水蛭、土鳖虫提取物对人肝癌细胞侵袭、转移能力的影响及机制研究
|
摘要
目的：探讨破血逐瘀类中药水蛭提取物(Extract of Leech,ELC)、土鳖虫提取物(Extract of Eupolyphaga,EEP)对人肝癌细胞侵袭、转移的影响及机制。
方法：采用液氮快速冻融法结合丙酮沉淀法制取ELC、EEP。采用细胞划痕愈合法、Transwell侵袭小室法观察两味中药提取物对人肝癌HCCLM3细胞侵袭力的影响。建立人肝癌细胞HCCLM3裸鼠肝脏原位移植瘤模型,造模成功后分组行ELC、EEP干预,观察药物对裸鼠肝脏肿瘤生长及肝内外转移的影响。采用免疫细胞化学法观察粒细胞集落刺激因子受体(G-CSFR)在不同转移潜能人肝癌HCCLM3、 Bel-7402、HepG2细胞中表达。采用免疫组织化学法、荧光实时定量PCR法检测G-CSFR蛋白在经ELC、EEP干预后的裸鼠肝脏肿瘤组织中的表达。
结果：①ELC、EEP均以剂量依赖性方式促进HCCLM3细胞迁移,愈合率随药物浓度升高而增加。经EC50浓度药物作用HCCLM3细胞,划痕愈合结果显示ELC、EEP组愈合率明显高于对照组,差异有显著统计学意义(P<0.01)。Transwell侵袭小室结果同样显示,ELC、EEP组HCCLM3细胞穿过Transwell膜的细胞数明显多于对照组,差异有显著统计学意义(P<0.01)。②ELC、EEP组裸鼠肝脏肿瘤的相对体积、肿瘤重量均大于对照组,差异有统计学意义(P<0.05)。ELC、EEP组总转移率均高于对照组,差异有显著统计学意义(P<0.01)。③免疫细胞化学法结果显示G-CSFR在不同转移潜能人肝癌细胞中表达,随着转移潜能的降低阳性表达率依次下降。其中HCCLM3细胞组与Bel-7402细胞组间比较差异具有显著统计学意义(P<0.01),Bel-7402细胞组与HepG2细胞组间比较差异具有显著统计学意义(P<0.01),HCCLM3细胞组与HepG2细胞组间比较差异具有显著统计学意义(P<0.01)。④裸鼠肝脏肿瘤免疫组化结果显示：ELC、EEP组G-CSFR阳性表达率均明显高于对照组,差异有显著统计学意义(P<0.01)；ELC组阳性表达率与EEP组比较,差异无统计学意义(P>0.05)。⑤荧光实时定量PCR结果显示：ELC、EEP均能上调裸鼠肝脏肿瘤G-CSFR mRNA表达,与对照组比较差异有统计学意义(P<0.05)；EEP组与ELC组G-CSFR mRNA表达的比较,差异无统计学意义(P>0.05)。
结论：①ELC、EEP体外能促进人肝癌HCCLM3细胞迁移及侵袭。②ELC、EEP体内能促进人肝癌细胞HCCLM3裸鼠肝脏原位移植瘤的生长及肝内外转移。③ELC、EEP通过上调G-CSFR蛋白表达水平从而促进肝癌细胞的侵袭、转移,可能是破血逐瘀类中药促进肝癌转移的重要机制之一
Objective:To analyse the effectts and mechanism of leech extract (ELC) and eupolyphaga extract (EEP) on invasion and metastatic ability of human hepatoma carcinoma cell.
Methods:The ELC and EEP were prepared by methods of rapid freezen-thaw with liquid nitrogen,and following precipitation with acetone. The invasive ability was assessed with a transwell cell culture chamber in HCCLM3treated with these drugs. Cell scratch experiment was used to detecte the cell moving ability. To set up model of orthotopic grafting carcinoma for HCCLM3in nude mouse, and observe its tumor growth and metastasis by intragastric method of ELC and EEP. Using immunocytochemistry methods to detect G-CSFR in different metastatic potential of human hepatoma cell. To detect G-CSFR in nude mouse tumor tissue with Immunohistochemical and real-time quantitative PCR.
Result:①The ELC and EEP can promote the migration of HCCLM3. The promotion effect is dose-dependent. The healing rate increased with increasing drug concentration. Using EC50drug interfere with HCCLM3, scratches healing results show that ELC and EEP group healing rate was significantly higher than control group, the difference was statistically significant (P<0.05). Transwell invasion chamber results also showed that ELC and EEP group HCCLM3cells through transwell membrane cell numbers was much more than control group. The difference was notable statistically significant (P<0.01).②The relative size of nude mice liver tumors and tumors weiht in ELC and EEP group are bigger than control group, the difference is statistically significant (P<0.05). The metastasis rate of ELC and EEP group are higher than the control group. The difference was notable statistically significant (P<0.01).③The expression of G-CSFR in different metastatic potentials of three HCC cell lines was determined by immunocytochemistry and the results showed that the the positive expression rate of G-CSFR was all significantly decreased with the decline of metastatic potentials. Difference between HCCLM3and Bel-7402(P<0.01) or Bel-7402and HepG2(P<0.01) or HCCLM3and HepG2(P<0.01) were notable statistically significant.④The expression of G-CSFR in nude mouse tumor tissue was determined by Immunohistochemistry and the results showed that the positive expression rate of G-CSFR was all significantly increased in the ELC group and the EEP group than that in control group (P<0.01). The positive expression rate of the ELC group and the EEP group, the different was no statistically significant (P>0.05).⑤The ELC and EEP group can up-regulate the expression of G-CSFR mRNA, compared with control group, the difference was statistically significant (P<0.05). The expression of G-CSFR mRNA in the ELC and the EEP group, differences was no statistically significant (P>0.05).
Conclusion:①ELC、EEP are both promote the motility and invasion of HCCLM3cell in vitro.②ELC、EEP are both promote the metastasis of nude mice liver tumor in vivo.③By detecting the expression of G-CSFR protein, we find that the ELC and the EEP increase the G-CSFR protein expression levels in order to promote hepatoma cells invasion and metastasis. There may be an important molecular mechanism of expelling blood stasis of Chinese medicine for liver metastasis.
