人肝门部胆管癌裸鼠肝门移植模型的建立及CD、HSV-tk双自杀基因治疗实验研究
摘要
目的 建立人肝门部胆管癌裸鼠肝门移植模型,对其生物学特性进行初步探讨。
方法 收集第90代肝门部胆管癌细胞系FRH细胞(2×10~6)接种于6只Balb/c裸鼠后肢背侧皮下,两周左右,有5只Balb/c裸鼠后肢背侧皮下移植瘤长至约8mm×6mm,切取裸鼠皮下移植瘤边缘直径约1mm组织块植入Balb/c裸鼠肝门部肝实质内,共32只,观察裸鼠生活情况。每周取两只裸鼠在麻醉下剖腹探查,观察肝门部肿瘤生长状况,测量肿瘤直径,计算肿瘤体积,绘制裸鼠肿瘤生长曲线。待裸鼠出现黄疸,全身衰竭时,予以处死,抽血检查肝功能;解剖肝门部肿瘤及观察周围浸润情况,有、无远处脏器转移并行病理检查。裸鼠肝门部移植瘤与人肝门部胆管癌标本进行比较,观察肿瘤病理结构;透射电镜观察肿瘤细胞超微结构;免疫组化检测VEGF、bFGF、PCNA、bcl-2、Fas、P53、P21、MMP-9、TIMP-1、TIMP-2、CD34表达情况,测定肿瘤微血管密度(MVD),。
结果 32只裸鼠建成30只肝门部肿瘤移植模型,成瘤率94%;建成的30只模型中28只出现黄疸,黄疸出现率为93%。接种后2周左右可触及肝门部有小结节,平均4周左右出现黄疸,6周左右裸鼠开始出现恶液质。解剖发现肝门部有肿瘤生长,肿瘤生长较快,长径1.8～2.8cm,包膜不完整,向周围组织浸润。主要向肝实质内生长,8例侵及胆囊,6例浸及肝门血管,3例腹腔淋巴结转移。肾脏、脾脏、胃肠、肺脏未见转移灶。肝功能检查显示:ALT 86±28.7U/L,AST 98±31.3U/L,GGT 122±41.6U/L,ALP 168±65.8U/L,TBIL 35±13.6μmol/L,DBIL 31±11.5μmol/L,均明显高于正常对照组。肝门肿瘤HE染色呈低分化胆管腺癌表现,与人肝门部胆
OBJECT To establish a xenograft model in porta hepatis of human hilar cholgangiocarcinoma in nude mice, and observe its biological characteeristics. METHOD The 90~(th) generation FRH cells(2 × 10~6) were subcutaneously transplanted into the back of the hindlimbs of 6 Balb/c-nu/nu mice. When the tumors reached 8mm × 6mm in diameter two weeks late, the tumors were cut into 1mm~3 pieces and retransplanted into porta hepatis of 32 new nude mice.The mice were observed every day. Each week the belly of two mice were laparotomize to observe the growth of xenograft tumor by measuring its diameter and volum, and the growth curves of the tumor were figured. The mice were killed when jaundice appeared and they were on the brink of death.They were laparotomized to exmine metastatic tumors in nearby and distants organs and pathologic examanition was made. Electron microscope was used to observe the ultrastructure. VEGF, bFGF, PCNA, bcl-2, Fas , P53, P21, MMP-9, TIMP-1, TIMP-2, CD34were examined with imrnunohistochemical methods. Microvessel density (MVD) was counted also to observe the expression of these tumor marks.RESULT 30 out of 32 nude mice xenograft model of human hilar cholangiocarcinoma were successfully established. The rate of tumor occurring was 94%.28 out of 30 nude mice had , The rate of jaundice occurring was 93%. About two weeks later, small nodes in porta hepatis were found; and about four weeks later, jaundice occurred. Dyscrasia appeared in six weeks or so. The xenograft tumor reached 1.8 to 2.8cm in major diameter.
