摘要
目的：原发性肝癌是目前发病率高、治疗困难、死亡率高的恶性肿瘤。迄今已建立的一系列人肝癌细胞系(cell line)和人肝癌细胞系的动物模型，为肝癌的发病机理和治疗研究奠定了良好的基础。由于每一种肝癌细胞的染色体特性和癌基因位点均有所不同，而且体外建立的人肝癌细胞系会发生转化，包括基因的丢失、基因的漂移等，和体内的实体瘤之间有显著的差异，同一种细胞系经过反复传代后也会发生转化，这样就给实验的结果带来不确定性。临床及实验资料说明现有的肝癌细胞系资源还远远不能满足研究的需要。因此建立新型人肝癌细胞系有助于研究肝癌发病、进展、转移机制的共同点和不同点。同时可以对多种肝癌细胞系进行药物敏感性实验研究，有望筛选出对某一类肝癌有效的药物，指导临床治疗。
方法：
1．人肝癌细胞系的建立
组织块培养法对活体人肝癌标本进行原代培养；采用自然纯化和反复贴壁法行单一肝癌细胞纯化；光学显微镜观察细胞生长情况；胰蛋白酶法消化传代培养细胞；采用慢冻和快融进行细胞的冻存和复苏。
第四军医大学硕士学位论文
2.人肝癌细胞系FHCC一98的生物学特性鉴定
细胞形态学观察采用HE染色，光镜观察;超微结构用电
镜观察;胎盘蓝染色计数检测冻存后复苏存活率;细胞计数法
测定贴壁率;MTT法测细胞生长期曲线;流式细胞仪分析细
胞周期和DNA组成;常规制备染色体标本，拍照分析核型;
软琼脂克隆形成实验检测细胞克隆形成率;ConA凝集实验检
测细胞的聚集特性;Westem一blotting和免疫组织化学法分析
CK、AFP和肝癌相关抗原HAb 1 SG/CD147在细胞中的表达;
采用PAGE检测FHCC一98的乳酸脱氢酶同工酶谱，拍照分
析;裸鼠皮下接种FHCC一98细胞悬液，接种后观察成瘤情
况，计算异种移植率;透射电子显微镜鉴定细胞系的污染情
况。
3.药物对肝癌细胞的敏感性研究
3.1 MTT法测定药物对肝癌细胞生长的抑制率
用taxotere、ADM作用于FHee一95和MHee一97一L肝癌细
胞，在不同时间点和不同浓度在ELISA仪上测定吸光值，计
算药物抑制率。
3.2侵袭实验测定药物对肝癌细胞运动能力的抑制
应用Millieell一PCF小室置于24孔板，实验组加Taxotere
和ADM，对照组不加药，小室滤膜HE染色后在100倍显微
镜下观察照相，图像分析仪计数后计算抑制率。
3.3 SCGE测定药物对肝癌细胞FHCC一98 DNA的损伤
采用两层制胶法，细胞裂解后在碱性条件下电泳，莹光显
微镜观察Taxotere和ADM对细胞核的损伤。
结果:
1.细胞原代培养早期，生长不稳定，细胞传代培养时需要较
高的接种密度，而且细胞倍增时间不同。到第十二代时细胞生
长已非常稳定。细胞呈上皮样，大小不一，以多角形、多边形
第4页
第四军医大学硕士学位论文
为主，占95%以上。细胞在高密度时(>l xlo。/mL)可见重叠
及堆积生长。传代时间一般为2一3天。
2.FHCC一98细胞生物学特性鉴定
透射电镜下见细胞大小不等，以不规则型为主，细胞表面
有丰富微绒毛，细胞间有桥粒紧密连接等，有少量连接复合
体;细胞间有形成腺结构及条索结构倾向，可形成类似胆小管
样结构;细胞质丰富，有大量糖原颗粒形成糖原湖样结构;线
粒体、粗面内质网较发达，有丰富的游离核糖体;细胞核增
大，核浆比例增大，核不规则，有多核细胞，核膜明显凹陷，
核仁1一5个不等;核异型性明显，核内以常染色质为主，异
染色质较少，可见有核分裂像，偶见异常核分裂。冻存后复苏
存活率为95%。细胞生长曲线显示上皮样恶性肿瘤生长特性，
细胞群体倍增时间为ZI.4h，sh后贴壁率已超过90%，细胞
能在软琼脂中生长，克隆形成率为32%。DNA倍体分析为为
四倍体，DNA周期分析:G:为75%，GZ为7%，S为18%，
DNA指数为1 .8 57。染色体众数以三倍体为主。LDH同工酶
谱显示四条带，缺少LDH;，LDH;和LDHS含量较高。FHCC-
98细胞的肝癌相关抗原HAb 18G/C D 147和细胞角蛋白均显示
强阳性，而AFP阴性。FHCC一98细胞在低浓度ConA中出现
聚集。FHCC一98细胞在裸鼠中的成瘤性为100%。透射电镜检
测FHCC一98细胞无支原体污染。
3.抗癌药物对不同肝癌细胞的抑制率
3.1经 Taxotere处理的FHCC一98细胞的增殖有明显的抑制，且
随时间和浓度的增加其抑制率增加，药物在SX10一Zog/mL浓
度作用FHCC一98细胞48h后其增殖抑制率达到30%以上，而
对MHCC一97一L细胞无作用。ADM对两株肝癌细胞均有作用，
较Taxotere的作用为强，新建细胞系FHCC一98对ADM的敏感
性较MHCC一97一L强，经Prism统计软件分析不同浓度药物的
第5页
第四军医大学硕士学位论文
抑制率均有统计学意义(P<0.05)。
3.2侵袭小室实验显示Taxotere对FHCC一98的穿膜能力有明显
的抑制，且呈浓度依赖性。ADM结果不确定。
3.3 SCGE显示阿霉素对FHCC一98细胞的DNA有一定程度的损
伤。细胞核出现部分碎裂，细胞核电泳出现彗星现象。而泰索
帝组没有出现彗星想象。
结论:
1.由人肝癌组织培养的FHCC一98细胞系能稳定生长，具有无
限生长的能力。该细胞系保持了来源组织的一些特性，AFP和
HBsAg阴性，细
