摘要
目的 研究中药复方制剂肠复康对人大肠癌的抗癌效应,并初步探讨其作用机制。
方法 (1)建立人大肠癌HT-29裸鼠移植瘤模型。设立肠复康低、中、高剂量组和空白对照组、5Fu+CF阳性对照组。(2)肠复康灌胃给药8周后比较各组抑瘤效应的强弱。(3)采用病理形态学、免疫组化、图象分析技术定量检测并比较各组移植瘤细胞核形态、凋亡细胞数、微血管密度(MVD)以及Ki-67、iNOS、MMP-2的基因表达情况。
结果 (1)肠复康能显著抑制移植瘤生长,低、中、高剂量组瘤重均明显小于空白组(P<0.05、P<0.01、P<0.001),其中高剂量组瘤重小于5Fu+CF组(P<0.05)。(2)肠复康可改善移植瘤细胞及细胞核形态,减少核内物质含量,高剂量组作用最为明显。肠复康各组的Ki-67计数较空白组减少,高剂量组最少并与空白组、5Fu+CF组比较有显著差异(P<0.001、P<0.05)。(3)肠复康低、中、高剂量组的凋亡细胞数均多于空白组(P<0.05、P<0.01、P<0.001),iNOS表达亦呈渐增趋势,其中高剂量组iNOS表达显著高于空白组(P<0.01)。肠复康用药组凋亡细胞数与iNOS表达水平呈显著正相关(r=0.7761,P=0.0001)。(4)肠复康可降低移植瘤MVD值和MMP-2表达水平,其中高剂量组与空白组比较差异显著(P<0.001、P<0.01)。
结论 (1)肠复康对人大肠癌具有确切的抑瘤效果,并呈量效依赖关系。(2)肠复康具有抑制人大肠癌细胞增殖的作用,可能与改善细胞核形态,降低核内物质合成代谢有关。(3)肠复康对人大肠癌细胞具有诱导凋亡的作用,可能与促进肿瘤组织表达高水平iNOS有关。(4)肠复康具有抗人大肠癌侵袭转移的潜在作用,可能与抗肿瘤血管生成和抑制细胞外基质及基底膜降解有关。
Objective: To study the anticancer effect of Chinese Compound Recipe Changfukang against humam large intestinal cancer and preliminarily explore its mechanism.
Methods: (l)Model rats of transplantation rumor of human large intestinal cancer HT-29 were established. The low, medium and high dose groups of Changfukang ,the blank control group and 5Fu+CF positive control group were set up. (2)After 8 weeks medication of Changfukang through gastrogavage, the antitumor effect of each group was compared. (3)The transplantation tumor karyon morphology, number of apoptotic cells, micro vascular density (MVD) and the gene expression of Ki-67, iNOS, MMP-2 in each group were quantitatively detected and compared by the application of pathomorphology, immunohistochemistry and image analysis technique.
Results: (1)Changfukang could significantly inhibit the growth of transplantation tumor, and the tumor's weight in the low, medium and high dose groups was obviously lower than that in the blank group(P<0.05, P<0.01, P<0.001), and that in the 5Fu+CF group (P<0.05). (2)Changfukang could improve the cell and karyon morphology of transplantation tumor, and reduce the physical content in nucleus. This effect was most significant in the high dose group. The Ki-67 count in each Changfukang group was lower than that in the blank group, among which the high dose group had the lowest and the difference was significant compared with the blank group and 5Fu+CF group(P<0.001, P<0.05). (3)The number of apoptotic cells in Changfukang low, medium and high dose groups was separately higher than that in the blank group(P<0.05, P<0.01, P<0.001), and iNOS expression showed a upward tendency, among which the iNOS expression in high dose group was significantly , higher than that in the blank
group(P<0 01) There was a significant positive correlation between the number of apoptotic cells and the level of iNOS expression in Changfukang treated groups(r = 0.7761, P=0.0001). (4)Changfukang could lower the MVD value of transplantation tumor and the llevel of MMP-2 expresson. And this difference was significant between the high dose group and the blank group(P<0.001, P<0.01).
Conclusion: (1)Changfukang has definite antitumor effect against human large intestinal cancer and this effect is dose-dependent .(2)Changfukang has the function of inhibitingthe cell proliferation of human large intestinal cancer, which may be correlated with its effect of improving karyon morphology and reducing the physical anabolism in nucleus. (3)Changfukang has the function of inducing cell apoptosis of human large intestinal cancer, which may be correlated with its promotion of tumor tissue to express high level iNOS. (4)Changfakang has the potential function of anti-invasion and transferation of human large intestinal cancer, which may be correlated with its anti-tumor angiogenesis effect and its inhibition of the degradation of extracellular matrix and basement membrane.