方法 收集第90代肝门部胆管癌细胞系FRH细胞(2×10~6)接种于6只Balb/c裸鼠后肢背侧皮下,两周左右,有5只Balb/c裸鼠后肢背侧皮下移植瘤长至约8mm×6mm,切取裸鼠皮下移植瘤边缘直径约1mm组织块植入Balb/c裸鼠肝门部肝实质内,共32只,观察裸鼠生活情况。每周取两只裸鼠在麻醉下剖腹探查,观察肝门部肿瘤生长状况,测量肿瘤直径,计算肿瘤体积,绘制裸鼠肿瘤生长曲线。待裸鼠出现黄疸,全身衰竭时,予以处死,抽血检查肝功能;解剖肝门部肿瘤及观察周围浸润情况,有、无远处脏器转移并行病理检查。裸鼠肝门部移植瘤与人肝门部胆管癌标本进行比较,观察肿瘤病理结构;透射电镜观察肿瘤细胞超微结构;免疫组化检测VEGF、bFGF、PCNA、bcl-2、Fas、P53、P21、MMP-9、TIMP-1、TIMP-2、CD34表达情况,测定肿瘤微血管密度(MVD),。
结果 32只裸鼠建成30只肝门部肿瘤移植模型,成瘤率94%;建成的30只模型中28只出现黄疸,黄疸出现率为93%。接种后2周左右可触及肝门部有小结节,平均4周左右出现黄疸,6周左右裸鼠开始出现恶液质。解剖发现肝门部有肿瘤生长,肿瘤生长较快,长径1.8～2.8cm,包膜不完整,向周围组织浸润。主要向肝实质内生长,8例侵及胆囊,6例浸及肝门血管,3例腹腔淋巴结转移。肾脏、脾脏、胃肠、肺脏未见转移灶。肝功能检查显示:ALT 86±28.7U/L,AST 98±31.3U/L,GGT 122±41.6U/L,ALP 168±65.8U/L,TBIL 35±13.6μmol/L,DBIL 31±11.5μmol/L,均明显高于正常对照组。肝门肿瘤HE染色呈低分化胆管腺癌表现,与人肝门部胆
OBJECT To establish a xenograft model in porta hepatis of human hilar cholgangiocarcinoma in nude mice, and observe its biological characteeristics. METHOD The 90~(th) generation FRH cells(2 × 10~6) were subcutaneously transplanted into the back of the hindlimbs of 6 Balb/c-nu/nu mice. When the tumors reached 8mm × 6mm in diameter two weeks late, the tumors were cut into 1mm~3 pieces and retransplanted into porta hepatis of 32 new nude mice.The mice were observed every day. Each week the belly of two mice were laparotomize to observe the growth of xenograft tumor by measuring its diameter and volum, and the growth curves of the tumor were figured. The mice were killed when jaundice appeared and they were on the brink of death.They were laparotomized to exmine metastatic tumors in nearby and distants organs and pathologic examanition was made. Electron microscope was used to observe the ultrastructure. VEGF, bFGF, PCNA, bcl-2, Fas , P53, P21, MMP-9, TIMP-1, TIMP-2, CD34were examined with imrnunohistochemical methods. Microvessel density (MVD) was counted also to observe the expression of these tumor marks.RESULT 30 out of 32 nude mice xenograft model of human hilar cholangiocarcinoma were successfully established. The rate of tumor occurring was 94%.28 out of 30 nude mice had , The rate of jaundice occurring was 93%. About two weeks later, small nodes in porta hepatis were found; and about four weeks later, jaundice occurred. Dyscrasia appeared in six weeks or so. The xenograft tumor reached 1.8 to 2.8cm in major diameter.
引文
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[26] Bouvet M,Elilis LM,Nishizaki M, et al. Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular andothelial growth factor expression and inhibits angiogenesis in human colon cancer[J]. Cancer Res, 1998, 58(1): 2288-2292.
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[31] 闫庆国,王文亮,黄高升。胆管癌中抗凋亡基因Bcl-2的表达及意义,临床肝胆病杂志,1997,13(4):194-196。
[32] 欧阳迪平,杨株林,李庆国。胆管癌中P21,p53,bcl-2癌基因蛋白的表达及生物学意义。中华实验外科杂志,1996,13(4):201-202。
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[2] 祝学光.胆管癌发病机理研究进展.中华实验外科杂志,2002,19(9)391-392。
[3] 李兆亭,阮长乐主编,实用普通外科学,济南:山东科学技术出版社,2000,308-315。
[4] 高进,李敏民.中国人良恶性肿瘤在免疫缺陷动物体内的模型建立及现有的问题[J].中华肿瘤杂志,1999,21(1)69-71。
[5] 王曙光,韩立本,殷恒春,等.外胆管癌细胞系的建立.中华实验外科杂志,1997,14(2)67。
[6] Elmore LW, Simica AE. "Intestinal-type" of dednocurinoma preferontialy induced in night/candute liver lobes of rats treated with furan [J].Cancer1993, 53: 254-5.
[7] Kinami Y, Ashida Y, Set K, et al. Influence of inermplete bile duct obstruction or the occunerue of cholangiocarcinma inducted by disionpropanot inhamster [J]. Oncoluge, 1990, 327: 170-176.
[8] Ansato H.Experimental inducted of biliary tract carcinoma associated with preliferative cholangotis in syrian hamster [J]. Hokkajdu Lgaku Zusshi, 1996, 71 (5): 623-636.
[9] Braakhuis BJM.The potential of the nude mice model for the study of head and neck cancer. Arch Otolaryngol. 1984, 229: 69-74.
[10] 范毓东,李开宗,孙岚,等。人胆管细胞癌荷瘤裸鼠模型的建立。中华肝胆外科杂志,2000,6(6):420。
[11] Fidler IJ, Gritial factors in the biology of human cancer metastasis: twentyeight GHA clowes memorial award lecture[J]. Cancer Res, 1990, 50: 6130.