Aims: Hepatocellular carcinoma (HCC) is a kind of malignant disease that is difficult to treat and has a high incidence and a high mortality at present. The established cell lines and animal models of human liver cancer have settled a good foundation for study of pathogenesis and treatment of HCC. There are variations of chromosomes and genie location among HCC cell lines, and the established HCC cell lines in vitro could be transformed, include loss and drift of genes, etc, also there is obvious difference between cell lines and their original tissue. Yet the same cell line could be transformed after passaged repeatedly, so the test results are uncertainty. Clinic and laboratory data shows limit resource of cell lines can't satisfy the needs of study. More kinds of new hum-
an HCC cell lines are needed, and can be help to study the common
and different points of pathogenesis, progress and metastasis of
liver cancer. Drug sensitivity can be tested through kinds of HCC
cell lines, so that some efficient drugs can be screened against
certain liver cancer and provide instruction on clincic treatment.
Methods:
1.Establishment of human HCC cell line
The sample of human HCC was primary cultured by tissue culturing. The single HCC cell were obtained by natural purification and anchoring repeatedly. The cells were passaged with trypsin. Growth status was observed under light microscope.The cells were freezed with temperature down slowly in liquid nitrogen and resuscitated quickly at 37C. 2.Characteristics of human HCC cell line FHCC-98.
FHCC-98 cell morphology was observed under light and electron microscope. The survival rate was measured by counting cells stained with protamine Blue.Seeding efficiency was measured by counting cell number every 2 h. Cell growth curve was plotted with MTT assay. Studies of cell cycle and stage were performed by flow cytometry.Chromosomes was made by traditional method and analyzed by photographed. Cloning efficiency was measured by counting clone number growing in soft agar. The coagulation characteristics was detected by ConA test. Expressions of tumor markers such as AFP, CK and HAbl8G/CD147 were detected by SP immunocytochemistry or Western blotting. LDH isoenzymes were detected by polyacrylamid gel PAGE and photographed analyzed. Xenograft was performed by inoculating FHCC-98 cells
into the flanks of the nude mice s.c and the tumors were observed, thus xenograft rate was counted. Cell contamination was detected by transmission electron microscopy.
