AMA, Cole KE, Smoot DT, et al Simian Virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens Proc Natl Acad Sci USA 1993;90:5123-5127
18. Ishida T, Ando H, Nomura s, et al Establishment and characterization of human fetal liver epithelial cell line??transfected with SV40 T antigen Exp Bio Med. 1995;251 -256
19. Berthon P, Goubin G, Dutrillaux B, et al A single-step transformation of human breast epithelial cells by SV40 large T oncogene Int J Cancer 1992;52:92-97
20. Gao QS, Hauser SH; Liu XL, et al Mutant p53-induced immortalization of primary human mammary epithelial cells Cancer Res 1996;56:3129-3133
21. KulKarni PS,Sundgvist K,Betsholtz C,et al Characterization of human buccal epithelial cells transfected with the simian virus 40 T antigen gene Carcinogenesis 1995;16:2515-2521
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35. Steenbergen CJLM, Walboomers RDM, Meijer JMM, et al Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: Activation of telomerase and allele losses at 3p, 10, 1 lq and/or 18q Oncogene 1996;13:1249-125736. Trott DA, Cuthbert AP, Overell RW, et al Mechanisms involved in the immortalization of mammalian cells by ionizing radiation and chemical carcinogens Carcinogenesis 1995;16: 193-204
37. Kulkami Schmidt-Kastner PS, Heiden T, Tribukait B, et al Non-tumorigenic SV40 T immortalized human buccal epithelial cells show aneuploidy and genetic instability Anticancer Res 1996;16:2681-2686
39. Andrew Ray F, Kraemer PM Iterative chromosome mutation and selection as a mechanism of complete transformation of human diploid fibroblasts by SV40 T Oncogene 1993;14:1511-1516
38. Woods C, LeFervre C, Stewart N, et al Induction of genomic instability in SV40 transformed human cells: sufficiency of the N-terminal 147 amino acids of large T antigen and role of pRB and p53 Oncogene 1994;9:2943-2950
40. Reddle RR, de Silva R, Duncan EL, et al SV-40 induced immortalization and ras-transformation of human bronchial epithlial cells Int J Cancer 1995;61:199-205
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42．柯杨，宁涛，王冰等 人胃粘膜上皮细胞系GES-1的建立及其生物学特性 中华肿瘤杂志 1994；16：7-10
43．王冰，苏秀兰，宁涛等 MNNG 诱导人胃粘膜上皮细胞系GES-1 及正常胃粘膜标本原癌基因的激活 中华肿瘤杂志 1996； 18：6-944. Garrett LR, Perez-Reyes N, Smith PP, and Mc Dougall JK Interaction of HPV-18 and nitrosomethylurea in the induction of squamous cell carcinoma Carcinogenesis 1993;14:329-332
45. Farin FM, Bigler LG, Oda D, et al Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial ceils immortalized by human papillomavirus type 16 E6/E7 genes Carcinogenesis 1995;16:1391-1401
2. Warshaw AL and Fernandez-Del Castillo C Pancreatic carcinomas New Eng J Med 1992;32b:455-465
3. Scarpelle DG, Rao MS, and Reddy JR Studies of pancreatic carcinogenesis in different animal models Enveron Health Perspect 1984;56:219-227
4. Pour PM Hamster model for carcinogenesis of the pancreas Int J Pancreatoi 1994;16:288-289
5．柯杨 DNA肿瘤病毒SV40的细胞转化特性 生物化学与生物物理进展1991：18：186-188
6．柯杨，宁涛，王冰等 人胃粘膜上皮细胞系GES-1的建立及其生物学特性 中华肿瘤杂志 1994；16：7-10
7．吴根生，陈秋燕，孙宗棠，CC．Harris SV40 T抗原介导的人胚肝细胞的体外永生化及其机制探讨 中华肿瘤杂志1993；(6)：415-418
8．徐新来 鼠尾胶原的制备 组织培养技术 修订本，鄂征主编，人民卫生出版社，北京，1988，pp63-64
9. Sambrook JJ, Fritsch EF,Maniatis J eds Molecular Cloning-A Laboratory Manual, 2nd edition, 金冬雁, 黎孟枫等译 科学出版社, 北京 1992, p908, p91010. Molecular Cloning, pp19-21, pp26-27
11. Molecular Cloning, pp55-56
12. Molecular Cloning, pp28-32
