永生化人支气管上皮细胞恶性转化过程中不同形态集落来源细胞的研究
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摘要
评价化学物致癌性,主要依靠哺乳动物长期诱癌实验。但其实验周期长,饲养条件要求严格,受试动物的敏感性和给药剂量常影响实验结果。而哺乳动物细胞的体外恶性转化实验既能反映化学物的致癌性,又能弥补动物诱癌实验的不足。因此,作为对长期诱癌实验的补充其结果具有重要参考价值。细胞体外恶性转化实验的起始点是致癌物导致细胞遗传物质改变,终点是细胞成瘤能力的改变,转化过程中则伴随有细胞形态、细胞生长能力、生化表型等的变化。本实验就以致癌物作用人支气管上皮细胞后传代细胞集落的形态学改变为切入点,寻找人源细胞体外转化的早期形态学标志,持续观察由不同形态集落而来的两株细胞随传代其生长特性、细胞构成、细胞遗传物质及细胞成瘤能力的变化,进而探讨肺肿瘤组织发生、人源细胞体外转化的形态学标志和基因组学改变等问题。期望建立一种以人源细胞为实验材料的化学物致癌性评价简化模型。
本研究在我室袁素波、陈光宇、周喆等人先后进行的赛替派(Thiotepa,TEPA)、环磷酰胺(Cyclophosphamide,CP)诱发永生化人支气管上皮细胞株BEAS-2B恶性转化、裸鼠接种成瘤及转化细胞、肿瘤细胞生物学特性和转化机理研究的基础上,分别以遗传毒性致癌物赛替派、丝裂霉素C(Mitomycin C,MMC)、环磷酰胺和非遗传毒性致癌物6-巯基嘌呤(6-mercaptopurine,6-MP)、丁基羟甲苯(butylated hvdroxytoluene,BHT)对BEAS-2B细胞进行染毒,克隆接种。一方面观察比较所产生的集落形态学上的差异,另一方面以电镜、免疫细胞化学染色、核型分析、基因芯片、裸鼠接种成瘤等技术对赛替派染毒BEAS-2B后两种不同形态集落来源的细胞的生长特性、细胞构成、遗传物质改变及其致癌性进行研究。主要实验结果如下:
在遗传毒性致癌物赛替派、环磷酰胺和丝裂霉素C染毒BEAS-2B细胞一定时间后,其传代细胞可形成两种形态差异很大的集落(克隆):一种集落与BEAS-2B细胞集落相似,细胞呈纺锤形、长梭形,胞核卵圆,胞浆少而淡染,细胞排列松散,呈放射状或略呈车轮状,我们称之为松散型集落(S-colony);另一种集落为数比较少,胞体大而圆,胞浆丰富,核嗜碱深染,细胞排列紧密,集落呈圆形边
The long-term mammalian induced cancer test is the most classical and reliable method to evaluate the carcinogenicity of chemicals. But it has some disadvantages: the experimental period lasts too long; animal raising condition is strict; the dosage and sensibility of subject animals often affect experimental results. Whereas, the in-vitro mammalian cellular malignant transformation experiment can make up these shortcomings besides evaluating the carcinogenicity of chemicals. So it is an important and valuable complementarity to long-term mammalian induced cancer test. Its initiation point is the change of cellular genetic material induced by carcinogen; its end point is the change of cellular tumorigenicity. The changes of cellular morphology, growth ability and biochemistry phenotype et.al accompany with the transforming process. So, I began my study from the cellular morphologic changes induced by carcinogen, searching for its early-stage morphologic marker.Then,I continuously observe the changes of cellular growth ability,cellulosity,genomics and tumorigenicity of the two cell strains derived from different morphologic colonies.I hope to build a simplified experimental model which use human-originated cells to evaluate chemical's carcinogenicity.Befor my study, Yuan-subo, Chen-guangyu and Zhou-zhe et.al had made some related researches.They had used thiotepa (TEPA) and cyclophosphamide (CP) to transform the immortalized human bronchial epithelial cell strain BEAS-2B, had inoculated the transformed cells into naked mice which then grew cancer,had researched their cell biology and transformation mechanism.Basing their study,I used the genotoxic carcinogens TEPA, CP,mitomy(?)n C(MMC) and non-genotoxic carcinogens 6-mercaptopurine(6-MP),butylated hydroxytoluene(BHT) to transform the BEAS-2B cells.Then,on the one hand I observed and compared their morphologic differences of transformed colonies;on the other hand I used electron microscope, immunocytochemical stain, karyotype analysis,genechip, inoculation naked mouse et.al techniques to research the two cell strains' growth properties,cellularity,genomics and
carcinogenicity which derived from different morphologic colonies.The study results as follow:After the genotoxic carcinogens TEPA,CP and MMC induced BEAS-2B cells.its early passage cells can form two different morphologic colonies:One is similar to BEAS-2B's colony.The cell is fusiform or long-fusiform,nucleus is orbicular-ovate, kytoplasm is poor stain.The colony is loose,scattered and wheel-shaped.So,we named it scattered-type colony(S-colony).The other colony is lesser than S-colony.Its shape is big and round,its kytoplasm is abundant,its nucleus is strong baso-stain.The colony is round and compact.Some cells of this colony grow in ambolayer.So,we named it compact-type colony(C-colony).Whereas,the non-genotoxic carcinogens 6-MP and BHT didn't induce these changes.The possible reason is that C-colony related to cellular genetic material damage.After the HE stain,we compared the morphologic differences of S-colony cells,C-colony cells and BEAS-2B cells through Mias-300 microimage analytic system.We discovered that the C-colony cells are more larger and more rounder than BEAS-2B cells and S-colony cells.We separated the S-colony and C-colony, subcultured, established two cell strains, and named them BEAS-S and BEAS-C respectively.Their differences exist not only in the cytomorphology but also in the growth properties.The growth rate, cloning efficiency and cellular saturation density of BEAS-2B and BEAS-S cells are bigger than BEAS-C cells'.But only the BEAS-C cells can produce compact-type colonies and transformed focis.Their cell cycle exist differences, too.Coomassie blue stain of the three cell strains showed that the cytoskeleton of BEAS-S and BEAS-C.cells was disordered.lt reflected that thiotepa can change the cellular and colony morphology by impacting their cytoskeleton system.The electron microscope results showed that thiotepa has obvious cytotoxicity.The cellular damage was severe.Edge-collected chromatin and vacuoles existed in nucleus and kytoplasm.The human bronchial epithelial basal cell's special marker-cytokeratin 340E12 antibody,the goblet cell's special marker-mucin 5AC(MUC5AC) antibody,the neuroendocrine cell's special marker— Chromogranin A(ChrA) antibody were used to PowerVision? two step immunocytochemical stain. We discovered that the ChrA didn't express in the three cell strains.Meanwhile, the MUC5AC was weakly expressed.With the passage,the cytokeratin 34(3E12 expressed increasely in BEAS-C cells as its cellular agglutination and tumorigenicity.But they didn't change obviously in the BEAS-2B and