方法：采用液氮快速冻融法结合丙酮沉淀法制取ELC、EEP。采用细胞划痕愈合法、Transwell侵袭小室法观察两味中药提取物对人肝癌HCCLM3细胞侵袭力的影响。建立人肝癌细胞HCCLM3裸鼠肝脏原位移植瘤模型,造模成功后分组行ELC、EEP干预,观察药物对裸鼠肝脏肿瘤生长及肝内外转移的影响。采用免疫细胞化学法观察粒细胞集落刺激因子受体(G-CSFR)在不同转移潜能人肝癌HCCLM3、 Bel-7402、HepG2细胞中表达。采用免疫组织化学法、荧光实时定量PCR法检测G-CSFR蛋白在经ELC、EEP干预后的裸鼠肝脏肿瘤组织中的表达。
结果：①ELC、EEP均以剂量依赖性方式促进HCCLM3细胞迁移,愈合率随药物浓度升高而增加。经EC50浓度药物作用HCCLM3细胞,划痕愈合结果显示ELC、EEP组愈合率明显高于对照组,差异有显著统计学意义(P<0.01)。Transwell侵袭小室结果同样显示,ELC、EEP组HCCLM3细胞穿过Transwell膜的细胞数明显多于对照组,差异有显著统计学意义(P<0.01)。②ELC、EEP组裸鼠肝脏肿瘤的相对体积、肿瘤重量均大于对照组,差异有统计学意义(P<0.05)。ELC、EEP组总转移率均高于对照组,差异有显著统计学意义(P<0.01)。③免疫细胞化学法结果显示G-CSFR在不同转移潜能人肝癌细胞中表达,随着转移潜能的降低阳性表达率依次下降。其中HCCLM3细胞组与Bel-7402细胞组间比较差异具有显著统计学意义(P<0.01),Bel-7402细胞组与HepG2细胞组间比较差异具有显著统计学意义(P<0.01),HCCLM3细胞组与HepG2细胞组间比较差异具有显著统计学意义(P<0.01)。④裸鼠肝脏肿瘤免疫组化结果显示：ELC、EEP组G-CSFR阳性表达率均明显高于对照组,差异有显著统计学意义(P<0.01)；ELC组阳性表达率与EEP组比较,差异无统计学意义(P>0.05)。⑤荧光实时定量PCR结果显示：ELC、EEP均能上调裸鼠肝脏肿瘤G-CSFR mRNA表达,与对照组比较差异有统计学意义(P<0.05)；EEP组与ELC组G-CSFR mRNA表达的比较,差异无统计学意义(P>0.05)。
结论：①ELC、EEP体外能促进人肝癌HCCLM3细胞迁移及侵袭。②ELC、EEP体内能促进人肝癌细胞HCCLM3裸鼠肝脏原位移植瘤的生长及肝内外转移。③ELC、EEP通过上调G-CSFR蛋白表达水平从而促进肝癌细胞的侵袭、转移,可能是破血逐瘀类中药促进肝癌转移的重要机制之一
Objective:To analyse the effectts and mechanism of leech extract (ELC) and eupolyphaga extract (EEP) on invasion and metastatic ability of human hepatoma carcinoma cell.
Methods:The ELC and EEP were prepared by methods of rapid freezen-thaw with liquid nitrogen,and following precipitation with acetone. The invasive ability was assessed with a transwell cell culture chamber in HCCLM3treated with these drugs. Cell scratch experiment was used to detecte the cell moving ability. To set up model of orthotopic grafting carcinoma for HCCLM3in nude mouse, and observe its tumor growth and metastasis by intragastric method of ELC and EEP. Using immunocytochemistry methods to detect G-CSFR in different metastatic potential of human hepatoma cell. To detect G-CSFR in nude mouse tumor tissue with Immunohistochemical and real-time quantitative PCR.
Result:①The ELC and EEP can promote the migration of HCCLM3. The promotion effect is dose-dependent. The healing rate increased with increasing drug concentration. Using EC50drug interfere with HCCLM3, scratches healing results show that ELC and EEP group healing rate was significantly higher than control group, the difference was statistically significant (P<0.05). Transwell invasion chamber results also showed that ELC and EEP group HCCLM3cells through transwell membrane cell numbers was much more than control group. The difference was notable statistically significant (P<0.01).②The relative size of nude mice liver tumors and tumors weiht in ELC and EEP group are bigger than control group, the difference is statistically significant (P<0.05). The metastasis rate of ELC and EEP group are higher than the control group. The difference was notable statistically significant (P<0.01).③The expression of G-CSFR in different metastatic potentials of three HCC cell lines was determined by immunocytochemistry and the results showed that the the positive expression rate of G-CSFR was all significantly decreased with the decline of metastatic potentials. Difference between HCCLM3and Bel-7402(P<0.01) or Bel-7402and HepG2(P<0.01) or HCCLM3and HepG2(P<0.01) were notable statistically significant.④The expression of G-CSFR in nude mouse tumor tissue was determined by Immunohistochemistry and the results showed that the positive expression rate of G-CSFR was all significantly increased in the ELC group and the EEP group than that in control group (P<0.01). The positive expression rate of the ELC group and the EEP group, the different was no statistically significant (P>0.05).⑤The ELC and EEP group can up-regulate the expression of G-CSFR mRNA, compared with control group, the difference was statistically significant (P<0.05). The expression of G-CSFR mRNA in the ELC and the EEP group, differences was no statistically significant (P>0.05).
Conclusion:①ELC、EEP are both promote the motility and invasion of HCCLM3cell in vitro.②ELC、EEP are both promote the metastasis of nude mice liver tumor in vivo.③By detecting the expression of G-CSFR protein, we find that the ELC and the EEP increase the G-CSFR protein expression levels in order to promote hepatoma cells invasion and metastasis. There may be an important molecular mechanism of expelling blood stasis of Chinese medicine for liver metastasis.
引文
1. El-Serag HB, Rudolph KL. Hepatocellular carcinoma:epidemiology and molecular carcinogenesis[J]. Gastroenterology,2007; 132(7):2557-2576.
2. Tung-Ping Poon R, Fan ST, Wong J, et al. Risk factors, prevention, and managemet of postoperative recurrence after resection of hepatocellular carcinoma[J].Ann Surg, 2000; 232(1):10-24.