3.Study of drug sensitivity against HCC cell lines
3.1 MTT assay was used to detect the inhibition rate of HCC cell growth
Taxotere and ADM were used to effect on FHCC-98 and MHCC-97-L cells. OD was measured by ELISA at different time and concentration, and inhibition rate was counted.
3.2 Invasion test was used to investigate the mobility inhibition of FHCC-98
Millicell-PCF transwell chambers were put in 24-plate, taxotere and ADM were added to test group, and no drugs in controlled one. Transwellchamber's membrane was HE stained and photographed. Inhibition rate was measured with imaging analyser.
3.3 DNA damage of FHCC-98 cell was dectected by SCGE
Two layers of gel was made, and FHCC-98 cells were el-ectrophoresised in alkaline condition after lysis, then nucleus damage by taxotere and ADM was observed under fluorescence microscope. Results:
1. Cells grew instably at the early stage of primary culture and needed a high density when passaged. Population doubling time was not the same. But the cells grew very stably at 12th passage. Cells were mainly polygonal epithelial-like in various size over 95%. Cells grew in overlap and piled up in high density (>1 X 106/mL). Passaging time was 2 -3 days average.
2. Transmission electron-microscopy showed cells were in
various size, mainly irregular type. There were abundant microvilli on the cell surface. Desmosomes and gap junction could be seen between cells, also with a few junctional complexs. There was a tendency to form structure of gland and strand alike. St
引文
1. Yu AS, Keeffe EB. Management of hepatocellular carcinoma. Rev Gastroenterol Disord 2003; 3:8-24.
2. El Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Meal 1999;340:745-750.
3. Kiyosawa K,Tanaka E.Characteristics of hepatocellular carcinoma in Japan. Ontology 2002;62 Suppl 1:5-7.
4. Tang ZY.Hepatocellular carcinoma-cause,treatment and metastasis. World J Gastroenterol 2001;7:445-454.
5．陆建华，陈建国，倪正平，等．HBs 携带状态与肝癌关系的十年随访研究[J)．中华预防医学杂志，1988，22(5)：259-262．
6．陆东东．肝炎病毒与肝癌[J]．中国肿瘤与临床杂志，1997，4(1)：90-93．
7. Jayshree RS, Sridhar H, Devi GMSurface, core, and X genes of hepatitis B virus in hepatocellular carcinoma: an in situ hybridization study. Cancer. 2003 Feb 25;99(1):63-7.
8. Nijhara R, Jana SS, Goswami SK, Rana A, Majumdar SS, Kumar V, Sarkar DP. Sustained activation of mitogen-activated protein kinases and activator protein 1 by the hepatitis B virus X protein in mouse hepatocytes in vivo. J Virol. 2001 Nov;75(21): 10348-58.
9．金顺钱，詹启敏．肿瘤分子生物学是基础医学研究的前沿．世界华人消化杂志，2002；10：678-680．
10. Lakhtakia R, Kumar V, Reddi H, Mathur M, Dattagupta S, Panda SK. Hepatocellular carcinoma in a hepatitis B 'x' transgenic mouse model:A sequential pathological evaluation. J Gastroenterol Hepatol. 2003 Jan;18(1):80-91.
11. Chun E,Lee J,Cheong HS, Lee KY.Tumor eradication by hepatitis B virus X antigen-specific CD8+ T cells in xenografted nude mice. J Immunol. 2003 Feb 1;170(3):1183-90.
12. Greenblatt MS, Feitelson MA, Zhu M, Bennett WP, Welsh JA, Jones R, Borkowski A, Harris CC. Integrity of p53 in hepatitis B x antigenpositive and -negative hepatocellular carcinomas. Cancer Res. 1997 Feb 1;57(3):426-32.