本研究在我室袁素波、陈光宇、周喆等人先后进行的赛替派(Thiotepa,TEPA)、环磷酰胺(Cyclophosphamide,CP)诱发永生化人支气管上皮细胞株BEAS-2B恶性转化、裸鼠接种成瘤及转化细胞、肿瘤细胞生物学特性和转化机理研究的基础上,分别以遗传毒性致癌物赛替派、丝裂霉素C(Mitomycin C,MMC)、环磷酰胺和非遗传毒性致癌物6-巯基嘌呤(6-mercaptopurine,6-MP)、丁基羟甲苯(butylated hvdroxytoluene,BHT)对BEAS-2B细胞进行染毒,克隆接种。一方面观察比较所产生的集落形态学上的差异,另一方面以电镜、免疫细胞化学染色、核型分析、基因芯片、裸鼠接种成瘤等技术对赛替派染毒BEAS-2B后两种不同形态集落来源的细胞的生长特性、细胞构成、遗传物质改变及其致癌性进行研究。主要实验结果如下:
在遗传毒性致癌物赛替派、环磷酰胺和丝裂霉素C染毒BEAS-2B细胞一定时间后,其传代细胞可形成两种形态差异很大的集落(克隆):一种集落与BEAS-2B细胞集落相似,细胞呈纺锤形、长梭形,胞核卵圆,胞浆少而淡染,细胞排列松散,呈放射状或略呈车轮状,我们称之为松散型集落(S-colony);另一种集落为数比较少,胞体大而圆,胞浆丰富,核嗜碱深染,细胞排列紧密,集落呈圆形边
The long-term mammalian induced cancer test is the most classical and reliable method to evaluate the carcinogenicity of chemicals. But it has some disadvantages: the experimental period lasts too long; animal raising condition is strict; the dosage and sensibility of subject animals often affect experimental results. Whereas, the in-vitro mammalian cellular malignant transformation experiment can make up these shortcomings besides evaluating the carcinogenicity of chemicals. So it is an important and valuable complementarity to long-term mammalian induced cancer test. Its initiation point is the change of cellular genetic material induced by carcinogen; its end point is the change of cellular tumorigenicity. The changes of cellular morphology, growth ability and biochemistry phenotype et.al accompany with the transforming process. So, I began my study from the cellular morphologic changes induced by carcinogen, searching for its early-stage morphologic marker.Then,I continuously observe the changes of cellular growth ability,cellulosity,genomics and tumorigenicity of the two cell strains derived from different morphologic colonies.I hope to build a simplified experimental model which use human-originated cells to evaluate chemical's carcinogenicity.Befor my study, Yuan-subo, Chen-guangyu and Zhou-zhe et.al had made some related researches.They had used thiotepa (TEPA) and cyclophosphamide (CP) to transform the immortalized human bronchial epithelial cell strain BEAS-2B, had inoculated the transformed cells into naked mice which then grew cancer,had researched their cell biology and transformation mechanism.Basing their study,I used the genotoxic carcinogens TEPA, CP,mitomy(?)n C(MMC) and non-genotoxic carcinogens 6-mercaptopurine(6-MP),butylated hydroxytoluene(BHT) to transform the BEAS-2B cells.Then,on the one hand I observed and compared their morphologic differences of transformed colonies;on the other hand I used electron microscope, immunocytochemical stain, karyotype analysis,genechip, inoculation naked mouse et.al techniques to research the two cell strains' growth properties,cellularity,genomics and
carcinogenicity which derived from different morphologic colonies.The study results as follow:After the genotoxic carcinogens TEPA,CP and MMC induced BEAS-2B cells.its early passage cells can form two different morphologic colonies:One is similar to BEAS-2B's colony.The cell is fusiform or long-fusiform,nucleus is orbicular-ovate, kytoplasm is poor stain.The colony is loose,scattered and wheel-shaped.So,we named it scattered-type colony(S-colony).The other colony is lesser than S-colony.Its shape is big and round,its kytoplasm is abundant,its nucleus is strong baso-stain.The colony is round and compact.Some cells of this colony grow in ambolayer.So,we named it compact-type colony(C-colony).Whereas,the non-genotoxic carcinogens 6-MP and BHT didn't induce these changes.The possible reason is that C-colony related to cellular genetic material damage.After the HE stain,we compared the morphologic differences of S-colony cells,C-colony cells and BEAS-2B cells through Mias-300 microimage analytic system.We discovered that the C-colony cells are more larger and more rounder than BEAS-2B cells and S-colony cells.We separated the S-colony and C-colony, subcultured, established two cell strains, and named them BEAS-S and BEAS-C respectively.Their differences exist not only in the cytomorphology but also in the growth properties.The growth rate, cloning efficiency and cellular saturation density of BEAS-2B and BEAS-S cells are bigger than BEAS-C cells'.But only the BEAS-C cells can produce compact-type colonies and transformed focis.Their cell cycle exist differences, too.Coomassie blue stain of the three cell strains showed that the cytoskeleton of BEAS-S and BEAS-C.cells was disordered.lt reflected that thiotepa can change the cellular and colony morphology by impacting their cytoskeleton system.The electron microscope results showed that thiotepa has obvious cytotoxicity.The cellular damage was severe.Edge-collected chromatin and vacuoles existed in nucleus and kytoplasm.The human bronchial epithelial basal cell's special marker-cytokeratin 340E12 antibody,the goblet cell's special marker-mucin 5AC(MUC5AC) antibody,the neuroendocrine cell's special marker— Chromogranin A(ChrA) antibody were used to PowerVision? two step immunocytochemical stain. We discovered that the ChrA didn't express in the three cell strains.Meanwhile, the MUC5AC was weakly expressed.With the passage,the cytokeratin 34(3E12 expressed increasely in BEAS-C cells as its cellular agglutination and tumorigenicity.But they didn't change obviously in the BEAS-2B and
