摘要
第一部分结肠荧光原位移植瘤模型构建与生物学功能鉴定
目的建立稳定、高表达绿色荧光蛋白标记的裸鼠结肠原位移植瘤模型。
方法采用绿色荧光转染的人结肠癌HCT-116细胞建立裸鼠的皮下瘤模型,活体荧光影像观察到绿色荧光标志的肿瘤生长至1Omm×10mm,选取15只BALB/C裸鼠进行结肠原位移植。利用荧光影像系统在不同时间位点观察裸鼠原位移植瘤的生长、转移情况。
结果15只裸鼠结肠原位荧光移植瘤模型均成功构建,未发生与手术相关的并发症和死亡。在第3周时,所有动物模型在荧光下观察到肿瘤。肿瘤体积随着时间推移逐步增大,术后3、4、5、6、7周不同时间点整体荧光影像测量原位移植肿瘤计算体积大于体表游标卡尺测量计算的体积,但差异无统计学意义(t=-1.280,—1.115,0.718,—0.199,—0.386,P>0.05)：测量方式与时间的交互作用,两种测量方法的结果差异有统计学意义(F=29.546,P<0.05)。实验终点时存活8只动物模型,其中6/8的动物模型发生肿瘤转移。
结论结肠原位荧光移植瘤模型技术可行,成活率高,能够进行体内实时和无创地动态观察和分析肿瘤细胞的生长与转移情况,是研究结直肠肿瘤转移模式和分析药物治疗效果较理想的动物模型。
第二部分黄芩苷抑制错配修复基因功能缺失的裸鼠结肠原位移植瘤的实验研究
目的研究黄芩苷抑制错配修复基因hMLHl功能缺失的人结肠癌HCT-116细胞裸鼠原位移植瘤生长、转移的作用。
方法建立人结肠癌HCT-116细胞裸鼠荧光原位移植瘤模型。60只荷瘤裸鼠随机分为阴性对照组(G1),50mg、100mg、200mg/kg黄芩苷组(G2-G4),每组15只。治疗组分别给予相应剂量的黄芩苷灌胃,2/日；阴性对照组采用等量5％NaHCO3灌胃。荧光影像系统下观察原发肿瘤的体积、血管密度以及肿瘤转移情况,并分析实验动物的体重、生存状况和死亡率。
结果药物干预后的第14、21、28天,G2-G4组肿瘤生长速度低于G1组,荧光测量的肿瘤体积(mm3)明显小于G1(14d：832±637；21d：2012±1566;28d：2494±1557),差异具有统计学意义(F=4.433,P<0.05),但治疗组间比较差异无统计学意义。实验终点(第28天)时,G2、G3组实验动物的存活率分别为9/15、13/15,生存状态分析显示G3组明显优于G1组和G4组,差异具有统计学意义(x=4.665、3.980,P<0.05);体内开放观察结果显示各组间原发肿瘤的重量、表面血管密度和肿瘤转移情况比较,差异均无统计学意义。
结论黄芩苷对错配修复基因hMLHl功能缺失的结肠癌HCT-116细胞裸鼠原位移植瘤生长具有明显的抑制作用；100mg/kg可能是一个比较理想的治疗剂量。
第三部分黄芩苷诱导HCT-116-GFP肿瘤细胞凋亡相关蛋白表达差异影响的研究
目的探讨黄芩苷抑制错配修复基因功能缺失的裸鼠结肠原位移植瘤的分子机理。
方法建立绿色荧光蛋白标记的裸鼠人结肠癌HCT-116中瘤细胞荧光原位移植瘤模型,三种浓度的黄芩苷溶液(50mg/kg、100mg/kg、200mg/kg)进行干预,检测肿瘤细胞细胞周期、微卫星不稳定变化、相关凋亡基因。
结果黄芩苷干预后的肿瘤细胞主要在G2/M期阻滞,与对照组比较差异有统计学显著性意义；黄芩苷具有诱导肿瘤细胞凋亡的作用,与对照组相比差异有统计学意义(G1vs.G2P=0.000, Glvs.G3P=0.004, Glvs.G4P=0.028);实验组与对照组的MSI未见明显变化；与对照组比较,黄芩苷干预后肿瘤细胞的PCNA、P53、hMLH1、hMSH2、TGF-β1、 IGF-IIR基因的蛋白表达发生了明显改变,差异有统计学意义(P<0.05),而凋亡基因Bcl-2以及Bax基因蛋白表达差异无统计学意义(P>0.05)。
结论黄芩苷抑制HCT-116细胞移植瘤的作用主要是通过调节PCNA、P53基因、hMLH1基因、hMSH2基因、TGF-β1基因、IGF-IIR基因的蛋白表达,诱导HCT-116肿瘤细胞凋亡,促使肿瘤细胞在G2/M期通路阻滞,但是其功能之间的联系需要做进一步的研究。
Part I:Establishment of orthotiopic model of human colon cancer marked by green fluorescent protein and its biological characteristics
Objective To establish a stable orthotiopic model with high green fluorescent protein (GFP) expressing in nude mice.