13. Sheu JC, Huang GT, Shih LN, Lee WC, Chou HC, Wang JT, Lee PH, Lai MY, Wang CY, Yang PM, et al. Hepatitis C and B viruses in hepatitis B surface antigen-negative hepatocellular carcinoma. Gastroenterology. 1992 Oct; 103(4): 1322-7.
14. Paterlini P, Gerken G, Nakajima E, Terre S, D'Errico A, Grigioni W, Nalpas B, Franco D, Wands J, Kew M, et al. Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen. N Engl J Med. 1990 Jul 12;323(2):80-5.
15. Tamoria A et al. J Med Virol,2003;69(4):475-481.
16. Munoz N, Bosch X. Epidemiology of hepatocellular carcinoma. Neoplasms of the liver. 1987:3
17．姚登福，孟宪镛，伊东进，等．丙型肝炎病毒核心蛋白基因表达及其核苦酸序列的分析．江苏医药，1995，21：366．
18. Shimizu I,Yao DF,Horie C,et al.Mutations in a hydrophilic part of the core gene of hepatitis C:virus from patients hepatocellular carcinoma. Shanghai International Conference of Gastroentrology. 1996.A272.
19. Suga M,Senota A,Arima K,Kodama T,Ikuta S,Sakamoto H,Ohe Y, Sugai S,Yachi A.Prevalence of HBV and HCV infection in Japanese patients with hepatocellular carcinoma. Hepatogastroenterology. 1994;41 (5):438-441.
20. Liang TJ,Jeffers LJ,Reddy KR,De Medina M,Parker IT, Cheinquer H,Idrovo V,Rabassa A,Schiff ER.Viral pathogenesis of hepatocellular carcinoma in the United States. Hepatology, 1993; 18(6): 1326-1333.
21．葛向华，王法印．HGV 感染与原发性肝癌的关系．临床肝脏病杂志．2000，16(3)：187-188．
22．涂文升，等．广西肝癌高低发区儿童摄入黄曲霉素B1与尿中排出黄曲霉素M1的关系研究．中华预防医学杂志．1993，27(4)：218．
23. Turner PC,SylIa A,Diallo MS,Castegnaro JJ,Hall AJ,Wild CP.The role of aflatoxins and hepatitis viruses in the etiopathogenesis of hepatocellular carcinoma:A basis for primary prevention in GuineaConakry,West Africa.J Gastroenterol Hepatol.2002 Suppl: S441-8.
24. Hsu IC,Metcalf RA,Sun T,Welsh JA,Wang NJ,Harris CC. Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature. 1991 Apr 4;350(6317):427-8.
25. Bressac B,Kew M,Wands J,Ozturk M.Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature. 1991 Apr 4;350(6317):429-31.
26．班克臣，苏建家，杨春，黄国华，覃柳亮，李瑗，罗小玲，段小娴，严瑞琪．人乙型肝炎病毒与黄曲霉毒B1诱发树Qu肝癌过程中肝组织p21表达与肝癌发生的关系中华肿瘤杂志，1998；20(3)：111．
27．陆培新，张启南，王金兵，吴燕．乙肝病毒和黄曲图毒素与肝癌发生的关系．中国肿瘤，1999；8(7)：305-306．
28. Martini GA.The role of alcohol in the etiology of cancer of the liver. Prevention and detection of cancer.. 1980;2163.
29. Adami HO,Hsing AW, McLaughlin JK, Trichopoulos D, Hacker D, Ekbom A, Persson Ⅰ. Alcoholism and liver cirrhosis in the etiology of primary liver cancer. Int J Cancer. 1992 Jul 30;51 (6):898-902.
30．陈君石．癌症的预防．中国肿瘤，19943(6)：3-6．
31. O' Neil IK.Eds.N-Nitroso compounds,occurrence,biological effects and relevance to human cancer.IARC Scientific Publications, No1984,57.