引文
1 李连第,张思维,鲁凤珠,等.中国恶性肿瘤死亡谱及分类构成特征研究.中华肿瘤杂志 1997;19(5):323~328.
2 陆志范,涂小煌.实用肿瘤诊疗手册.北京:人民军医出版社.2000:454~464.
3 汤钊猷.现代肿瘤学.北京:人民卫生出版社.1993:521~555.
4 Seymour MT, Stenning SP, Cassidy J. Attitudes and pratice in the mane-gement of metastic cotoretal cancer in Britain. Cotoretal cancer working part of the UK medical research council[J]. Clin Oncol, 1997, 9(4): 248-251
5 徐燮渊,俞受程,曾逖闻,等,现代肿瘤放射冶疗学.北京:人民军医出版社,2000:519
6 任刚,蔡嵘,许幼如,等.结直肠癌化疗新观念.国外医学·肿瘤学分册.2000,20(3):156~158
7 吴雪梅.姚德蛟.肠复康胶囊治疗原发性中晚期大肠癌近期疗效观察.成都中医药大学学报.2001,24(2):12
8 熊绍权,刘碧清,王柏丁,等.肠复康胶囊治疗大肠癌临床疗效探析.中药药理与临床.2003.19(1):45~46
9 Lud GD, Baylin SB. Successful treatment with DL-α-difluo-romethyi-ornithine in established human small cell variang luang carcinoma impl-ants in athymic mice[J]. Cancer Res, 1983, 43: 4239~4243
10 王伯云,李玉松,黄高升 病理学技术 北京:人民卫生出 2000:354-437
11 许良中.实用肿瘤病理方法学 上海:上海医科大学出版 1997:161-175
12 Weidner N. Current pathologic methods for measuring intratumoral mic-rovessel density within breast carcinoma and other solid tumors[J]. Breast Cancer Res Treat, 1995, 36 (2): 169~180
13 杨万才,王立东,周琦,等.细胞凋亡与食道贲门癌组织病理学特征的关系 新乡医学院学报 1996,13(4):326~329.
14 刘国廉主编.细胞毒理学.北京:军事医学科学出版社.2001:97~194.
15 申洪,沈忠英 实用生物体视学技术广州:中山大学出版 1991:64-152
16 田令培.癌症病人心理状态与生存期的关系[J].浙江中医杂志 1990,(6):25
17 贾建义,等.恐吓影响小鼠恶性肿瘤侵袭与转移的实验研究[J].中国中医基础医学杂志.2001,7(6):36
18 金嫣莉摘译.关于生药抗肿瘤作用研究:鸦胆子对人体癌细胞增殖的抑制作用.国外医学中医中药分册.1995,17:33
19 汤涛,孟凌华,陈陵际等.鸦胆子乳油具有多药耐药逆转和拓扑异构酶Ⅱ抑制作用.中药药理学通报.2001,17(5):534~539
20 潘启超,胥彬.肿瘤药理与化学治疗学.郑州:河南医科大学出版社.2000:191~194,303,630~633
21 绉丽娟.王丽,卢步峰,等.不同剂型的β-榄香烯类药物抗癌疗效及其机制的研究.中国肿瘤临床.2001,28(5):344~346.
22 杨骅,王仙平,郑树等.康莱特抗肿瘤的研究论文集[M].浙江:浙江大学出版社.1998,107~109
23 姜晓玲,张良,郭成浩.康莱特注射液对血管生成的影响[J].肿瘤杂志,2000,20(4):313~314
24 陈大富,赵杨冰,白绍槐,等.人参皂甙Rg3对裸鼠原位种植人乳腺侵润性导管癌的抗癌作用及其机制的研究.华西医学.2002,17(3):296~298.
25 邢建华,陈永芹,纪明霞,等.人身皂甙诱导直肠癌细胞凋亡的临床观察.中国中西医结合杂志.2001,21(4):260~261.