3. Kuper H, Ye W, Broome U, et al. The risk of liver and bileduct cancer in patients with chronic viral hepatitis,alcoholism, or cirrhosis [J]. Hepatology,2001; 34(4): 714-718.
4.李佩文,邹立琰,程建华,等.中西医临床肿瘤学[M].北京：中国中医药出版社,1996：54.
5.闫洪飞.活血化瘀法则治疗肿瘤与展望[J].中国肿瘤,2001；10(10)：595-597.
6.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J].中成药,2003；25(6)：496-498.
7.张娟,张媛.浅谈水蛭在肿瘤疾病中的应用[J].湖南中医杂志,2006；22(4)：69.
8. Fitch MJ,Campagnolo L,Kuhnert F, et al. Egfl7, a novel epidermal growth factordomain gene expressed in endothelial cells[J]. DevDyn,2004; 230(2): 316-324.
9. Araki K, Kishihara F, Takahashi K, et al.Hepatocellular carcinoma producing a granulocyte colony-stimulating factor:report of a resected case with a literature review[J]. Liver Int,2007,27(5):716-721.
10. Inoue C, Kato S, Higuchi K, et al.A case of malignant pleural mesothelioma with elenation of G-CSF and CYFRA in the serum and pleural fluid[J]. Nihon Kokyuki Gakkai Zasshi,2007,45(3):243-247.
11. Takahashi H, Yasuda A, Ochi N, et al. Granulocyte-colony stimulating factor producing rectal cancer[J]. World J Surg Oncol,2008,6:70.
12.杨敏,胡春宏.粒细胞集落刺激因子及其受体在肺癌中表达与作用的研究进展[J].肿瘤,2009；29(8)：812-817.
13.李桂源.现代肿瘤学基础[M].北京：科学出版社,2011；179-180.
14.郭永良，田雪飞,肖竺.水蛭提取物对人肝癌HepG2细胞体外抑制作用研究[J].中国中医药信息杂志.2009,16(8)：30-31.
15.许亚平,林俊兵.蛋白提纯研究进展[J].天津化工,2006,20(4)：9-11.
16. Ou DP, Tao YM, Tang FQ,et al. The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases [J]. Int J Cancer,2007; 120(6):1208-1214.
17. Ou DP, Tao YM, Chang ZG, et al. Hepatocellular carcinoma cells containing hepatitis B virus Xprotein have enhanced invasivepotential conditionally[J]. Dig Liver Dis,2006; 38(4):262-267.
18. Wang W, Yang LY, Yang ZL, et al. Elevated expression of autocrine motility factor receptor correlates with overcxpression of RhoC and indicates poor prognosis in hepatocellular carcinoma[J]. Dig Dis Sci,2007; 52(3):770-775.
19. Li WC, Ye SL, Sun RX, et al. Inhibition of growth and metastasis ofhuman hepatocellular carcinoma by antisense oligonucleotide targeting signal transducer and activator of transcription 3[J]. Clin Cancer Res,2006; 12(23):7140-7148.
20. Shimizu M,Saitoh Y, Itoh H.Immunohistochemical staining of Ha-ras oncogeneproduct in normal,benign,and malignant human pancreatic tissues [J].Hum Pathol.1990; 21(6):607-612.
21.管冬元,方肇勤.健脾理气法防治肝癌的研究进展[J].上海中医药大学学报.2005,02：60-63.
22.朱希,管冬元,方肇勤.健脾益气法下调大鼠肝癌组织信号转导相关基因的研究进展[J].辽宁中医药大学学报,2007,4：47-50.
23. Psaila B, Lyden D. The metastatic nich:adapting the foreign soil[J]. Nat Rev Cancer 2009,9(4):285-293.
24. Morales-Arias J, Meyers PA, Bolontrade MF, et al.Expression of granulocyte-colony-stimulating factor and its receptor in humam Ewing sarcoma cells and patient tumor specimens [J]. Cancer,2007; 110(7):1568-1577.
25.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J].中成药,2003,25(6)：496-498.
26.张娟,张媛.浅谈水蛭在肿瘤疾病中的应用[J].湖南中医杂志,2006,22(4)：69.
27.张伯臾,董建华,周仲英,等.中医内科学[M].上海：上海科技出版社,2007：187.
28.苑艳娟,王智森,刘亚娴.活血化瘀药物在治疗恶性肿瘤过程中的作用[J].辽宁中医药大学学报,2008；10(6)：26-27.
29.黄静,马兰花,刘锦霞,等.浅谈活血化瘀中药抗肿瘤转移的思路与应用[J].国医论坛,2001；16(4)：11-13.
30.丁罡,宋明志,于尔辛.丹参、赤芍对大鼠walker256癌肝转移影响机制的研究[J].中国癌症杂志,2001；11(4)：364.
31.李俊德,杨通礼,孙永章主编.中医药防治肿瘤特技集成[M].北京：北京科学技术出版社,2007：335.
32.张培彤,裴迎霞,祁鑫,等.活血化瘀药对人肺癌细胞黏附和侵袭的影响.中国中西医结合杂志,2009；19(2)：103-105.
33. Fitch MJ, Campagnolo L, Kuhnert F, et al. Egfl7, a novel epidermal growth factordomain gene expressed in endothelial cells[J]. DevDyn,2004; 230(2): 316-324.
34. Psaila B, Lyden D. The metastatic niche:adapting the foreign soil[J]. Nat Rev Cancer,2009; 9(4):285-293.
35.顾宇,陆枫林VEGF促进肝癌SMMC-7721:细胞侵袭性的自分泌机制[J].世界华人消化杂志,2009；17(32)：3292-3295.
36.朱倩,夏羽佳,涂炜,等.过表达连接蛋白32对人肝癌细胞株MHCC-97H体外侵袭能力的影响[J].华中科技大学学报：医学版,2010；39(4)：439-441.
37. Holder J W, Elmore E, Barrett J C. Gap junction function and cancer[J]. Cancer Res,2003; 53(15):3475-3485.
38.王开阳,熊爱珍,蒋星星,等.细胞周期依赖性蛋白激酶对体外培养人肝癌细胞侵袭力的影响[J].世界华人消化杂志,2010(2)：119-124.