32．艾兆伟，查锡良，汤华，等．维甲酸对人肝癌细胞一些表型的逆转作用．中华肿痛杂志，1991，13(1)：9-12．
33. Ng IO,Liu CL,Fan ST,et al.Expression of p-glycoprotein in hepatocellular carcinoma:a determinant of chemotherapy response [J].Am J Clin Pathol, 2000,113:335.
34. Seno H,Ito K,Kojima K,Nakajima N,Chiba T.Efficacy of an im planted drug delivery system for advanced hepatoceilular carcinoma using 5-fluorouracil,epirubicin and mitomycin C.J Gastroenterol Hepatol. 1999 Aug;14(8):811-6.
35．石学涛，李胜，衣龙海．吉西他滨肝动脉灌注治疗晚期肝癌．中国癌症杂志．2003，13(3)．-280-280，286．
36．涂水平，吴达明．肝动脉灌注羟基喜树碱和栓塞治疗原发性肝癌．世界华人消化杂志．1999,7(2)．-158-160．
37. Matsuda I,Konno H,Tanaka T,Nakamura S.Antimetastatic effect of hepatotropic liposomal adriamycin on human metastatic liver tumors. Surg Today. 2001 ;31 (5):414-20.
38．汤钊猷主编．现代肿瘤学上海．上海医科大学出版社，1986，177-188．
39．陈瑞铭．一株人体肝癌细胞株的建立和一些初步的观察．肿瘤研究论文集．1962，39-47．
40. Alexander JJ, Bey EM, Geddes EW, Lecatsas G.Establishment of a continuously growing cell line from primary carcinoma of the liver. S Afr Med J. 1976 Dec 18;50(54):2124-2128.
41. Daemer RJ, Feinstone SM, Alexander JJ, Tully JG, London WT, Wong DC, Purcell RH.PLC/PRF/5 (Alexander) hepatoma cell line:
further characterization and studies of infectivity. Infect Immun. 1980 Nov;30(2):607-611.
42. Marion PL, Salazar FH, Alexander JJ, Robinson WS.Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line. J Virol. 1979 Dec;32(3):796-802.
43. Morris KM,Aden DP,Knowles BB,Colten HR.Complement biosynthesis by the human hepatoma-derived cell line HepG2. J Clin Invest. 1982 Oct;70(4):906-913.
44. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J.Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982 Sep;42(9):3858-3863.
45．郭秀婵，滕智，等．乙型肝炎病毒和黄曲霉素协同作用诱发人肝细胞癌细胞株的建立．病毒学报．2001，17(3)．-205-209．
46．王金兵，朱德厚，叶秀珍，陈瑞铭．启东肝癌细胞系QGY-7703的建立及其特征．中华肿瘤杂志，1981，3(4)：243．
47．董荣春，周荣华，吕发度，陶文照 SMMC-7721 人肝癌细胞株的建立及其生物学特性的初步观察．第二军医大学学报，1980,(1)：5-9．
48. Aoki Y,Aizaki H,Shimoike T,Tani H, Ishii K,Saito I, Matsuura Y, Mi yamura T.A human liver cell line exhibits efficient translation of HCV RNAs produced by a recombinant adenovirus expressing T7 RNA polymerase. Virology, 1998; 250: 140-150.
49. Tian J,Tang ZY,Ye SL,Liu YK,Lin ZY,Chen J, Xue Q.New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97) and its expressions of the factors associated with metastasis. Br J Cancer. 1999 Nov; 81(5): 814-821.
50. Li Y, Tang ZY, Ye SL, Liu YK, Chen J, Xue Q, Chen J, Gao DM, Bao WH.Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol. 2001 Oct; 7 (5):630-636.
51．李雁，汤钊猷，叶胜龙，刘银坤，陈洁，薛琼，黄晓武，陈军，鲍卫华，杨炯，高东梅．体内连续筛选法建立自发性肺转移人肝癌细胞系．中华医学杂志，82(9)：601-605．
52．外生型肝癌细胞系的建立及生物学特性研究．吴小鹏，王占民，刘博，刘军，高英茂，李兆亭，刘春生．中华外科杂志，2002，40(8)：616-617．
53. Murakami T,Yano H,Maruiwa M,Sugihara S,Kojiro M. Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice. Hepatology, 1987;7:551-556.