Methods Human HCT-116colon cancer cells transfected with GFP were used to build a subcutaneous tumor model in nude mice. Fifteen BALB/C nude mice were selected to undergo orthotiopic transplantation of colon when the GFP-labeled tumor grew to lOmm×lOmm as observed by in vivo fluorescent microscopy. The growth and metastasis of orthotopically implanted colon cancer cells were observed with fluorescent imaging system at different time points.
Results GFP-labeled colon cancer models were successfully established in all the15nude mice, and there was no surgery-related complications or death. Tumors marked by GFP were observed under fluoroscope in week3. The size of the tumors progressively increased with time. The volumes of the orthotopically transplanted tumors obtained from global measurement using fluorescent imaging system was larger than those measured by peripheral vernier calipers at postoperative week3,4,5,6,7, while no statistically significant difference was observed (t=-1.280,-1.115,0.718,-0.199,-0.386, P>0.05); There was a significant difference in the interation of measure method and different time points (F=29.546, P(?)0.05). Eight nude mice survived at the end of the experiment, and tumor metastasis was observed in6mice.
Conclusion It is technically feasible to construct GFP-labeled colon cancer orthotiopic transplantation model with high survival rate. The mice model could be used for real-time, in vivo, no-invasive and dynamic observation and analysis of the growth and metastasis of tumor cells.
PartⅡ:Effects of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer
Objective To study the anticancer effects of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer.
Methods Sixty orthotopic transplantation mice model of human colon cancer cell line HCT-116expressing GFP were established, which were divided randomly into negative controlled group (5%NaHCO3) and50,100,200mg/kg Baicalin groups. The nude mice were treated with intragastric infusion twice a day. Nude mice growth state, average weigh, inhibition rate of transplanted tumor, tumor metastasis and survival state were observed.
Results At14,21and28days after treatment with different dose of Baicalin, tumor growth velocity was significantly slower in the treatment groups, and tumor volume was significantly smaller than the controlled group (there were832±637,2012±1566and2494±1557mm3respectively in14,21and28days)(F=4.433, P<0.05). At the end point of study, survival state of100mg/kg group(13/15)was superior to controlled group (8/15) and200mg/kg group (8/15)(x2=4.665and3.980, P<0.05). However, there were no significant differences in tumor metastasis and tumor surface vessel density.
Conclusions Baicalin has statistically significant effects in inhibiting tumor growth in an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer, and100mg/kg may be an ideal treatment dose.
PartⅢ:Mechanism of inhibitation of Baicalin on an orthotopic transplantation mouse model of mismatch repair gene deficient colorectal cancer
Objective To study the mechanism of Baicalin on an orthotopic transplantation mice model of mismatch repair gene deficient colorectal cancer.
Methods Sixty orthotopic transplantation mice model of human colon cancer cell line HCT116expressing GFP were established, which were divided randomly into four equal-sized groups, including a negative control group (G1) and three treatment groups that received50mg/kg (G2),100mg/kg (G3), or200mg/kg (G4) of baicalin. The cell cycle, apoptosis, microsatellite, and relative gene were detected.