39.周艳艳,周南翔,霍继荣.siRNA靶向抑制HDGF基因对人肝癌细胞株增殖和侵袭力的影响[J].胃肠病学,2009；14(10)：585-588.
40.郭昱,姚金锋,武京学.黄芩甙对人肝癌BEL-7402细胞恶性行为的影响及分子基础研究[J].中药药理与临床,2008；24(4)：15-18.
41. Baskova I P, Zavalova L L, Basanoval A V. Protein profiling of the medical leech salivary gland secretion[J]. Biochemisrty(Moscow),2004,69(7):770-775.
42.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J]. 中成药,2003,25(6)：496-498.
43.韩雅莉,李张伟.地鳖纤溶活性蛋白的纯化及性质研究[J].生物工程学报,2006,22(4)：639-643.
44.李满.眼肿瘤裸鼠模型的应用进展[J].四川医学,2009,30(3)：419-420.
45.马玉杰,梁辉.中国组织工程研究与临床康复[J].粒细胞集落刺激因子与血液病,2009；13(14)：2755-2758.
46. Uemura Y, Kobayashi M, Nakata H, et al.Effects of granulocyte colony-stimulation factor and granulocyte-macrophage colony-stimulating factor on lung cancer:roles of cyclooxygenase-2[J]. Oncol Rep,2007;17(4):955-961.
47. Chen M, Fu YY, Lin CY, et al.Prostasin induces protease-dependent and independent molecular changes in the human prostate carcinoma cellline PC-3[J].BiochimBiophysActa,2007; 1773(7):1133-1140.
48. Morales-Arias J, Meyers PA, Bolontrade MF, et al.Expression of granulocyte-colony-stimulating factor and its receptor in humam Ewing sarcoma cells and patient tumor specimens[J]. Cancer,2007; 110(7):1568-1577.
1.杨惠玲,潘景轩,吴伟康,等.高级病理生理学.北京：科学出版社,2009：307-311.
2.管冬元,方肇勤.健脾理气法防治肝癌的研究进展[J].上海中医药大学学报.2005,2：60-63.
3.朱希,管冬元,方肇勤.健脾益气法下调大鼠肝癌组织信号转导相关基因的研究进展[J].辽宁中医药大学学报,2007,4：47-50.
4. Kuper H, Ye W, Broome U, et al. The risk of 1 iver and bileduct cancer in patients with chronic viral hepatitis, alcoholism, or cirrhosis[J]. Hepatology,2001, 34(4):714-718.
5. Psaila B, Lyden D. The metastatic niche:adapting the foreign soil[J].Nat Rev Cancer 2009,9(4):285-293.
6.顾宇,陆枫林VEGF促进肝癌SMMC-7721细胞侵袭性的自分泌机制[J].世界华人消化杂志,2009,17(32)：3292-3295.
7.朱倩,夏羽佳,涂炜,等.过表达连接蛋白32对对人肝癌细胞株MHCC-97H体外侵袭能力的影响[J].华中科技大学学报：医学版,2010,39(4)：439-441.
8. Holder J W,Elmore E,Barrett J C.Gap junction function and cancer[J].Cancer Res,1993,53(15):3475-3485.
9.王开阳,熊爱珍,蒋星星,等.细胞周期依赖性蛋白激酶对体外培养人肝癌细胞侵袭力的影响[J].世界华人消化杂志,2010(2)：119-124.
10.周艳艳,周南翔,霍继荣siRNA靶向抑制1HDGF基因对人肝癌细胞株增殖和侵袭力的影响[J].胃肠病学,2009,14(10)：585-588.
11.郭昱,姚金锋,武京学.黄芩甙对人肝癌BEL-7402细胞恶性行为的影响及分子基础研究[.J].中药药理与临床,2008,24(4)：15-18.
12. Folkman J. Role of angiogenesis in tumor growth and metastasis[J]. Semin Oncol, 2002,29(6):15-18.
13. Yance DR Jr,Sagar SM. Targeting angiogenesis with integrative cancer therapies [J].Integr Cancer Ther,2006,5(1):9-29.
14.杨莉,何奇,王晓.Ki-67、P53蛋白在乳腺癌中的表达及其临床意义[J].医学诊断与实践杂志,2009,8(2)：87-91.
15.黄修燕,黄自丽,晁愚,等.中药“松友饮”对高转移人肝癌侵袭性及小鼠免疫功能的影响[J].肿瘤.2008,28(6)：489-493.
16.唐印华,梁桃,尚国印,等.三氧化二砷抑制裸鼠肝癌侵袭及转移的体内实验研究[J].哈尔滨医科大学学报,2010,44(5)：447-449.
17.刘细平,钟德许,王劲,等.蜈蚣提取液对裸鼠移植肝癌治疗作用研究[J].现代中西医结合杂志,2010,19(15)：1842-1844.
18. Jean M.Angiogenesis:A boost for tumor starvation[J].Science,2003,301:452-454.
19.李佩文,邹立琰，程建华,等.中西医临床肿瘤学.北京：中国中医药出版社,1996：54.
20.张伯臾,董建华,周仲英,等.中医内科学.上海：上海科技出版社,2007：187.
21.苑艳娟,王智森,刘亚娴.活血化瘀药物在治疗恶性肿瘤过程中的作用[J].辽宁中医药大学学报,2008,10(6)：26-27.
22.房广星,耿晖.蜈蚣临床应用研究进展[J].山东中医杂志,2004,23(8)：509-511.
23.蔺爽,曲晓波,李娜,等.动物药整理研究-蜈蚣[J].吉林中医药2009,29(7)：615-616.
24.尹华荣,张刚.蜈蚣临床应用举偶[J].中国中医药信息杂志,2000,7(10)：66.
25. Weon-Kyoo You, Young-Doug Sohn, et al,Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipe smutilans, Insect Biochemistry and Molecular Biology,2004,(34):239-250.
26.张艳慧,司秋菊,王鑫国.蜈蚣抗家兔动脉样硬化的实验研究[J].中药药理与临床.2005,21(1)：26-27.
27.庄海峰,姚芳,王伟涛,等.全蝎对人白血病HL-60细胞作用的研究.天津中医药.2005,22(1)：65-67.