54. Sing GK, Pace R, Prior S, Scott JS, Shield P, Martin N, Searle J, Batt ersby C, Powell LW, Cooksley WG. Establishment of a cell line from a hepatocellular carcinoma from a patient with hemochromatosis. Hepatology, 1994; 20:74-81
55. Dippold WG,Dienes HP,Knuth A,Sachsse W,Prellwitz W,BitterSuermann D,Meyer zum Buschenfelde KH.Hepatocellular carcinoma after thorotrast exposure: establishment of a new cell line (Mz-Hep-1). Hepatology. 1985 Nov-Dec;5(6):1112-1119.
56. Tsurimoto T,Fujiyama A,Matsubara K.Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA. Proc Natl Acad Sci U S A. 1987 Jan; 84 (2):444-448.
57．谭龙益，王皓，孔宪涛．表达HBV的SMMC—7721 人肝癌细胞系模型的建六．第二军医大学学报，1998，19(2)：120-122．
58. Song ZQ,Hao F,Min F,Ma QY,Liu GD.Hepatitis C virus infection of human hepatoma cell line 7721 in vitro. World J Gastroenterol. 2001 Oct;7(5):685-689.
59. Dash S,Halim AB,Tsuji H,Hiramatsu N,Gerber MA.Transfection of HepG2 cells with infectious hepatitis C virus genome. Am J Pathol. 1997 Aug; 151 (2):363-373.
60. Yoo BJ,Selby MJ,Choe J,Suh BS,Choi SH, Joh JS, Nuovo GJ, Lee HS, Houghton M, Han JH.Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transcribed hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. J Virol. 1995 Jan;69(1):32-38.
61．杨俊霞，汤为学．人肝癌顺铂耐药细胞系的建立及其生物学特征．癌症，2002，21(8)：872-876．
62．张洪新，刘燕，等．耐药人肝癌细胞模型—7721／Adm的建立．第四军医大学学报．2000，21(4)．425-429．
63. Ebinuma H,Saito H,Kosuga M,Wakabayashi K, Saito Y,Takagi T, Nakamoto N,Okuyama T, Ishii H.Reduction of c-myc expression by an antisense approach under Cre/loxP switching induces apoptosis in human liver cancer cells. J Cell Physiol. 2001 Jul;188(1):56-66.
64. Farshid M,Tabor E.Expression of oncogenes and tumor supperssor genes in human hepatocellular carcinoma and hepatoblastoma cell lines. J Med Virol. 1992 Dec;38(4):235-9.
65. Saito H,Morizane T,Watanabe T,Kagawa T,Miyaguchi S, Kumagai N, Tsuchimoto K, Tsuchiya M.Proto-oncogene expression in three
human hepatoma cell lines, HCC-M, HCC-T and PLC/PRF/5. Keio J Med. 1991 Sep;40(3):139-45.
66. Li MS,Li PF,He SP,Du GG,Li G.The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Be17402 cell line. World J Gastroenterol. 2002 Jun;8(3):469-75.
67. Obora A,Shiratori Y,Okuno M,Adachi S,Takano Y, MatsushimaNishiwaki R,Yasuda I,Yamada Y,Akita K,Sano T,Shimada J, Kojima S,Okano Y,Friedman SL,Moriwaki H.Synergistic induction of apoptosis by acyclic retinoid and interferon-beta in human hepatocellular carcinoma cells. Hepatology. 2002 Nov;36(5):1115-1124.
68. Wang Z,Ruan YB,Guan Y,Liu SH.Expression of IGF-Ⅱ in early experimental hepatocellular carcinomas and its significance in early diagnosis. World J Gastroenterol. 2003 Feb;9(2):267-70.