引文
1. WHO, Prevention of Cancer, Technical Rep.Series No.276, Genf (1964).
2.张敏磊,方福德.转导GST-pi基因表达抑制甲基丙烯酸环氧丙酯的致癌作用.科学通报.1999,13(1):1414-1418.
3.覃兆海,金淑惠,李楠编著.基础有机化学.北京:科学技术文献出版社.2004,1.
4.刘毓谷主编.卫生毒理学基础(第二版).北京:人民卫生出版社.1998:116-137.
5. Charles Heidelberger, et al.Cell transformation by chemical agents—a review and analysis of the literature,A report of the United States Environmental Protection Agency.Mutation Research. 1983; 114-283.
6. Engelhardt G, Schwind KR,Mubler B.The testing of chemicals in the Syrian hamster embryo (SHE) cell transformation assay for assessment of carcinogenic potential.Toxicology in Vitro. 18(2004)213-218.
7.刘国廉主编.细胞毒理学.北京:军事医学科学出版社.2001,287-313.
8. Berwald Y, Sachs L.In vitro cell transformation with chemical carcinogens.Nature, 1963; 200:1182.
9. Harris CC.Human tissues and cells in carcinogenesis research. Cancer Res.1987, 47(1):110.
10. Reddle RR,Ke Y, Breneda I,et al.Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 1988; 48:1904-1909.
11. Iizasa T, Momiki S,Harris CC,et al.Invasive tumors derived from xenotransplanted,immortalized human cells after in vivo exposure to chemical carcinogens.Carcinogenesis. 1993 Sep; 14(9): 1789-1794.
12. Van Agen B, Maas LM, Zwingmann IH, et al. B[a] P-DNA adduct formation and induction of human epithelial lung cell transformation. Environ Mol Mutagen. 1997; 30(3):287-92.
13. Bonfil RD,Momiki S,Harris CC.Enhancement of the invasive ability of a transformed human bronchial epithelial cell line by 12-o-tetradecanoyl-phorbol-13-acetate and diacylglycerol.carcinogenesis. 1989 Dec; 10(12):2335-2338.
14.袁雄,叶常青。重离子照射致人支气管上皮细胞突变效应。中华放射医学与防护杂志。1999,19(5):300-302.
15. Hornberg C,Maciuleviciute L,Seemayer NH.Human bronchcepithelial cells in vitro as a tool for detection of genotoxic activity of airborne particulates. Aerosol Sci.1996, Suppl 1:s35-s36.
16. Amstad P, Reddel RR, Pfeifer A, et al. Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras. Mol Carcinog. 1988; 1(3):151-160.
17. Pfeifer AM, Jones RT, Bowden PE, et al. Human bronchial epithelial cells transformed by the c-raf-1 and c-myc protooncogenes induce multidifferentiated carcinomas in nude mice: a model for lung carcinogenesis. Cancer Res. 1991 Jul 15; 51(14):3793-3801.
18. Reddel RR, Ke Y, Kaighn ME, et al. Human bronchial epithelial cells neoplastically transformed by v-Ki-ras: altered response to inducers of terminal squamous differentiation. Oncogene Res. 1988; 3(4):401-408.
19.袁素波,王治乔,叶常青,廖明阳.环磷酰胺、赛替派诱发人支气管上皮细胞的染色体畸变.癌变·畸变·突变.2000;12(4):205-211.
20.袁素波,王治乔,叶常青,廖明阳.环磷酰胺或塞替派诱导人支气管上皮细胞恶性转化的形态计量学.中国药理学与毒理学杂志.2000;14(5):346-351.
21.袁素波,王治乔,叶常青,廖明阳.环磷酰胺及塞替派诱导人支气管上皮细胞恶性转化的超微结构.中国药理学与毒理学杂志.2000;14(5):341-345.
22.袁素波,王治乔,叶常青,廖明阳.环磷酰胺诱导永生化人支气管上皮细胞恶性转化过程中的基因突变.中国药理学与毒理学杂志.2001;15(6):447-452.
23.袁素波,王治乔,叶常青,廖明阳.环磷酰胺诱发永生化人支气管上皮细胞的恶性转化.中国药理学与毒理学杂志.2001;15(6):441-446.
24.袁素波,王治乔,叶常青,廖明阳.塞替派诱发永生化人支气管上皮细胞的恶性转化(一)、(二).卫生毒理学杂志 2001;15(4):207-215.
25.陈光宇,袁素波,马华智,廖明阳.环磷酰胺及塞替派诱导的人支气管上皮恶性转化细胞的p16INK4a及p15INK4b基因突变.中国药理学与毒理学杂志.2002;16(2):133-137.