28.朱灵,韦晓谋,杨春旭,等.蝎毒对人肝癌细胞Bel-7404凋亡及端粒酶逆转录酶基因的作用[J].广西医学,2008,30(2)：158-160.
29.房德芳,王明艳.集中有毒动物药材及其有效成分抗癌作用的研究进展[J].江苏中医药.2007,39(11)：92-93.
30.陈国光.马琴.全蝎生物酶制剂对小鼠H22肝癌、S180肉瘤移植瘤的抑制生长作用.世界中医药[J].2010,5(3)：208-209.
31.彭延古.李路丹，付灿云,等.全蝎提取液对家兔实验性动脉血栓的影响[J].微循环学杂志,2006,16(4)：59-60.
32.朱灵,韦晓谋,杨春旭.蝎毒对人肝癌细胞Bel-7404凋亡及端粒酶逆转录酶基因的作用[J].广西医学,2008,30(2)：158-160.
33.汤耀光,陈斌,梁业玲,等蛇毒多肽和蝎毒多肽对小鼠肝癌移植瘤的抑制作用和Caspase-3表达的影响[J].广州医学院学报,2006,34(5)：18-21.
34.张维东,崔亚洲,姚成芳,等.蝎毒多肽提取物抗肿瘤血管生成作用的实验研究[J].中国药理学通报,2005,21(6)：708-711.
35.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J].中成药,2003,25(6)：496-498.
36.韩雅莉,李张伟.地鳖纤溶活性蛋白的纯化及性质研究[J].生物工程学报,2006,22(4)：639-643.
37.张微,邹玺,魏嘉,等.土鳖虫含药血清与化疗药物联合对人肝癌细胞增殖的影响[J].中西结合肝病杂志,2007,17(6)：361-363.
38.林静华,吴映娥,蔡应木,等.地鳖虫纤溶活性蛋白组分的提取及对肿瘤细胞的抑制作用[J].国际检验医学杂志,2007,28(12)：1088-1090,1093.
39.胡文静,钱小平,邹玺,等.重楼、土鳖虫对人肝癌SMMC-7721细胞增殖抑制的协同作用[J].南京中医药大学学报,2007,23(4)：234-238.
40.张娟,张媛.浅谈水蛭在肿瘤疾病中的应用[J].湖南中医杂志,2006,22(4)：69
41.王本祥.现代中药药理学[M].天津：天津科学技术出版社,1994：926.
42. Baskova I P,Zavalova L L,Basanoval A V.Protein profiling of the medical leech salivary gland secretion[J].Biochemisrty(Moscow),2004,69(7):770-775
43.任青华,贾金秋,范延英,等.复方水蛭丸对体内外肿瘤的影响[J].中华中西医杂志,2005,6(12)：46-48.
44.于瑞发,王杰,张博,等.复方水蛭素提取物抗移植性肿瘤的验研究[J]实用医药杂志,2007,24(5)：599-601.
45.任青华,牟忠祥,耿延君,等.中药水蛭素对荷瘤鼠Ki-67与VEGF表达的影响[J].医学研究杂志,2010,39(6)：46-48,136.
46.任凤芝,刘刚叁,张丽,牛桂云.白花蛇舌草黄酮类化学成分研究[J].中国药学杂志2005,40：502-504.
47.吴杨,周忆新,吴银生.白花蛇舌草总黄酮的提取以及体外对肝癌细胞的作用[J].抗感染药学.2008,5(3)：150-152.
48.胡玲,罗晓韵,谢宇晖,等.白花蛇舌草诱导HSP70表达对H22肝癌细胞移植瘤细胞凋亡的影响[J].中药新药与临床药理,2009,20(6)：536-539.
49.Kim HE,Jiang X,Du f,et al.PHAPI,CAS and Hsp70 promote apoptosome formation by preventing Apaf-1 aggregation and enhancing nucleotide exchange on Apaf-1 [J].Mol Cell,2008,30(2):239-247.
50.王春涛,袁晓环,王晓东.白花蛇舌草体外诱导人肝癌HEPG2细胞凋亡的初步研究[J].牡丹江医学院学报,2008,29(4)：4-5.
51.王顺启,陈力,倪虹,等.白花蛇舌草对肝癌细胞SMMC-7721基因表达的影响[J].中药材,2007,30(10)：1285-1289.
52. Tian Z, Yang M, Huang F, et al. Cytotoxicity of three cycloartane triterpenoids from Cimicifuga dahurica[J]Cancer Lett,2005,226(1):65-75.
53. Cheng J, Imanishi H, AmuroY, HadaT. NS-398, a selective cyclooxygenase 2 inhibitor, inhibited cell growth and induced cell cycle arrest in human hepatocellular carcinoma cell lines[J]. Int J Cancer,2002,99(5):755-761.
54.唐渊,李晓辉.莪术提取物对肝癌细胞系HepG2的抗癌作用及机制研究[J].中国药理学通报,2007,23(6)：790-794.
55.梁朝晖,张维彬,胡少为.莪术油对小鼠原位移植HepA肝癌细胞影响的研究[J].陕西中医,2009,30(1)：107-109.
56.梁朝晖,潘智然,李娟.抑癌基因PTEN和肿瘤增殖抗原Ki67在肝癌细胞中表达的实验研究[J].中国医疗前沿,2008,3(20)：9-10.
57.李学臣,张涛,魏晓东.三棱提取物对H22荷瘤小鼠的抑制作用[J].黑龙江医药科学,2010,33(5)：78.
58.胡宗涛,董六一,高杉,等.三棱祁甲合剂的体内外抗肿瘤作用[J].安徽医科大学学报,2009,44(1)：85-87.
59.刘少宾,陈学彬,张建荣.《金匮要略》治癥方药探讨[J].长春中医药大学学报,2007,2(1)：4-5.
60.杨爱萍,何虹.张锡纯治癥瘕用药特点浅论[J].辽宁中医杂志,2005,32(4)：294.
61.卓鹏伟,吴鸿洲.浅析叶天士癥瘕证治[J].中国中医急症,2010,19(7)：1171,1204.
2. Tung-Ping Poon R, Fan ST, Wong J, et al. Risk factors, prevention, and managemet of postoperative recurrence after resection of hepatocellular carcinoma[J].Ann Surg, 2000; 232(1):10-24.