69．万军，王孟薇．肝癌细胞系，肝癌组织及癌旁肝硬化组织中异常凝血酶原水平的研究．中华消化杂志．1994，14(5)．259-261．
70. Favoulet P, Cercueil JP, Faure P, Osmak L, Isambert N, Beltramo JL, Cognet F, Krause D, Bedenne L, Chauffert B.Increased cytotoxicity and stability of Lipiodol- pirarubicin emulsion compared to classical doxorubicin-Lipiodol: potential advantage for chemoembolization of unresectable hepatocellular carcinoma. Anticancer Drugs. 2001 Nov; 12(10):801-6.
71. Abiru S, Nakao K, Ichikawa T, Migita K, Shigeno M, Sakamoto M, Ishikawa H, Hamasaki K, Nakata K, Eguchi K.Aspirin and NS-398 inhibit hepatocyte growth factor-induced invasiveness of human hepatoma cells. Hepatology. 2002 May;35(5): 1117-24.
72．韩雨生，刘予川．p53 转染对肝癌细胞系 Hep3B 的作用．癌症．1999，18(6)．652-656．
73. Kao ST, Yeh CC, Hsieh CC, Yang MD, Lee MR, Liu HS, Lin JG. The Chinese medicine Bu-Zhong-Yi-Qi-Tang inhibited proliferation of hepatoma cell lines by inducing apoptosis via G0/G1 arrest. Life Sci. 2001 Aug 17;69(13):1485-96.
74. Ofuchi T,Suzuki M,Kase Y,Ando K, Isono K,Ochiai T.Chromo-some breakage and cell lethality in human hepatoma cells irradiated with X rays and carbonion beams. J Radiat Res (Tokyo). 1999 Jun;40(2):125-33.
75. Bouchard MJ, Puro RJ, Wang L, Schneider RJ.Activation and inhibition of cellular calcium and tyrosine kinase signaling pathways
identify targets of the HBx protein involved in hepatitis B virus replication. J Virol. 2003 Jul;77(14):7713-9.
76. Sureau C, Romet-Lemonne JL, Mullins JI, Essex M.Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell. 1986 Oct 10;47(1):37-47.
77. Alisi A, Giambartolomei S, Cupelli F, Merlo P, Fontemaggi G, Spaziani A, Balsano C.Physical and functional interaction between HCV core protein and the different p73 isoforms. Oncogene. 2003 May 1;22(17):2573-80.
78. Wood RP, Katz SM, Ozaki CF, Monsour HP, Gislason GT, Kelly JH, Sussman NL.Extracorporeal liver assist device (ELAD): a preliminary report. Transplant Proc. 1993,Aug;25(4 Suppl 3):53-54.
79. Albini A,Iwamoto Y,Kleinman HK,Martin GR,Aaronson SA, Kozlowski JM,McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 1987;47 (12): 3239-3245.
80. Erkell LJ,Schirrmacher V.Quantitative in vitro assay for tumor cell invasion through extracellular matrix or into protein gels. Cancer Res 1988 ;48(23):6933-7.
81. Singh NP,McCoy MT,Tice RR,Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988 Mar;175(1):184-91.
82．张建平，田源，陈道达．单细胞凝胶电泳制胶方法的改进．癌变．畸变．突变．1998，10(3)．-189-190
83. Knowles BB, Howe CC, Aden DP Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980 Jul 25;209(4455):497-499.
84. Bagnarelli P, Devescovi G, Manzin A, Bearzi I, Bonazzi P, Carloni G, Clementi M. Growth-factor independence of a new differentiated hepatitis B virus DNA-negative human hepatoma cell line. Hepatology, 1990;11:1024-1032.
85. Saito H, Morizane T, Watanabe T, Kagawa T, Iwabuchi MN, Kumagai N, Ina gaki Y, Tsuchimoto K, Tsuchiya M. Establishment of a human cell line (HCC-T) from a patient with hepatoma bearing no evidence of hepatitis B or A virus infection. Cancer, 1989;64:1054-1060.