26 刘希永,郑树.人参对癌化学预防的研究进展.肿瘤防治研究.2000,27(5):420~421
27 韩锐主编.肿瘤化学预防及药物治疗.北京医科大学中国协和医科大学联合出版社.1991:16~26
28 徐叔云.卞如濂,陈修主编.北京:人民卫生出版社.药物实验方法学.2002:1757~1768
29 陈陵际.运用人癌裸小鼠移植瘤模型进行抗癌新药评价.上海实验动物科学.2001,21(4):247~250
30 潘启超,胥彬编著.肿瘤药理学与化学治疗学.郑州:河南医科大学出版社.2000:627
31 刘爱武,李玉山.大肠癌化疗方案选择 山东医药.2001.41(4):54
32 周志伟 结直肠癌化疗的研究进展.癌症.2001,(11):1322
33 朱世能,陆世伦.肿瘤基础理论.上海:上海医科大学出版社.2000:1~2.
34 陈泽雄,张诗军,陈雯.中药复方对人肿瘤细胞凋亡及增殖影响的实验研究.中药材.2000.23(8):477~478.
35 袁淑兰,黄光琦,黄韧敏,等.丹参酮体外诱导分化作用及其机制的研究.癌症.1996,15(6):408~409
36 Bruno S, Darzynkiewicz Z. Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cell[J]. Cell Frolif, 1992, 25(1): 31~40.
37 Van-Oijie MG, Medema RH, Slootweg, et al. Positivity of the proli-feration maker Ki-67 in noncycling cells [J]. Am J Clin Pathol, 1998, 110(1): 24~31
38 Ramires M, David L, Leitao D, et al. Ki-67 labelling index in gastric carcinomas. An immunohistochemical study using double staining for the evaluation of the proliferative activity o diffuse-type carcinomas. J Pathol, 1997, 182(1): 62~63.
39 彭挺生,丘钜世,吴惠茜,等.CD44s、MMP-9、Ki-67的表达与骨肉瘤侵袭、转移、复发的关系.癌症.2002.21(7):745~750.
40 张军,王严庆,吴凯南.甲酰四氢叶酸钙5-Fu的增效作用及其在大肠癌化疗中的应用.国外医学外科学分册 1996.23(6):340~344
41 Togo S, Kasumi F, Kuno K, et al. Morphometric analysis of structural atypism of adenoma and carcin(?)na of the large intestine with special reference to borderline lesion between benignancy and malignance[J]. Stomach Intestine, 1983, 18: 423
42 Riddell RH, Goldman H, Ransohoff DF, et al. Dyplasia in inflammatory bowel disease[J]. Hum Pathol, 1983, 14: 931.
43 晋雯,高美钦,杨代兴.结肠癌及肠腺瘤细胞核形态计量指标的逐步判别分析 福建医科大学学报.2000,34(3):240~242.
44 Evans R. The steroied and thyoid hormone receptor superfamily Science, 1998, 244: 889~895.
45 艾兆伟,查锡良,陈惠黎.视黄酸对人肿瘤细胞一些表型的逆转作用.中华肿
瘤杂志.1991,13:9.
46 艾兆伟,查锡良,陈惠黎.视黄酸对人肝癌细胞的逆转作用—生物学研究.生物化学杂志.1990,6:163.
47 Ai ZW, Zha XL, Chen HL. Effect of retinic acid on hepato-carcinoma cell line. J Tumor Marker Oneol, 1990, 5: 59.
48 彭仲生,谢礼雄,梁湛聪.细胞凋亡与大肠癌.国外医学内科学分册.1997.24(4):141~144
49 陈冠林,陈蔚文.中医药诱导肿瘤细胞凋亡的研究进展.天津中医.2002.19(2):77~78
50 Kerr JF, Winterfod CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy[J]. Cancer, 1994, 73(8): 2013~2026
51 郭玉庆,朱兆华,黎锦芳.细胞凋亡的检测方法.国外医学内科学分册.1997, 24(4):145~148
52 杨万才,王立东,周琦,等.细胞凋亡与食道贲门癌组织病理学特征的关系.新乡医学院学报 1996,13(4):326~329.
53 Bedi A, Pasricha PJ, Adhlar AJ, et al. Inhibition of apoptosis during development of colorectal cancer. Cancer Res. 1995, 55: 1811
54 Inada T, Ichikawa A, Kubota T, et al. 5-Fu-induced apoptosis correlates with efficacy against human gastric and colon cancer xenografts in nude mice[J] Anticance Res, 1997, 17(3c): 1965~1971
55 谢德金,赵行远,吴建平,等.大肠癌诱导一氧化氮合酶与血管内皮生长因子表达及与临床预后的关系.中国误诊学杂志.2002,2(12):1776~1779.