3. Kuper H, Ye W, Broome U, et al. The risk of liver and bileduct cancer in patients with chronic viral hepatitis,alcoholism, or cirrhosis [J]. Hepatology,2001; 34(4): 714-718.
4.李佩文,邹立琰,程建华,等.中西医临床肿瘤学[M].北京：中国中医药出版社,1996：54.
5.闫洪飞.活血化瘀法则治疗肿瘤与展望[J].中国肿瘤,2001；10(10)：595-597.
6.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J].中成药,2003；25(6)：496-498.
7.张娟,张媛.浅谈水蛭在肿瘤疾病中的应用[J].湖南中医杂志,2006；22(4)：69.
8. Fitch MJ,Campagnolo L,Kuhnert F, et al. Egfl7, a novel epidermal growth factordomain gene expressed in endothelial cells[J]. DevDyn,2004; 230(2): 316-324.
9. Araki K, Kishihara F, Takahashi K, et al.Hepatocellular carcinoma producing a granulocyte colony-stimulating factor:report of a resected case with a literature review[J]. Liver Int,2007,27(5):716-721.
10. Inoue C, Kato S, Higuchi K, et al.A case of malignant pleural mesothelioma with elenation of G-CSF and CYFRA in the serum and pleural fluid[J]. Nihon Kokyuki Gakkai Zasshi,2007,45(3):243-247.
11. Takahashi H, Yasuda A, Ochi N, et al. Granulocyte-colony stimulating factor producing rectal cancer[J]. World J Surg Oncol,2008,6:70.
12.杨敏,胡春宏.粒细胞集落刺激因子及其受体在肺癌中表达与作用的研究进展[J].肿瘤,2009；29(8)：812-817.
13.李桂源.现代肿瘤学基础[M].北京：科学出版社,2011；179-180.
14.郭永良，田雪飞,肖竺.水蛭提取物对人肝癌HepG2细胞体外抑制作用研究[J].中国中医药信息杂志.2009,16(8)：30-31.
15.许亚平,林俊兵.蛋白提纯研究进展[J].天津化工,2006,20(4)：9-11.
16. Ou DP, Tao YM, Tang FQ,et al. The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases [J]. Int J Cancer,2007; 120(6):1208-1214.
17. Ou DP, Tao YM, Chang ZG, et al. Hepatocellular carcinoma cells containing hepatitis B virus Xprotein have enhanced invasivepotential conditionally[J]. Dig Liver Dis,2006; 38(4):262-267.
18. Wang W, Yang LY, Yang ZL, et al. Elevated expression of autocrine motility factor receptor correlates with overcxpression of RhoC and indicates poor prognosis in hepatocellular carcinoma[J]. Dig Dis Sci,2007; 52(3):770-775.
19. Li WC, Ye SL, Sun RX, et al. Inhibition of growth and metastasis ofhuman hepatocellular carcinoma by antisense oligonucleotide targeting signal transducer and activator of transcription 3[J]. Clin Cancer Res,2006; 12(23):7140-7148.
20. Shimizu M,Saitoh Y, Itoh H.Immunohistochemical staining of Ha-ras oncogeneproduct in normal,benign,and malignant human pancreatic tissues [J].Hum Pathol.1990; 21(6):607-612.
21.管冬元,方肇勤.健脾理气法防治肝癌的研究进展[J].上海中医药大学学报.2005,02：60-63.
22.朱希,管冬元,方肇勤.健脾益气法下调大鼠肝癌组织信号转导相关基因的研究进展[J].辽宁中医药大学学报,2007,4：47-50.
23. Psaila B, Lyden D. The metastatic nich:adapting the foreign soil[J]. Nat Rev Cancer 2009,9(4):285-293.
24. Morales-Arias J, Meyers PA, Bolontrade MF, et al.Expression of granulocyte-colony-stimulating factor and its receptor in humam Ewing sarcoma cells and patient tumor specimens [J]. Cancer,2007; 110(7):1568-1577.
25.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J].中成药,2003,25(6)：496-498.
26.张娟,张媛.浅谈水蛭在肿瘤疾病中的应用[J].湖南中医杂志,2006,22(4)：69.
27.张伯臾,董建华,周仲英,等.中医内科学[M].上海：上海科技出版社,2007：187.
28.苑艳娟,王智森,刘亚娴.活血化瘀药物在治疗恶性肿瘤过程中的作用[J].辽宁中医药大学学报,2008；10(6)：26-27.
29.黄静,马兰花,刘锦霞,等.浅谈活血化瘀中药抗肿瘤转移的思路与应用[J].国医论坛,2001；16(4)：11-13.
30.丁罡,宋明志,于尔辛.丹参、赤芍对大鼠walker256癌肝转移影响机制的研究[J].中国癌症杂志,2001；11(4)：364.
31.李俊德,杨通礼,孙永章主编.中医药防治肿瘤特技集成[M].北京：北京科学技术出版社,2007：335.
32.张培彤,裴迎霞,祁鑫,等.活血化瘀药对人肺癌细胞黏附和侵袭的影响.中国中西医结合杂志,2009；19(2)：103-105.
33. Fitch MJ, Campagnolo L, Kuhnert F, et al. Egfl7, a novel epidermal growth factordomain gene expressed in endothelial cells[J]. DevDyn,2004; 230(2): 316-324.
34. Psaila B, Lyden D. The metastatic niche:adapting the foreign soil[J]. Nat Rev Cancer,2009; 9(4):285-293.
35.顾宇,陆枫林VEGF促进肝癌SMMC-7721:细胞侵袭性的自分泌机制[J].世界华人消化杂志,2009；17(32)：3292-3295.
36.朱倩,夏羽佳,涂炜,等.过表达连接蛋白32对人肝癌细胞株MHCC-97H体外侵袭能力的影响[J].华中科技大学学报：医学版,2010；39(4)：439-441.
37. Holder J W, Elmore E, Barrett J C. Gap junction function and cancer[J]. Cancer Res,2003; 53(15):3475-3485.
38.王开阳,熊爱珍,蒋星星,等.细胞周期依赖性蛋白激酶对体外培养人肝癌细胞侵袭力的影响[J].世界华人消化杂志,2010(2)：119-124.