86．黄利华，谢志萍，童福易，等．HBsAg 阴性／HBV—DNA阳性与S基因变异的关系一附8例报告．传染病信息杂志,2001，14(4)：165-167．
87．陈瑞铭．体外培养三个人体肝癌细胞系的建立及其特征．中国科学1979，12：1225．
88. Robson SC,Kirsch RE.Liver function tests and tumor markers. In:Terblanche J eds.Hepatobiliary malignancy:its multidisciplinary management.London,CreatBritain: Edward Arnold,1994;61-64.
89. Chedid D.Antigenic markers of hepatic cellular carcinoma. Cancer,1990;65:84-87.
90．任江波尹珊珊 贾继东甲胎蛋白阴性和阳性原发性肝癌的临床分析中华实用医学．2002，4(14)．-22-24．
91．司徒镇强吴军正．细胞培养．[M]．西安．世界图书出版公司，1996：68-71．
92．骞爱荣，商澎，陈志南，等．HAb18G／CD147拮抗肽与肝癌细胞表面抗原HAb18G的亲和性研究．细胞与分子免疫杂志,2003，19(2)：127-129．
93．叶克龙，任世玲，周联娣，林玉尊，徐志一，苏德隆，俞永富，马曾辰．体肝癌细胞系 YY-8103 若干生物学特征的初步观察上海第一医学院学报．1984，11；(6)：423-429．
94. Doi I et al. Establishment and some biological characteristics of human hepatoma cell line. Gann 1975,66:385.
95. Tammen H, Kreipe H, Hess R, Kellmann M, Lehmann U, Pich A, Lamping N, Schulz-Knappe P, Zucht HD, Lilischkis R. Expression profiling of breast cancer cells by differential peptide display. Breast Cancer Res Treat 2003;79:83-93.
96. Nelson SJ, Cha S.Imaging glioblastoma multiforme.Cancer J 2003;9:134-145.
97. Kim GJ,Cho SJ,Won NH,Sung JM,Kim H,Chun YH,Park SH.Genomic imbalances in Korean hepatocellular carcinoma. Cancer Genet Cytogenet 2003;142:129-133.
98. Liu H,Wang Y,Zhou Q,Gui SY,Li X.The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non HCC prevalent area in China.World J Gastroenterol 2002;8: 480-482.
99. Zhao P,Song X,Nin YY,Lu YL,Li XH.Loss of fragile histidine triad protein in human hepatocellular carcinoma. World J Gastroenterol.2003;9:1216-1219.
100. Qin LX,Tang ZY,Ye SL,Liu YK,Ma ZC,Zhou XD,Wu ZQ,Lin ZY, Sun FX,Tian J,Guan XY,Pack SD,Zhuang ZP.Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared. J Cancer Res Clin Oncol. 2001 Aug; 127(8):482-8.
101. Yang J,Qin LX,Ye SL,Liu YK,Li Y,Gao DM,Chen J,Tang ZY. The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential. J Cancer Res Clin Oncol,2003 [Epub ahead of print].
102. Liao C, Zhao MJ, Zhao J, Song H, Pineau P, Marchio A, Dejean A, Tiollais P, Wang HY, Li TP. Mutation analysis of novel human liverrelated putative tumor suppressor gene in hepatocellular carcinoma. World J Gastroenterol 2003 ;9:89-93.
103．马瑾瑜，朱世能．人体肝癌细胞的生物学特性观察．上海第一医学院学报．1984，11(2)：107-113．
104. Itsubo M,Ishikawa T,Toda G,Tanaka M.Immunohistochemical study of expression and cellular localization of the multidrug resistance gene product P-glycoprotein in primary liver carcinoma. Cancer. 1994 Jan 15;73(2):298-303.
105．韩雨生，明文玉，等．p53对肝癌P-糖蛋白的表达和耐药表型的影响．中华肝脏病杂志．2001，9(4)：237-239．
106. Fairbairn DW,Olive PL,O'Neill KL.The comet assay: a comprehensive review. Mutat Res. 1995 Feb;339(1):37-59