26.陈光宇,袁素波,马华智,廖明阳.环磷酰胺及塞替派诱导的人支气管上皮恶性转化细胞的细胞周期相关蛋白表达.中国药理学与毒理学杂志.2002;16(2):138-142.
27.周喆,袁素波,廖明阳.塞替派、环磷酰胺诱发的人支气管上皮恶性转化细胞的致瘤性.中国药理学与毒理学杂志.2002;16(3):216-219.
28.周喆,袁素波,廖明阳.塞替派诱发人支气管上皮恶性转化成瘤细胞的染色体畸变.中国药理学与毒理学杂志.2002;16(4):302-305.
29. IARC/NCI/TEPA Working Group.Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for prediction of carcinogenic chemicals.Cancer Res, 1985, 45(1):2395-2399.
30.张煦,赵俊生.肺癌组织学类型的光镜、电镜比较观察[J].兰州大学学报(自然科学版),1998,34(1):85-89.
31. Weinberger JH, Williams GM.Carcinogen testing: Current problems and new approaches.Sience, 1981 (214), 23:401-407.
32. Tomatis L. Cell proliferation and carcinogenesis: a brief history and current view based on an IARC workshop report. International Agency for Research on Cancer.Environment health perspective. 1993, 101 (suppl 5): 149-51.
33. Folyd RA.The role of 8-hydroxyguanine in carcinogenesis.Carcinogenesis.1990, 11(9): 1447-50.
34. Seleznera TG, Korman NP.Analysis of chromosomes of somatic cells in patients treated with antitumor drugs.Sov Genet. 1973, 9:1575-1579.
35. Myhr BC.Validation studies with muta mouse: A transgenic mouse model for detecting mutations in vivo.Environ Mol Mutagen. 1991, 18:308.
36. Sheu CW, Lee LK, Rodriguez I, et al.The use of uninduced rat liver s-9 to supplement BALB/3T3 cells in the in vitro transformation assay.Drug and chemical toxicology. 1991, 14:113-126.
37. Stoner GD, Shimkin MB, Kniazeff AJ, et al.Test for carcinogenicity of food additives and chemotherapeutic agents by the pulmonary tumor response in stain A mice.Cancer Res. 1973, 33:3069-3085.
38. Siberberg JM, Hosein Zarrabi M.Acute nonlymphocytic leukemia after thiotepa instillation into the bladder: report of 2 cases and review of the literature.J Urol. 1987, 138:402-403.
39. IARC. Thiotepa study in pharmaceutical drugs[C]. Lyon. IARC Scientific Publications. 1990, 50: 123-141.
40. Milic M, Kopjar N.Evaluation of in vitro genotoxic activity of bleomycin and mitomycin C in human lymphocytes using the alkaline comet assay. Arh Hig Rada Toksikol. 2004.55(4):249-259.
41. Kimball RF.The development of ideas about the effect of DNA repairs on the induction of gene mutation and chromosomal aberrations by radiation and by chemicals.Mutat Res. 1987, 186:1-34.
42. Ueda K,Komano T. Sequence-specific DNA damage induced by reduced mitomycin C and 7-N-(p-hydroxyphenyl)mitomycin C. Nucleic Acids Res. 1984; 12(17): 6673-6683.
43.袁伯俊,王治乔,廖明阳,等。新药临床前安全性评价与实践。北京:军事医学科学出版社,1997:101-102.
44. Dedrich RL, Morrison PF.Carcinogenic potency of alkylating agents in rodent and human.Cancer Res. 1992, 52:2464.
45. Radis CD, Kahl LE, Baker GL, et al.Effects of cyclophosphamide on the development of malignancy and on long-term survival of patients with rheumatoid arthritis.A 20 years follow up study.Arthritis Rheum. 1995.38:1120.
46. Stein JP, Skinner EC,Boyd SD,et al.Squamous cell carcinoma of the bladder associated with cyclophosphamide therapy for Wegener's granulomatosis:a report of 2 case.J Urol. 1993.149:588.
47. Ortiz A, Gonzelez-parra E, Alvarez-Costa, et al.Bladder cancer after cyclophosphamide therapy for lupus nephritis.Nephron. 1992, 60:378.
48. Miyamae Y, Zaizen K, Ohara K, et al.Detection of DNA lesions induced by chemical mutagens by the single cell electrophoresis (Comet) assay. 1. Relationship between the onset of DNA damage and the characteristics of mutagens. Mutat Res. 1998 Jul 31; 415(3):229-35.
49. Morales RP, Vallarino KT, Anguiano OG. Pharmacokinetic parameters of genotoxic activity inferred from the comparison of the kinetics of MN-PCE induced by chemical agents and ionizing radiation. Mutat Res. 1997 Jul 14; 391(3):127-34.
50. IARC (IARC (1986) IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 40. Some NaturallyOccurring and Synthetic Food Components, Furocoumarins and Ultraviolet Radiation. Butylated hydroxyanisole (BHA). International Agency for Researchon Cancer, Lyon. 123-206.
51. Witschi, H.P., Malkinson, A.M. and Thompson, JA.Metabolism and pulmonary toxicity of butylated hydroxytoluene.In:T.E.Gram, (Ed), metabolic activation and toxicity of chemical agents to lung tissue and cells, Pergamon Press, Ltd, UK, 1993, 185-212.
52. A.M. Malkinson, K.M. Koski, W.A. Evans, M.F. Festing, Butylated hydroxytoluene exposure is necessary to induce lung tumors in BALB mice treated with 3-methylcholanthrene,Cancer Res. 57 (1997) 2832-2834.
53. Williams G. M., Iatropoulos M. J. and Whysner J. Safety assessment of butylated hydroxyanisole and butylated hydroxytoluene as antioxidant food additives. Food and Chemical Toxicology, 1999, 37, 1027-1938.