56 常卫红,曹雪涛一氧化氮与细胞凋亡的调控 癌症 2001,20(9):998~1000
57 Jun CD, Cho BM, Hoon-Ryu, et al. Synergistic cooperation between phorbolester and IFN-gramma for induction of nitric oxide synthesis in murine peritoneal macrophages[J]. J Immunol, 1994, 153: 3684~3689
58 Jenkins DC, Charles IG, Thomsen LL, et al. Role of nitric oxide in tumor growth. Proc Natl Acad Sci USA, 1995, 92: 4392~4396.
59 李学龙,周军,丁彦青,等.一氧化氮合酶在大肠癌细胞中的表达及意义.中
国肿瘤临床 2001,28(2):95~98.
60 Ambes S, Merriam WG, Bennet WP, et al. Frequent oxide synthase-2 expression in human colon adenocarcinoma implication for tumor anglo- genesis and colon caner propression. Cancer Res, 1998, 58: 334~341
61 Drapir JC, Wi(?)rbin J, Hibbs HB Jr. Interferon-gamma and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages[J]. J Immunol, 1998, 18: 1587~1592.
62 Juang SH: Xie KL. Suppression of lumorigenicity and melaslas is of human renal carcinoma cells by infection with retroviral vectors harboring the murine inducible nitric oxide synthase gene. Hnm Gene Ther, 1998, 9(9): 845~854
63 Ishigami SI, Arii S, Furutani M, et al. Predictive value of vascular endothelial growth factor in metastasis and prognosis of human eolore-ctal cancer[J]. Br J Cancer, 1998, 78(10): 1379
64 顾康,许正昌,殷广福.大肠癌侵袭转移基机制研究进展.临床肿瘤学杂志.2001,6(2):183~186.
65 粟超跃,银平章.孔令非.等 胶原酶A与大肠癌侵袭转移性的影响.实用癌症杂志.2000.15(4):367~369
66 Ambriu S, Miyazaki M, Ito H, et al. A prospective study of prognostic value of type Ⅳ collagenase activity in colorector cancer tissue[J]. Dig Dis Sci, 1997, 42(8): 1660~1665
67 Hideki M, Hisaguki Host expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in normal colon tissue affects mectum, 1998. 42: 383~402
68 Liabakk Nm, Talbot I, Smith RA, et al. Matrix Metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) type Ⅳ collag-enase in colorectal cancer, Cancer Res. 1996, 56: 190~206
69 张莉.秦北宁,张忠明,等.大肠癌患者明胶酶A基因表达的研究 肿瘤研究与临床.2002,14(3):173~174
70 Liu DH, Zhang XY, Huang YX, FanDM, VEGF165 antisense gene and biological
characteristics of human gastric cancer cell[J]. Di-Si Junyi Daxue Xuebao(J Fourth Mil Med Univ), 2001,22(9): 821~824
71 Liu CI, DouKF, LiB Expression and significance of vascular endothelial growth factor and it's receptors in hepatocellular carcinoma cell lines[J]. Di-Si Junyi Daxue Xuebao (J Fourth Mil Med Univ), 2000, 21(6): 770~772.
72 Zolota V, Gerokosta A, Melachrinou M, et al. Microvessel density, proliferating activity, P53 and bcl-2 expression in situ ductal carcinoma of the breast[J]. Fediatrics, 1999, 103: 1145~149
73 耿舰,申洪 微血管定量与大肠癌转移和预后的关系 国外医学生理病理科学与临床分册.1999,19(6):428~430
74 张国锋,王元和,张明敏.Endostatin对结肠癌生长和转移抑制作用的实验研究癌症 2002,21(1):50~53
75 O'Reilly MS, Boehm. Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell, 1997, 88: 277~278
76 Hanahan D, Folkman J. Patterms and emerging meehanisns of the angio-genic seich during tumorigegenesis[J]. Cell, 1996, 86(3): 353~36
77 Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocrine Rev, 1997, 18: 4~25
78 徐叔云,卞如濂,陈修 药理实验方法学 北京:人民卫生出版社.2002:1767~1768
79 Yano S, Nishioka Y, Nokihara H, et al. Macrophage coionv-stimulating factor gene transduction into human lung caner cells differentially regul-aes metatasis for, ations in various organ micoenvironments of natural killer cell-deplated SCID mice. Cancer Res, 1997, 57: 784
80 陈陵际.丁健.运用人癌裸小鼠移植瘤模型进行抗癌新药评价.上海实验动物科学、2001,21(4):247~250