Results The proportion of tumor cells in G2/M phase in experimental groups was higher than that in control group signigicantly (G2vs G1, p=0.000; G3vs G1, p=0.003; G4vs G1,p=0.004). The apoptotic rates were significantly increase in experimental groups when compared with the control group (G2vs Gl,p=0.000; G3vs G1,p=0.004; G4vs G1,p=0.028). There were higer significant differences in PCNA, P53, hMLH1, hMSH2, TGF-β1, and IGF-IIR genes, but no difference in MSI, Bcl-2, and Bax genes.
Conclusion The mechanism of the inhibitory effects of Baicalin is related to inhibit the expression of PCNA. P53、hMLH1、hMSH2、TGF-β1and IGF-ⅡR, induce apoptosis of tumor cells, and cell cycle progression is blocked at G2/M phase.
引文
1. Jemal A,Bray F,Center M,et al. Global cancer statistics[J]. CA Cancer J Clin 2011;61 (2):69-90.
2. Alencar H, King R, Funovics M, Stout C, Weissleder R, Mahmood U. A novel mouse model for segmental orthotopic colon cancer[J]. Int J Cance r2005:117,335-339.
3. Teicher BA. Tumor models for efficacy determination[J]. Mol Cancer Ther 2006;5:2435-2443.
4. Carlomagno F, Anaganti S, Guida T, Salvatore G, Troncone G, Wilhelm SM, Santoro M. BAY 43-9006 inhibition of oncogenic RET mutants[J]. J Natl Cancer Inst 2006; 98:326-334.
5. Taketo MM, Edelmann W. Mouse models of colon cancer[J]. Gastroenterology 2009; 136:780-798
6. Yang M, Jiang P, Sun FX, et al. A fluoreaeent orthotopic bone metaataais model of human prostate cancer[J]. Cancer Res 1999;59(4):781-786.
7. Taketo MM. Mouse models of gastrointestinal tumors[J]. Cancer Sci.2006; 97(5):355-361.
8. Tokairin Y, Kakinuma S, Arai M, Nishimura M, Okamoto M, Ito E, Akashi M, Miki Y, Kawano T, Iwai T, Shimada Y. Accelerated growth of intestinal tumours after radiation exposure in Mlhl-knockout mice: evaluation of the late effect of radiation on a mouse model of HNPCC[J]. Int J Exp Pathol. 2006;87(2):89-99.
9. Leclerc D, Deng L, Trasler J, Rozen R. ApcMin/+mouse model of colon cancer:geneexpression profiling in tumors[J]. Cell Biochem.2004; 15;93(6):1242-1254
lO.Teicher BA. Tumor models for efficacy determination[J]. Mol Cancer Ther 2006;5:2435-2443
11.Carlomagno F, Anaganti S, Guida T, Salvatore G, Troncone G, Wilhelm SM, Santoro M. BAY 43-9006 inhibition of oncogenic RET mutants[J]. Natl Cancer Inst 2006; 98:326-334.
12.Hoffman R. M. Orthotopic is orthodox. Why are orthotopic-transplant metastatic models different from all other models[J]. Cell. Biochem,1994;56:1-3.
13.Fu X., Besterman J. M., Monosov A., Hoffman R. M. Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically-intact patient specimens. Proc[J]. Natl. Acad. Sci. USA,1991;88:9345-9349
14.金黑鹰,刘秀芳,丁义江等.结肠造口结肠癌原位移植模型的建立[J].中华胃肠外科杂志2009；12(3)：294-296.
15.金黑鹰,王建东,张甦寅等.结肠皮下易位结肠癌荧光原位移植模型的建立[J]. 中华胃肠外科杂志2010：13(6)： 448-449.
16.Kubota T:Metastatic models of human cancer xenografted in the nude mouse:The importance of orthotopic transplantation[J]. J Cell Biochem 1994; 56:4-8.