39.周艳艳,周南翔,霍继荣.siRNA靶向抑制HDGF基因对人肝癌细胞株增殖和侵袭力的影响[J].胃肠病学,2009；14(10)：585-588.
40.郭昱,姚金锋,武京学.黄芩甙对人肝癌BEL-7402细胞恶性行为的影响及分子基础研究[J].中药药理与临床,2008；24(4)：15-18.
41. Baskova I P, Zavalova L L, Basanoval A V. Protein profiling of the medical leech salivary gland secretion[J]. Biochemisrty(Moscow),2004,69(7):770-775.
42.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J]. 中成药,2003,25(6)：496-498.
43.韩雅莉,李张伟.地鳖纤溶活性蛋白的纯化及性质研究[J].生物工程学报,2006,22(4)：639-643.
44.李满.眼肿瘤裸鼠模型的应用进展[J].四川医学,2009,30(3)：419-420.
45.马玉杰,梁辉.中国组织工程研究与临床康复[J].粒细胞集落刺激因子与血液病,2009；13(14)：2755-2758.
46. Uemura Y, Kobayashi M, Nakata H, et al.Effects of granulocyte colony-stimulation factor and granulocyte-macrophage colony-stimulating factor on lung cancer:roles of cyclooxygenase-2[J]. Oncol Rep,2007;17(4):955-961.
47. Chen M, Fu YY, Lin CY, et al.Prostasin induces protease-dependent and independent molecular changes in the human prostate carcinoma cellline PC-3[J].BiochimBiophysActa,2007; 1773(7):1133-1140.
48. Morales-Arias J, Meyers PA, Bolontrade MF, et al.Expression of granulocyte-colony-stimulating factor and its receptor in humam Ewing sarcoma cells and patient tumor specimens[J]. Cancer,2007; 110(7):1568-1577.
1.杨惠玲,潘景轩,吴伟康,等.高级病理生理学.北京：科学出版社,2009：307-311.
2.管冬元,方肇勤.健脾理气法防治肝癌的研究进展[J].上海中医药大学学报.2005,2：60-63.
3.朱希,管冬元,方肇勤.健脾益气法下调大鼠肝癌组织信号转导相关基因的研究进展[J].辽宁中医药大学学报,2007,4：47-50.
4. Kuper H, Ye W, Broome U, et al. The risk of 1 iver and bileduct cancer in patients with chronic viral hepatitis, alcoholism, or cirrhosis[J]. Hepatology,2001, 34(4):714-718.
5. Psaila B, Lyden D. The metastatic niche:adapting the foreign soil[J].Nat Rev Cancer 2009,9(4):285-293.
6.顾宇,陆枫林VEGF促进肝癌SMMC-7721细胞侵袭性的自分泌机制[J].世界华人消化杂志,2009,17(32)：3292-3295.
7.朱倩,夏羽佳,涂炜,等.过表达连接蛋白32对对人肝癌细胞株MHCC-97H体外侵袭能力的影响[J].华中科技大学学报：医学版,2010,39(4)：439-441.
8. Holder J W,Elmore E,Barrett J C.Gap junction function and cancer[J].Cancer Res,1993,53(15):3475-3485.
9.王开阳,熊爱珍,蒋星星,等.细胞周期依赖性蛋白激酶对体外培养人肝癌细胞侵袭力的影响[J].世界华人消化杂志,2010(2)：119-124.
10.周艳艳,周南翔,霍继荣siRNA靶向抑制1HDGF基因对人肝癌细胞株增殖和侵袭力的影响[J].胃肠病学,2009,14(10)：585-588.
11.郭昱,姚金锋,武京学.黄芩甙对人肝癌BEL-7402细胞恶性行为的影响及分子基础研究[.J].中药药理与临床,2008,24(4)：15-18.
12. Folkman J. Role of angiogenesis in tumor growth and metastasis[J]. Semin Oncol, 2002,29(6):15-18.
13. Yance DR Jr,Sagar SM. Targeting angiogenesis with integrative cancer therapies [J].Integr Cancer Ther,2006,5(1):9-29.
14.杨莉,何奇,王晓.Ki-67、P53蛋白在乳腺癌中的表达及其临床意义[J].医学诊断与实践杂志,2009,8(2)：87-91.
15.黄修燕,黄自丽,晁愚,等.中药“松友饮”对高转移人肝癌侵袭性及小鼠免疫功能的影响[J].肿瘤.2008,28(6)：489-493.
16.唐印华,梁桃,尚国印,等.三氧化二砷抑制裸鼠肝癌侵袭及转移的体内实验研究[J].哈尔滨医科大学学报,2010,44(5)：447-449.
17.刘细平,钟德许,王劲,等.蜈蚣提取液对裸鼠移植肝癌治疗作用研究[J].现代中西医结合杂志,2010,19(15)：1842-1844.
18. Jean M.Angiogenesis:A boost for tumor starvation[J].Science,2003,301:452-454.
19.李佩文,邹立琰，程建华,等.中西医临床肿瘤学.北京：中国中医药出版社,1996：54.
20.张伯臾,董建华,周仲英,等.中医内科学.上海：上海科技出版社,2007：187.
21.苑艳娟,王智森,刘亚娴.活血化瘀药物在治疗恶性肿瘤过程中的作用[J].辽宁中医药大学学报,2008,10(6)：26-27.
22.房广星,耿晖.蜈蚣临床应用研究进展[J].山东中医杂志,2004,23(8)：509-511.
23.蔺爽,曲晓波,李娜,等.动物药整理研究-蜈蚣[J].吉林中医药2009,29(7)：615-616.
24.尹华荣,张刚.蜈蚣临床应用举偶[J].中国中医药信息杂志,2000,7(10)：66.
25. Weon-Kyoo You, Young-Doug Sohn, et al,Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipe smutilans, Insect Biochemistry and Molecular Biology,2004,(34):239-250.
26.张艳慧,司秋菊,王鑫国.蜈蚣抗家兔动脉样硬化的实验研究[J].中药药理与临床.2005,21(1)：26-27.
27.庄海峰,姚芳,王伟涛,等.全蝎对人白血病HL-60细胞作用的研究.天津中医药.2005,22(1)：65-67.