13. Hansen MB, Nielsen SE, and Berg K Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell dill J Immunol Methods 1989;119: 203-210
14．鄂征 培养细胞染色体显示法 组织培养和分子细胞学技术，鄂征主编，北京出版社，北京，1995，pp164-167
15．鄂征 细胞转化及恶性程度检查--软琼脂培养 组织培养技术 修订本，鄂征主编，人民卫生出版社，北京，1989，pp156-158
16. Kahn SM, Jiang W, Culbertson TA, et al Rapid and sensitive nonradioactive detection of mutant K-ras genes via "enriched" PCR amplification Oncogene 1991;6:1079-1983
17. Urban T, Ricci S, Grange JD, et al Detection of c-K-ras mutation by PCR/RFLP analysis and diagnosis of pancreatic adenocarcinommnas J Natl Cancer Inst 1993;85:2008-2012
18．王志永，刘彤华，崔全才等 胰腺癌的基因诊断 中华病理学杂志 1994；23：270-273
19. Molecular Cloning pp474-488, pp503
20. Githens S Pancreatic duct cell cultrures Annu Rev Physio 1994;56:419-44321. Trautmann B, Schlitt HJ, Hahn EG, and Lohhr M Isolation, culture, and characterization of human pancreatic duct cells Pancreas 1993; 8:248-254
22. Hall PA and Lemoine NR Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas J Pathol 1992;166:97-103
23. Harris A and Coleman L Establishment of a tissue culture system for epithelial cells derived from human pancreas: a model for the study of cystic fibrosis J cell Sci 1987;87:695-703
24. Vila ME, Lloreta J, Real FX Normal human pancreas cultures display functional ductal characteristics Lab Invest 1994; 71: 423-431
25. Masue T,Wakefield LM, Lechner JF, et al Type β transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells Proc Natl Acad Sci 1986;83:2438
26. Bisgaard HC and Thorgeirsson SS Exiddence For a commnon cell of origin for primitive epithetial cells isolated from rat liver and pancrease J Cell Physiol 1991;147:333-43
27. Leclmer JF and La Veck MA A serum-free method for culturing mormal mormal human bronchial epithelial cells at clonal density J Tissue Culture Methods 1985;9:43-48
28．柯杨，宁涛，高燕等 人胃粘膜上皮细胞原代培养及其生物??学鉴定 解剖学报 1992, 23:384-390
29.Hayflick L The limited in vitro lifetime of human diploid cell strains Exp Cell Res 1965;37:615-636
30.Fausto N and Mead JE Regulation of liver growth: protooncogenes and transforming growth factors Lab Invest 1989;60:4-13
31 .Kulkarni PS, Sundgvist K, Betsholtz C, et al Characterization of human buccal epithelial cells transfected with the simian virus 40 T antigene gene Carcinogenesis 1995;16:2515-2512
32. Chang SE In vitro transformation of human epithelial cells Bioch Biophys Acta 1986;823:161-194
33. Harris CC Human tissues and cells in carcinogenesis research Cancer Res 1987;47:1-10
34.Almoguera C, Shibata D, Forresteer K, et al Most carecinomas of the exocrine pancreas contain mutant c-K-ras genes Cell 1988;53:549-559
35. Hrubuan RH,Van Mansfeld AOM, Offerhaus GJA, et al K-ras oncogene activation in adenocarcinomas of the human pancreas: a study of 82 carcinoma using a comnination of mutant-rich PCR analysis and allele oligonucleotide hybridization Am J Pathol 1993;143:545-554
36.Berthelemy P, Bouisson M, Escorror J, et al Identification of K- ras mutations in pancreatec juice in the early diagnosis of??pancreatic cancer Am Intern Med 1995;123:188-191
37. Iguchi H, Sugano K, Fukayama N, et al Analysis of the Ki-ras codon 12 mutations in the duodenal juice of patients with pancreatic cancer Gastroenterology 1996;110:221-116
38. Urrutia R and Di D Magno EP Genetic markers: The Key to early diagnosis and improved survival in pancreatic cancer Gastroenterology 1996;110:306-316
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