17.Pocard M, Tsukui H, Salmon RJ, Dutrillaux B, Poupon MF. Efficiency of orthotopic xenograft models for human colon cancers[J]. In Vivo 1996; 10:463-469
18.Bibby MC. Orthotopic models of cancer for preclinical drug evaluation:advantages and disadvantages[J]. Eur J Cancer 2004;40:852-857.
19.Nucera C, Nehs M A, Mekel M, et al. A novel orthotopic mouse model of human anaplastic thyroid carcinoma [J]. Thyroid 2009; 19(10):1077-1084.
20.Kishimoto H, Urata Y, Tanaka N, et al. Selective metastatic tumor labeling with green fluorescent protein and killing by systemic administration of telomerase-dependent adenoviruses[J]. Mol Cancer Ther 2009;8(11)3001-3008.
21.Kobaek-Larsen M, Thorup I, Diederichsen A, Fenger C, Hoitinga MR. Review of colorectal cancer and its metastases in rodent models:Comparative aspects with those in humans[J]. Comp Med 2000; 50:16-26.
22.Heijstek M, Kranenburg O, Rinkes B. Mouse models of colorectal cancer and liver metastases[J]. Dig Surg 2005;22:16-25.
23.Sun FX, Sasson AR, Jiang P, et al. An ultra-metastatic model of human colon cancer in nude mice[J]. Clin Exp Metastasis 1999; 17:41-48.
24.Cespedes MV, Espina C, Garcia-Cabezas MA, et al. Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites[J]. Am J Pathol 2007; 170:1077-1085.
1. Kohler B A,Ward E.Mccarthy B J.et al.Annual report to the nation on the status of cancer,1975—2007, featuring tumors of the brain and other nervous system[J].J Natl Cancer Inst.2011; 103(9):714-736.
2. Muller A, Fishel R. Mismatch repair and the hereditary non-polyposis colorectal cancersyndrome (HNPCC) [J]. Cancer Invest 2002; 20:102-109
3. Chen WC,Lin SC,Lee JC.A novel nonsense mutation of MS H2 gene in a Taiwanese family with hereditary nonpolyposis colorectal cancer[J].Kaohsiung J Med Sci.2011;27(2):68-71.
4. Christine M.Ribic, M.Sc, Daniel J.Sargent, et al. Tumor microsatellite-instability status as a predictor of benefit from Fluorouracil-Based adjuvant chemotherapy for colon cancer[J]. The New England Journal of Medicine 2003; 349(3):247-257.
5. John M.Carethers, E.Julieta Smith, Cynthia A.Behling, et al. Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer[J]. Gastroenterolog.2004;126(2),394-401.
6.杨柏霖,刘飞,谷云飞等.黄芩苷对错配修复因因功能缺失LoVo细胞的抑制作用及其机制[J].世界华人消化杂志2008；16(21)：2376-2380
7.刘秀芳,金黑鹰,丁义江等.抗突变中药对错配修复基因功能缺失结直肠癌细胞的抑制作用[J].世界华人消化杂志2007；15(20)：2201-2204.
8.杨柏林.微卫星不稳定性结直肠癌临床病理特征及hMLH1基因甲基化[J].大肠肛门病杂志2005；11(4)：312-314
9. Christine M.Ribic, M.Sc, Daniel J.Sargent, et al. Tumor microsatellite-instability status as a predictor of benefit from Fluorouracil-Based adjuvant chemotherapy for colon cancer[J]. The New England Journal of Medicine 2003; 349(3):247-257.
10.John M.Carethers, E.Julieta Smith, Cynthia A.Behling, et al. Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer[J]. Gastroenterology 2004;126(2):394-401.
11.National Comprehensive Cancer Network.. NCCN Clinical Practice Guidelines in Oncology:colon cancer. V.2.2011.http://www.uccn.org/professionals/physician_gls/pdf/eolon.pdf.
12.National Comprehensive Cancer Network.. NCCN Clinical Practice Guidelines in Oncology:rectal cancer. V.2.2011. http://www.nccn.org/professionals/physician gls/pdf/rectal.pdf.