28.朱灵,韦晓谋,杨春旭,等.蝎毒对人肝癌细胞Bel-7404凋亡及端粒酶逆转录酶基因的作用[J].广西医学,2008,30(2)：158-160.
29.房德芳,王明艳.集中有毒动物药材及其有效成分抗癌作用的研究进展[J].江苏中医药.2007,39(11)：92-93.
30.陈国光.马琴.全蝎生物酶制剂对小鼠H22肝癌、S180肉瘤移植瘤的抑制生长作用.世界中医药[J].2010,5(3)：208-209.
31.彭延古.李路丹，付灿云,等.全蝎提取液对家兔实验性动脉血栓的影响[J].微循环学杂志,2006,16(4)：59-60.
32.朱灵,韦晓谋,杨春旭.蝎毒对人肝癌细胞Bel-7404凋亡及端粒酶逆转录酶基因的作用[J].广西医学,2008,30(2)：158-160.
33.汤耀光,陈斌,梁业玲,等蛇毒多肽和蝎毒多肽对小鼠肝癌移植瘤的抑制作用和Caspase-3表达的影响[J].广州医学院学报,2006,34(5)：18-21.
34.张维东,崔亚洲,姚成芳,等.蝎毒多肽提取物抗肿瘤血管生成作用的实验研究[J].中国药理学通报,2005,21(6)：708-711.
35.杨耀芳,杨翊雯,王赛前,等.土鳖虫口服液镇痛、活血化瘀与红细胞免疫研究[J].中成药,2003,25(6)：496-498.
36.韩雅莉,李张伟.地鳖纤溶活性蛋白的纯化及性质研究[J].生物工程学报,2006,22(4)：639-643.
37.张微,邹玺,魏嘉,等.土鳖虫含药血清与化疗药物联合对人肝癌细胞增殖的影响[J].中西结合肝病杂志,2007,17(6)：361-363.
38.林静华,吴映娥,蔡应木,等.地鳖虫纤溶活性蛋白组分的提取及对肿瘤细胞的抑制作用[J].国际检验医学杂志,2007,28(12)：1088-1090,1093.
39.胡文静,钱小平,邹玺,等.重楼、土鳖虫对人肝癌SMMC-7721细胞增殖抑制的协同作用[J].南京中医药大学学报,2007,23(4)：234-238.
40.张娟,张媛.浅谈水蛭在肿瘤疾病中的应用[J].湖南中医杂志,2006,22(4)：69
41.王本祥.现代中药药理学[M].天津：天津科学技术出版社,1994：926.
42. Baskova I P,Zavalova L L,Basanoval A V.Protein profiling of the medical leech salivary gland secretion[J].Biochemisrty(Moscow),2004,69(7):770-775
43.任青华,贾金秋,范延英,等.复方水蛭丸对体内外肿瘤的影响[J].中华中西医杂志,2005,6(12)：46-48.
44.于瑞发,王杰,张博,等.复方水蛭素提取物抗移植性肿瘤的验研究[J]实用医药杂志,2007,24(5)：599-601.
45.任青华,牟忠祥,耿延君,等.中药水蛭素对荷瘤鼠Ki-67与VEGF表达的影响[J].医学研究杂志,2010,39(6)：46-48,136.
46.任凤芝,刘刚叁,张丽,牛桂云.白花蛇舌草黄酮类化学成分研究[J].中国药学杂志2005,40：502-504.
47.吴杨,周忆新,吴银生.白花蛇舌草总黄酮的提取以及体外对肝癌细胞的作用[J].抗感染药学.2008,5(3)：150-152.
48.胡玲,罗晓韵,谢宇晖,等.白花蛇舌草诱导HSP70表达对H22肝癌细胞移植瘤细胞凋亡的影响[J].中药新药与临床药理,2009,20(6)：536-539.
49.Kim HE,Jiang X,Du f,et al.PHAPI,CAS and Hsp70 promote apoptosome formation by preventing Apaf-1 aggregation and enhancing nucleotide exchange on Apaf-1 [J].Mol Cell,2008,30(2):239-247.
50.王春涛,袁晓环,王晓东.白花蛇舌草体外诱导人肝癌HEPG2细胞凋亡的初步研究[J].牡丹江医学院学报,2008,29(4)：4-5.
51.王顺启,陈力,倪虹,等.白花蛇舌草对肝癌细胞SMMC-7721基因表达的影响[J].中药材,2007,30(10)：1285-1289.
52. Tian Z, Yang M, Huang F, et al. Cytotoxicity of three cycloartane triterpenoids from Cimicifuga dahurica[J]Cancer Lett,2005,226(1):65-75.
53. Cheng J, Imanishi H, AmuroY, HadaT. NS-398, a selective cyclooxygenase 2 inhibitor, inhibited cell growth and induced cell cycle arrest in human hepatocellular carcinoma cell lines[J]. Int J Cancer,2002,99(5):755-761.
54.唐渊,李晓辉.莪术提取物对肝癌细胞系HepG2的抗癌作用及机制研究[J].中国药理学通报,2007,23(6)：790-794.
55.梁朝晖,张维彬,胡少为.莪术油对小鼠原位移植HepA肝癌细胞影响的研究[J].陕西中医,2009,30(1)：107-109.
56.梁朝晖,潘智然,李娟.抑癌基因PTEN和肿瘤增殖抗原Ki67在肝癌细胞中表达的实验研究[J].中国医疗前沿,2008,3(20)：9-10.
57.李学臣,张涛,魏晓东.三棱提取物对H22荷瘤小鼠的抑制作用[J].黑龙江医药科学,2010,33(5)：78.
58.胡宗涛,董六一,高杉,等.三棱祁甲合剂的体内外抗肿瘤作用[J].安徽医科大学学报,2009,44(1)：85-87.
59.刘少宾,陈学彬,张建荣.《金匮要略》治癥方药探讨[J].长春中医药大学学报,2007,2(1)：4-5.
60.杨爱萍,何虹.张锡纯治癥瘕用药特点浅论[J].辽宁中医杂志,2005,32(4)：294.
61.卓鹏伟,吴鸿洲.浅析叶天士癥瘕证治[J].中国中医急症,2010,19(7)：1171,1204.