54. Yude Sun, Dwyer-Nield L.D., Malkinson A.M., et al. Responses of tumorigenic and non-tumorigenic mouse lung epithelial cell lines to electrophilic metabolites of the tumor promoter butylated hydroxytoluene. Chemico-Biological Interactions 145 (2003) 41-51.
55.谭曾鲁,周柔丽,主编。医学细胞生物学。北京:北京医科大学出版社。1992:233-293.
56.张进华,丁彦秋,杨学皆。介绍一种细胞骨架蛋白改良染色发。诊断病理学杂志。1996,3(4):235-236.
57. Boers JE,Ambergen AW, Thunnissen FB.Number and proliferation of basal and parabasal cells in normal human airway epithelium.AM J Respir Crit Care Med, 1998;157:2000-2006.
58. Boers JE, Den Brok JLM, Koudstaal J, et al. Number and proliferation of neuroendocrine cells in normal human airway epithelium[J]. AM J Respir Crit Care Med, 1996, 154: 758-763.
59. Kirkham S,Sheehan JK,Knight D,et al.Heterogeneity of airways mucus:variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B.Bioche m J,2002,361 (Pt3):537-546.
60.董波,陈肖华,张浩,等。一种改进的用于测定细胞周期的细胞制备方法。军事医学科学院院刊。2002,26(2):124-129.
61.孙秀泓,罗自强,秦晓群,等.肺的非呼吸功能基础与临床[M].第1版.北京:人民卫生出版社,2003:222-223.
62. Boers JE, Ambergen AW, Thunnissen FB. Number and proliferation of Clara cells in normal human airway epithelium[J]. AM J Respir Crit Care Med, 1999, 159: 1 585-1 591.
63. Soderberg M. Structural characterization of bronchial mucosal biopsies from healthy volunteers: A light and electron microscopical study[J]. Eur Respir J, 1990, 3: 261.
64.熊敏,吴一龙,主编.现代肺癌病理与临床.北京:科学出版社,2003.109-110.
65. Otto W.R.Lung epithelial stem cells.J Pathol 2002,197: 527-535.
66. Barrett JC.Mechanisms of action of known human carcinogens.IARC Sci Publ.1992, 11(6):115-134.
67. Vogelstein B, Kinzler KW.The multistep nature of cancer.Trends Genet. 1993.9 (4): 138-141.
68.纪之莹.李桂兰.李凌凛,等。苯接触工人外周血淋巴细胞染色体畸变分析。卫生研究。2004
69. Olofsson K K, Mark J. Specificity of asbestos-induced chromosomal aberrations in short-term cultured human mesothelial cell. Cancer Genet Cytogenet, 1989; 41: 33-39.
70.赵永良,金璀珍,刘秀林,吴德昌。辐射诱发大鼠器官上皮细胞染色体非随机改变。致癌.致畸.致突变。1997,9(2):71。
71. Kildal W, Kaern J, Kraggerud SM,et al. Evaluation of genomic changes in a large series of malignant ovarian germ cell tumors--relation to clinicopathologic variables. Cancer Genet Cytogenet. 2004 Nov; 155(1): 25-32.
72.刘毓谷主编.卫生毒理学基础(第二版).北京:人民卫生出版社.1998:95-96.
73. Ochi H.Cytogenetic studies in primary gastric cancer. Cancer Genet Cytogenet. 1986, 22:295.
74. Weaver DA, Hei TK, Hukku B, et al.Cytogenetic and molecular genetic analysis of tumorgenic human bronchial epithelial cells induced by radon alpha particles.Carcinogenesis. 1997, 18(6): 1251-1257.
75.王辉云,关新元,方燕,等。肝癌染色体DNA拷贝数的变化及其与临床病理 和预后的关系。癌症。1999,18(5):509-513.
76. Lu YJ,Dong Xy, Guo SP, et al.2q-,a non-random chromosomal abnormality in human non-small-cell lung cancer.Carcinogenesis. 1996,179(18): 1589-1593.
77. Konnol I, Morishita K, Takano H, et al.Homozygous deletions at chromosome 2q33 in human small-cell lung carcinoma identified by arbitrarily primed PCR Genomic fingerprinting.Oncogene. 1994, 9:103-108.
78. Douglass EC,Valentire M,Etcubanas E,et al.A specific chromosomal abnormality in rhabdomuosarcoma.Cytogenet Cell Genet. 1987,45:147-155.
79. Haglund U,Juliusson G, Stellan B,et al.Hairy cell leukemia is characterized by clonal chromosome abnormalties clustered to specific regions. Blood. 1994, 83:2637-2645.
80. Tran TA, Kallakury BVS, Sheehan CE, et al. Expression of CD_(44) standard from and variant isoforms in non-small cell lung carcinomas. Human Pathol, 1997, 28:809-814.
81. Wimmel A, Schilli M, Kaiser U, et al. Preferential histiotypic expression of CD_(44)-isoforms in human lung cancer. Lung Cancer, 1997, 16:151-172.
82.赵惠儒,方军,杜光烨,等。CD44在肺癌中表达的临床研究。中华结核和呼吸感染 1998年第9期第21卷
83. Gunthert U,Hofmann M,Rudy W, et al.A new variant glycoprotein CD_(44) confers metastatic potential to rat carcinoma cells.Cell, 1991,65: 13-24.
84.范小玲,张祥生,郝元瑞,等。CD44和nm23在肾脏肿瘤中表达及其临床意义。中华泌尿外科杂志 1999年第9期第20卷。
85. Ikoma T, Takahashi T, Nagano S,et al.A definitive role of RhoC in metastasis of orthotopic lung cancer in mice.Clin Cancer Res. 2004 Feb 1; 10(3): 1192-1200.