[12] 从超,吴晓鹏,等.人肝门部胆管癌裸鼠皮下移植模型的建立。中华肝胆外科杂志,2004,待发表。
[13] Hamahan D, Folkman J.Pattems and emerging mechanisms of the angiogenesis switch during tumorgenesis. Cell, 1997, 86: 354-364.
[14] Folkman J.Clinical applications of research on angiogenesis. N Engl J Med, 1995, 333: 1757-1763.
[15] Senger DK,Connolly DT, Vandewater L,et al.Purification and NH2-terminal amino acid sequence of guines pig tumor-secreted vascular permeability factor[J]. Cancer Res, 1990, 50(6): 1774-1778.
[16] Neufeld G, Tessler S,Gitaygoren H,et al.Vascular endothelial growth factor and its recptors[J]. Prog Growth Factor Res, 1994, 5(1): 89-97.
[17] Jin-no K, Tanimizu M, Hyodo I, et al.Circulating vascular endothelial growth factor is a posible tumor marker for metasta-sis in human hepatocellular carcinoma. J Gastroenterology, 1998, 33 (3): 376-382.
[18] Pepper MS, Wasi W, Feriara N, et al. In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells[J]. Exp Cell Res, 1994, 210(2): 298-305.
[19] 陈汝福,邹声泉。肝门部胆管癌组织中VEGF和血管生成相关性的研究。肿瘤防治杂志 1999,6(2):99-101。
[20] 陈汝福,邹声泉。肝门部胆管癌组织内血管形成与细胞凋亡相关性的研究。中华肝胆外科杂志 1999,5(6):373-375。
[21] Hugbes SE,Hall PA.Overview of the fibroblast growth factor and receptor families: complexity, functional diversity and implication for future cardiovascular research. Cardiovascular Res, 1993, 27: 1199.
[22] Susada R, Kurokawa T, Iwane M, et al. Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor Cdna. Mol Cell Biol 1998, 8; 588.
[23] Tanigawa N, Lu G, Mitsui T, et al Quantitation of sinusoid-like vessels in hepatocellular carcinoma: its clinical and prognostic significance. Hepatology, 1997, 26: 1216-1223.
[24] 杨林,李永国,钟德珞,等。胆系恶性肿瘤VEGF,bFGF的表达与微血管计数的关系。中华普通外科杂志,2000,16(9):453-454。
[25] Su Wc,Shiesh SC,Liu HS,et al.Expression of oncogene products HER2/Neu and Ras and fibrosis-related growth factors bFGFTGFbeta and PDGF in bile from biliary malignancies and inflammatory discorders. Dig Dic Sci 2001, 46(7): 1387-92.
[26] Bouvet M,Elilis LM,Nishizaki M, et al. Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular andothelial growth factor expression and inhibits angiogenesis in human colon cancer[J]. Cancer Res, 1998, 58(1): 2288-2292.
[27] Blezinger P, Wang J,Gondo M,et al.Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene [J]. Nat Biotechnol, 1997, 17(4): 343-348.
[28] Gillen,P, et al.Proliferating cell nuclear antigen in the assessment of Barrett's mucosa. Br J Surg. 1994; 81 (12): 1766-8.
[29] 孔凡民,郭仁宣,等。增殖细胞核抗原在肝外胆管组织中的表达及临床意义。中华实验外科,1997,14(2):71-72。
[30] 杨林,钟德,李永国,等。胆系恶性肿瘤增殖细胞核抗原表达及其意义。中华外科杂志,1995,33(2):437-438。
[31] 闫庆国,王文亮,黄高升。胆管癌中抗凋亡基因Bcl-2的表达及意义,临床肝胆病杂志,1997,13(4):194-196。
[32] 欧阳迪平,杨株林,李庆国。胆管癌中P21,p53,bcl-2癌基因蛋白的表达及生物学意义。中华实验外科杂志,1996,13(4):201-202。
[33] Harhois DM,Que FG, Cell A,et al.Bcl-2 is overexpanssed and alter the threshold for apoptosis in a cholangiocarcinoma cell line. Hepatology, 1997, 26(4): 884.
[34] Xerri L, Devilard E, Hassoun J, et al. Fas ligand is not only expressed in immune privileged human organs but is also coexpressed with Fas in various epithelial tissues [J]. J ClinPathol, 1997, 50: 87～91.
[35] O' Connel 1B J, O' Sullivan GC, Collins JK, et al. The Fas counterattack: Fasmediated T cell killing by colon cancer cells expressing Fas ligand [J]. J Exp Med, 1996, 184: 1075～1082.
[36] Leithauser F, Dhein J, Mechtershimer G, et al. Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells [J]. Lab Invest, 1993, 69: 415～429.
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