13. Taketo MM, Edelmann W. Mouse models of colon cancer[J]. Gastroenterology 2009;136:780-798
14. Jin H, Yang Z, Wang J, Zhang S, Sun Y, Ding Y. A superficial colon tumor model involving subcutaneous colon translocation and orthotopic transplantation of green fluorescent protein-expressing human colon tumor [J]. Tumour Biol 2011;32(2):391-397.
15. Jin H, Liu X, Li VK, et al. A simple colostomy implantation model for evaluating colon cancer[J]. Int J Colorectal Dis 2009;24(1):41-47.
16.Bibby MC. Orthotopic models of cancer for preclinical drug evaluation:advantages and disadvantages[J]. Eur J Cancer 2004;40:852-857.
17. Nucera C,Nehs M A,Mekel M,et al. A novel orthotopic mouse model of human anaplastic thyroid carcinoma[J]. Thyroid 2009; 19(10):1077-1084.
18.Ruschoff J,Wallinger S,Dietmaier W,et al. Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection[J]. Genetics.1998,95:11301-11306.
19. Goel A,Chang DK,Ricciardiello L. A novel mechanism for aspirin mediated growth inhibition of Human colon cancer cells[J]. Clinical cancer Reaearch,2003,9:383-390.
2O.Raju R, Cruz-Correa M. Chemoprevention of colorectal cancer[J]. Dis colon rectum,2005; 49:113-125.
21.李玉祥,樊华,张劲松等.中药抗癌的体外研究[J].中国药科大学学报,1999；30：37-42
22.刘雄,高建德黄芩研究进展[J].甘肃中医学院学报,2007；24(2)：46-50.
23.顾正勤,孙颖浩,许传亮.黄芩苷诱导前列腺癌细胞株DU145凋亡的体外研究[J].中国中药杂志,2005;30：63-66.
24.王英俊,付莉,华海婴.黄芩苷诱导结肠癌细胞株SW-1116凋亡的研究[J].中医研究,2007；20(6)：22-23.
25. Acikaln M F, Oner L, Topeu L, et al. Tumour angiogenesis and mast cell density in the prognostic assessment of colorectal carcinomas[J]. Dig Liver Dis 2005;37(3):162-169.
26. Carron CP, Meyer DM, Pegg JA, et al. A peptidomimetic antagonist of the integrin alpha(v) btea3 inhibits leydigcell tumor growth and development of hypercalcemia of malignancy[J]. Cancer Res 2006;58(9):1930-1935.
1. Muller A, Fishel R. Mismatch repair and the hereditary non-polyposis colorectal cancer syndrome (HNPCC) [J]. Cancer Invest 2002; 20:102-109.
2. Barrow E, Alduaij W, Robinson L, et al. Colorectal cancer in HNPCC:cumulative lifetime incidence, survival and tumour distribution. A report of 121 families with proven mutations[J]. Clin Genet 2008;74(3):233-42.
3. ten Kate GL, Kleibeuker JH, Nagengast FM, et al. Is surveillance of the small bowel indicated for Lynch syndrome families[J]. Gut 2007;56(9):1198-20.
4.Vasen HF, Mecklin JP, Khan PM, et al. The international collaborative group on hereditary nonpolyposis colorectal cancer (ICG HNPCC) [J]. Dis Colon Rectum 1991;34(5):424-425.
5.Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for hereditary nonpolyposis colorectal cancer (H NPCC, Lynch syndrome) proposed by the international collaborative group on HNPCC[J]. Gastroenterology 1999; 116(6):1453-1456.
6. U.S. Preventive Services Task Force. Screening for colorectal cancer:U.S. Preventive Services Task Force recommendation statement[J]. Ann Intern Med.2008;149(9):627-37.
7. Baglietto L, Lindor NM, Dowty JG, et al. Risks of Lynch syndrome cancers for MSH6 mutation carriers[J]. Natl Cancer Inst.2010; 102(3):193-201.
8. Stoffel E, Mukherjee B, Raymond VM, et al. Calculation of risk of colorectal and endometrial cancer among patients with Lynch syndrome[J]. Gastroenterology.2009; 137(5):1621-7.