86. Shikada Y, Yoshino I, Okamoto T, et al.Higher expression of RhoC is related to invasiveness in non-small cell lung carcinoma.Clin Cancer Res. 2003 Nov 1; 9 (14): 5282-5286.
87. Zeng L, Cui W, Fang W.Differential expression and response of growth factors in metastatic variants of human pulmonary giant cell carcinoma cell line.Zhonghua Bing Li Xue Za Zhi. 1997 Apr; 26(2): 93-5.
88. Piyathilake CJ, Frost AR, Manne U, et al.Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung.Clin Cancer Res.
2.张敏磊,方福德.转导GST-pi基因表达抑制甲基丙烯酸环氧丙酯的致癌作用.科学通报.1999,13(1):1414-1418.
3.覃兆海,金淑惠,李楠编著.基础有机化学.北京:科学技术文献出版社.2004,1.
4.刘毓谷主编.卫生毒理学基础(第二版).北京:人民卫生出版社.1998:116-137.
5. Charles Heidelberger, et al.Cell transformation by chemical agents—a review and analysis of the literature,A report of the United States Environmental Protection Agency.Mutation Research. 1983; 114-283.
6. Engelhardt G, Schwind KR,Mubler B.The testing of chemicals in the Syrian hamster embryo (SHE) cell transformation assay for assessment of carcinogenic potential.Toxicology in Vitro. 18(2004)213-218.
7.刘国廉主编.细胞毒理学.北京:军事医学科学出版社.2001,287-313.
8. Berwald Y, Sachs L.In vitro cell transformation with chemical carcinogens.Nature, 1963; 200:1182.
9. Harris CC.Human tissues and cells in carcinogenesis research. Cancer Res.1987, 47(1):110.
10. Reddle RR,Ke Y, Breneda I,et al.Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 1988; 48:1904-1909.
11. Iizasa T, Momiki S,Harris CC,et al.Invasive tumors derived from xenotransplanted,immortalized human cells after in vivo exposure to chemical carcinogens.Carcinogenesis. 1993 Sep; 14(9): 1789-1794.
12. Van Agen B, Maas LM, Zwingmann IH, et al. B[a] P-DNA adduct formation and induction of human epithelial lung cell transformation. Environ Mol Mutagen. 1997; 30(3):287-92.
13. Bonfil RD,Momiki S,Harris CC.Enhancement of the invasive ability of a transformed human bronchial epithelial cell line by 12-o-tetradecanoyl-phorbol-13-acetate and diacylglycerol.carcinogenesis. 1989 Dec; 10(12):2335-2338.
14.袁雄,叶常青。重离子照射致人支气管上皮细胞突变效应。中华放射医学与防护杂志。1999,19(5):300-302.
15. Hornberg C,Maciuleviciute L,Seemayer NH.Human bronchcepithelial cells in vitro as a tool for detection of genotoxic activity of airborne particulates. Aerosol Sci.1996, Suppl 1:s35-s36.
16. Amstad P, Reddel RR, Pfeifer A, et al. Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras. Mol Carcinog. 1988; 1(3):151-160.
17. Pfeifer AM, Jones RT, Bowden PE, et al. Human bronchial epithelial cells transformed by the c-raf-1 and c-myc protooncogenes induce multidifferentiated carcinomas in nude mice: a model for lung carcinogenesis. Cancer Res. 1991 Jul 15; 51(14):3793-3801.
18. Reddel RR, Ke Y, Kaighn ME, et al. Human bronchial epithelial cells neoplastically transformed by v-Ki-ras: altered response to inducers of terminal squamous differentiation. Oncogene Res. 1988; 3(4):401-408.
19.袁素波,王治乔,叶常青,廖明阳.环磷酰胺、赛替派诱发人支气管上皮细胞的染色体畸变.癌变·畸变·突变.2000;12(4):205-211.
20.袁素波,王治乔,叶常青,廖明阳.环磷酰胺或塞替派诱导人支气管上皮细胞恶性转化的形态计量学.中国药理学与毒理学杂志.2000;14(5):346-351.
21.袁素波,王治乔,叶常青,廖明阳.环磷酰胺及塞替派诱导人支气管上皮细胞恶性转化的超微结构.中国药理学与毒理学杂志.2000;14(5):341-345.
22.袁素波,王治乔,叶常青,廖明阳.环磷酰胺诱导永生化人支气管上皮细胞恶性转化过程中的基因突变.中国药理学与毒理学杂志.2001;15(6):447-452.
23.袁素波,王治乔,叶常青,廖明阳.环磷酰胺诱发永生化人支气管上皮细胞的恶性转化.中国药理学与毒理学杂志.2001;15(6):441-446.
24.袁素波,王治乔,叶常青,廖明阳.塞替派诱发永生化人支气管上皮细胞的恶性转化(一)、(二).卫生毒理学杂志 2001;15(4):207-215.
25.陈光宇,袁素波,马华智,廖明阳.环磷酰胺及塞替派诱导的人支气管上皮恶性转化细胞的p16INK4a及p15INK4b基因突变.中国药理学与毒理学杂志.2002;16(2):133-137.
26.陈光宇,袁素波,马华智,廖明阳.环磷酰胺及塞替派诱导的人支气管上皮恶性转化细胞的细胞周期相关蛋白表达.中国药理学与毒理学杂志.2002;16(2):138-142.
27.周喆,袁素波,廖明阳.塞替派、环磷酰胺诱发的人支气管上皮恶性转化细胞的致瘤性.中国药理学与毒理学杂志.2002;16(3):216-219.
28.周喆,袁素波,廖明阳.塞替派诱发人支气管上皮恶性转化成瘤细胞的染色体畸变.中国药理学与毒理学杂志.2002;16(4):302-305.
29. IARC/NCI/TEPA Working Group.Cellular and molecular mechanisms of cell transformation and standardization of transformation assays of established cell lines for prediction of carcinogenic chemicals.Cancer Res, 1985, 45(1):2395-2399.
30.张煦,赵俊生.肺癌组织学类型的光镜、电镜比较观察[J].兰州大学学报(自然科学版),1998,34(1):85-89.
31. Weinberger JH, Williams GM.Carcinogen testing: Current problems and new approaches.Sience, 1981 (214), 23:401-407.
32. Tomatis L. Cell proliferation and carcinogenesis: a brief history and current view based on an IARC workshop report. International Agency for Research on Cancer.Environment health perspective. 1993, 101 (suppl 5): 149-51.
33. Folyd RA.The role of 8-hydroxyguanine in carcinogenesis.Carcinogenesis.1990, 11(9): 1447-50.
34. Seleznera TG, Korman NP.Analysis of chromosomes of somatic cells in patients treated with antitumor drugs.Sov Genet. 1973, 9:1575-1579.
35. Myhr BC.Validation studies with muta mouse: A transgenic mouse model for detecting mutations in vivo.Environ Mol Mutagen. 1991, 18:308.
36. Sheu CW, Lee LK, Rodriguez I, et al.The use of uninduced rat liver s-9 to supplement BALB/3T3 cells in the in vitro transformation assay.Drug and chemical toxicology. 1991, 14:113-126.
37. Stoner GD, Shimkin MB, Kniazeff AJ, et al.Test for carcinogenicity of food additives and chemotherapeutic agents by the pulmonary tumor response in stain A mice.Cancer Res. 1973, 33:3069-3085.
38. Siberberg JM, Hosein Zarrabi M.Acute nonlymphocytic leukemia after thiotepa instillation into the bladder: report of 2 cases and review of the literature.J Urol. 1987, 138:402-403.
39. IARC. Thiotepa study in pharmaceutical drugs[C]. Lyon. IARC Scientific Publications. 1990, 50: 123-141.
40. Milic M, Kopjar N.Evaluation of in vitro genotoxic activity of bleomycin and mitomycin C in human lymphocytes using the alkaline comet assay. Arh Hig Rada Toksikol. 2004.55(4):249-259.
41. Kimball RF.The development of ideas about the effect of DNA repairs on the induction of gene mutation and chromosomal aberrations by radiation and by chemicals.Mutat Res. 1987, 186:1-34.
42. Ueda K,Komano T. Sequence-specific DNA damage induced by reduced mitomycin C and 7-N-(p-hydroxyphenyl)mitomycin C. Nucleic Acids Res. 1984; 12(17): 6673-6683.
43.袁伯俊,王治乔,廖明阳,等。新药临床前安全性评价与实践。北京:军事医学科学出版社,1997:101-102.
44. Dedrich RL, Morrison PF.Carcinogenic potency of alkylating agents in rodent and human.Cancer Res. 1992, 52:2464.
45. Radis CD, Kahl LE, Baker GL, et al.Effects of cyclophosphamide on the development of malignancy and on long-term survival of patients with rheumatoid arthritis.A 20 years follow up study.Arthritis Rheum. 1995.38:1120.
46. Stein JP, Skinner EC,Boyd SD,et al.Squamous cell carcinoma of the bladder associated with cyclophosphamide therapy for Wegener's granulomatosis:a report of 2 case.J Urol. 1993.149:588.
47. Ortiz A, Gonzelez-parra E, Alvarez-Costa, et al.Bladder cancer after cyclophosphamide therapy for lupus nephritis.Nephron. 1992, 60:378.
48. Miyamae Y, Zaizen K, Ohara K, et al.Detection of DNA lesions induced by chemical mutagens by the single cell electrophoresis (Comet) assay. 1. Relationship between the onset of DNA damage and the characteristics of mutagens. Mutat Res. 1998 Jul 31; 415(3):229-35.
49. Morales RP, Vallarino KT, Anguiano OG. Pharmacokinetic parameters of genotoxic activity inferred from the comparison of the kinetics of MN-PCE induced by chemical agents and ionizing radiation. Mutat Res. 1997 Jul 14; 391(3):127-34.
50. IARC (IARC (1986) IARC Monographs on the Evaluation of Carcinogenic Risk to Humans. Vol. 40. Some NaturallyOccurring and Synthetic Food Components, Furocoumarins and Ultraviolet Radiation. Butylated hydroxyanisole (BHA). International Agency for Researchon Cancer, Lyon. 123-206.
51. Witschi, H.P., Malkinson, A.M. and Thompson, JA.Metabolism and pulmonary toxicity of butylated hydroxytoluene.In:T.E.Gram, (Ed), metabolic activation and toxicity of chemical agents to lung tissue and cells, Pergamon Press, Ltd, UK, 1993, 185-212.
52. A.M. Malkinson, K.M. Koski, W.A. Evans, M.F. Festing, Butylated hydroxytoluene exposure is necessary to induce lung tumors in BALB mice treated with 3-methylcholanthrene,Cancer Res. 57 (1997) 2832-2834.
53. Williams G. M., Iatropoulos M. J. and Whysner J. Safety assessment of butylated hydroxyanisole and butylated hydroxytoluene as antioxidant food additives. Food and Chemical Toxicology, 1999, 37, 1027-1938.
54. Yude Sun, Dwyer-Nield L.D., Malkinson A.M., et al. Responses of tumorigenic and non-tumorigenic mouse lung epithelial cell lines to electrophilic metabolites of the tumor promoter butylated hydroxytoluene. Chemico-Biological Interactions 145 (2003) 41-51.
55.谭曾鲁,周柔丽,主编。医学细胞生物学。北京:北京医科大学出版社。1992:233-293.
56.张进华,丁彦秋,杨学皆。介绍一种细胞骨架蛋白改良染色发。诊断病理学杂志。1996,3(4):235-236.