9.宋扬文,陈忻中药黄芩药理作用的研究进展[J].中国中医药科技2010；17(4)：375—376
10.郑海峰,段国辰 黄芩苷抑制肿瘤机制的研究进展[J].河北医科大学学报2012；33(3)：363--366
11.张淑英,余志金,甘爱华,等遗传性非息肉病性结直肠癌遗传易感基因的研究现状[J].胃肠病学和肝病学杂志2012；21(1)989-993。
12. Lynch PM. Prevention of colorectal cancer in high-risk populations:the increasing role for endoscopy and chemoprevention in FAP and HNPCC[J].Digestion 2007;76:68-76.
13. Raju R, Cruz-Correa M. Chemoprevention of colorectal cancer. [J].Dis colon rectum 2005; 49:113-125.
14.丁义江,吴崑岚 中医在结直肠癌综合治疗中的作用[J].中华消化外科杂志2010,9(6)：405-407.
15.李玉祥,樊华,张劲松,等 中药抗癌的体外研究[J].中国药科大学学报1999,30：37-42
16.刘雄,高建德黄芩研究进展[J].甘肃中医学院学报2007;24(2)：46-50.
17.顾正勤,孙颖浩,许传亮.黄芩苷诱导前列腺癌细胞株DU145凋亡的体外研究[J].中国中药杂志2005；30：63-66.
18.王英俊,付莉,华海婴.黄芩苷诱导结肠癌细胞株SW-1116凋亡的研究[J].中医研究2007；20(6)：22-23.
19.杨柏霖,刘飞,谷云飞,等.黄芩苷对错配修复基因功能缺失LoVo细胞的抑制作用及其机制[J].世界华人消化杂志2008；16(21)：2376-2380.
20.刘秀芳,金黑鹰,丁义江,等.抗突变中药对错配修复基因功能缺失结直肠癌细胞的抑制作用[J].世界华人消化杂志2007；15(20)：2201-2204.
21.花宝金.肿瘤的中医药多靶点治疗[J].癌症进展杂志2004,2(6)：441-445
22.Perucho M. Microsatellite instability:The mutator that mutates the other mutator[J].Nat Med,1996,2(6):630-631
23.Olschwang S, Laurent-Puig P, Eisinger F, et al.An alternative to prophylactic colectomy for colon cancer prevention in HNPCC syndrome[J]. Gut.2005;54(1):169-79
24.Ruschoff J, Wallinger S, Dietmaier W, et al. Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selection[J]. Genetics,1998,95:11301-11306.
25.Goel A, Chang DK, Ricciardiello L. A novel mechanism for aspirin mediated growth inhibition of Human colon cancer cells[J]. Clinical cancer Reaearch,2003,9:383-390.
26.杨柏霖,陈红锦,陈玉根,等.黄芩苷抑制错配修复基因功能缺失的结直肠癌裸鼠原位移植瘤的实验研究[J].中华外科杂志2012；50(9)；843-847.
27. Wang N,Tang LJ, Zhu GQ, et al. Apoptosis induced by baicalin involving up-regulation of p53 and bax in MCF-7 cells[J]. Asian Nat Prod Res 2008; 10(11-12):1129-1135.
28.洪铁,杨振,绳娟,等.黄芩苷抗肿瘤作用及机制的研究[J].中国药理学通报2008；24(12)：1676-1678.
29.任霞,李翠玲,王恒孝,等.黄芩苷诱导HL-60细胞凋亡的分子机制研究[J].中国实验血液学杂志2012；20(4)：847-851.
30.Liu ZQ, Dong ZH, Yang ZH, et al. Role of eIF3a (eLF3 p170) in intestinal cell differentiation and its association with early development[J]. Differentiation 2007;75(7):652-661.
31.Liu JJ,Huang TS,Cheng WF.et al.Baicalein and baicalin ale potent inhibitors of angiogenesis:Inhibition of endothelial cell proliferation,migration and differentiation[J].Int J Cancer 2003;106(4):559-565.