57. Boers JE,Ambergen AW, Thunnissen FB.Number and proliferation of basal and parabasal cells in normal human airway epithelium.AM J Respir Crit Care Med, 1998;157:2000-2006.
58. Boers JE, Den Brok JLM, Koudstaal J, et al. Number and proliferation of neuroendocrine cells in normal human airway epithelium[J]. AM J Respir Crit Care Med, 1996, 154: 758-763.
59. Kirkham S,Sheehan JK,Knight D,et al.Heterogeneity of airways mucus:variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B.Bioche m J,2002,361 (Pt3):537-546.
60.董波,陈肖华,张浩,等。一种改进的用于测定细胞周期的细胞制备方法。军事医学科学院院刊。2002,26(2):124-129.
61.孙秀泓,罗自强,秦晓群,等.肺的非呼吸功能基础与临床[M].第1版.北京:人民卫生出版社,2003:222-223.
62. Boers JE, Ambergen AW, Thunnissen FB. Number and proliferation of Clara cells in normal human airway epithelium[J]. AM J Respir Crit Care Med, 1999, 159: 1 585-1 591.
63. Soderberg M. Structural characterization of bronchial mucosal biopsies from healthy volunteers: A light and electron microscopical study[J]. Eur Respir J, 1990, 3: 261.
64.熊敏,吴一龙,主编.现代肺癌病理与临床.北京:科学出版社,2003.109-110.
65. Otto W.R.Lung epithelial stem cells.J Pathol 2002,197: 527-535.
66. Barrett JC.Mechanisms of action of known human carcinogens.IARC Sci Publ.1992, 11(6):115-134.
67. Vogelstein B, Kinzler KW.The multistep nature of cancer.Trends Genet. 1993.9 (4): 138-141.
68.纪之莹.李桂兰.李凌凛,等。苯接触工人外周血淋巴细胞染色体畸变分析。卫生研究。2004
69. Olofsson K K, Mark J. Specificity of asbestos-induced chromosomal aberrations in short-term cultured human mesothelial cell. Cancer Genet Cytogenet, 1989; 41: 33-39.
70.赵永良,金璀珍,刘秀林,吴德昌。辐射诱发大鼠器官上皮细胞染色体非随机改变。致癌.致畸.致突变。1997,9(2):71。
71. Kildal W, Kaern J, Kraggerud SM,et al. Evaluation of genomic changes in a large series of malignant ovarian germ cell tumors--relation to clinicopathologic variables. Cancer Genet Cytogenet. 2004 Nov; 155(1): 25-32.
72.刘毓谷主编.卫生毒理学基础(第二版).北京:人民卫生出版社.1998:95-96.
73. Ochi H.Cytogenetic studies in primary gastric cancer. Cancer Genet Cytogenet. 1986, 22:295.
74. Weaver DA, Hei TK, Hukku B, et al.Cytogenetic and molecular genetic analysis of tumorgenic human bronchial epithelial cells induced by radon alpha particles.Carcinogenesis. 1997, 18(6): 1251-1257.
75.王辉云,关新元,方燕,等。肝癌染色体DNA拷贝数的变化及其与临床病理 和预后的关系。癌症。1999,18(5):509-513.
76. Lu YJ,Dong Xy, Guo SP, et al.2q-,a non-random chromosomal abnormality in human non-small-cell lung cancer.Carcinogenesis. 1996,179(18): 1589-1593.
77. Konnol I, Morishita K, Takano H, et al.Homozygous deletions at chromosome 2q33 in human small-cell lung carcinoma identified by arbitrarily primed PCR Genomic fingerprinting.Oncogene. 1994, 9:103-108.
78. Douglass EC,Valentire M,Etcubanas E,et al.A specific chromosomal abnormality in rhabdomuosarcoma.Cytogenet Cell Genet. 1987,45:147-155.
79. Haglund U,Juliusson G, Stellan B,et al.Hairy cell leukemia is characterized by clonal chromosome abnormalties clustered to specific regions. Blood. 1994, 83:2637-2645.
80. Tran TA, Kallakury BVS, Sheehan CE, et al. Expression of CD_(44) standard from and variant isoforms in non-small cell lung carcinomas. Human Pathol, 1997, 28:809-814.
81. Wimmel A, Schilli M, Kaiser U, et al. Preferential histiotypic expression of CD_(44)-isoforms in human lung cancer. Lung Cancer, 1997, 16:151-172.
82.赵惠儒,方军,杜光烨,等。CD44在肺癌中表达的临床研究。中华结核和呼吸感染 1998年第9期第21卷
83. Gunthert U,Hofmann M,Rudy W, et al.A new variant glycoprotein CD_(44) confers metastatic potential to rat carcinoma cells.Cell, 1991,65: 13-24.
84.范小玲,张祥生,郝元瑞,等。CD44和nm23在肾脏肿瘤中表达及其临床意义。中华泌尿外科杂志 1999年第9期第20卷。
85. Ikoma T, Takahashi T, Nagano S,et al.A definitive role of RhoC in metastasis of orthotopic lung cancer in mice.Clin Cancer Res. 2004 Feb 1; 10(3): 1192-1200.
86. Shikada Y, Yoshino I, Okamoto T, et al.Higher expression of RhoC is related to invasiveness in non-small cell lung carcinoma.Clin Cancer Res. 2003 Nov 1; 9 (14): 5282-5286.
87. Zeng L, Cui W, Fang W.Differential expression and response of growth factors in metastatic variants of human pulmonary giant cell carcinoma cell line.Zhonghua Bing Li Xue Za Zhi. 1997 Apr; 26(2): 93-5.
88. Piyathilake CJ, Frost AR, Manne U, et al.Differential expression of growth factors in squamous cell carcinoma and precancerous lesions of the lung.Clin Cancer Res.