32.Chao JI, Su WC, Liu HF. Baicalein induces cancer cell death and proliferation retardation by the inhibition of CDC2 kinase and survivin associated with opposite role of p38 mitogen—activated protein kinase and AKT[J]. Mol Cancer Ther.2007;6(11):3039-3048.
33.Wu A, Gu J, Ji J,et al.Role of COX-2 in carcinogenesis of colorectal cancer and its relationship with tumor biological characteristics and patients prognosis[J]. World J Gastroenterol 2003;9:1990-1994
34张楠,徐江.口腔鳞状细胞癌中错配修复基因hMLH1、hMSH2、P53和PCNA表达的相关性及意义[J].石河子大学学报2009；27(6)：740-742.
35.Aubry MC,Hailing KC,et al. DNA mismateh repair genes hMLHl,hMSH2 and hMSH6 are not inactivated in Bronchioloalveolar carcinomas of the lung [J]. Cancer,2001,92(11):28982-29011.
36.Li M, Liu L, et al. Overexpression of hMSH2 and hMLHl protein in certain gastric cancers and their surrounding mucosae[J]. Oncol Rep,2008,19 (2):401-406.
37.Baserga R. Growth regulation of the PCNA gene [J]. J Cell Sci 1991; 98:433-436.
38.Xinarianos G, Scott FM, Liloglou T, et al.hMLHl and hMSH2 expression correlates with allelic imbalance on chromosome 3p in non-small cell lung carcinomas [J]. Cancer Res 2000; 60(15):4216-4221.
39.姚登福,姚敏,蔚丹丹.IGF-Ⅱ/IGF-Ⅰ R相关信号通路与肝细胞癌分子靶向治疗[J].世界华人消化杂志2011；19(29)：3009-3014.
40. Matsuyama M, Sugiura S, Kakita A, et al. Hepatocellular carcinoma arising from ectopic liver tissue in the spleen producing insulin-like growth factor 11 [J]. Pathol Res Pract 2011; 207:124-126.
41.Himpe E, Kooijman R.Insulin-like growth factor-I receptor signal transduction and the Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) pathway[J]. Biofactors. 2009;35(1):76-81.
42.Liou JM, Shun CT, Liang JT, et al. Plasma insulin-like growth factor-binding protein-2 levels as diagnostic and prognostic biomarker of colorectal cancer [J]. Clin Endocrinol Metab.2010;95 (4):1717-25.
43.Breuhahn K, Schirmacher P. Reactivation of the insulin-like growth factor-Ⅱ signaling pathway in human hepatocellular carcinoma[J]. World J Gastroenterol.2008; 14(11):1690-8.
44.苏燕,李宏伟,崔培林等.转化生长因子TGF-β1通过促进结缔组织生长因子表达抑制胰腺癌细胞PANC-1增殖[J].现代生物医学进展2007；7(12)：1761-1763.
45.崔澂,王润田,胡建军等.抗肿瘤中药制剂逆转结直肠癌NK细胞免疫抑制的靶分子探讨[J].江苏医药2011；37(5)：509-513.
1. Jemal A, Bray F, Center MM, et al. Global Cancer Statistics[J]. CA Cancer J Clin 2011;61(2):69-90.
2.Muller A, Fishel R. Mismatch repair and the hereditary non-polyposis colorectal cancer syndrome (HNPCC) [J]. Cancer Invest 2002; 20:102-109.
3. Barrow E, Alduaij W, Robinson L, et al. Colorectal cancer in HNPCC:cumulative lifetime incidence, survival and tumour distribution. A report of 121 families with proven mutations[J]. Clin Genet 2008;74(3):233-242.
4. ten Kate GL, Kleibeuker JH, Nagengast FM, et al. Is surveillance of the small bowel indicated for Lynch syndrome families[J]. Gut 2007;56(9):1198-20.
5.Vasen HF, Mecklin JP, Khan PM, et al. The international collaborative group on hereditary nonpolyposis colorectal cancer (ICG HNPCC) [J]. Dis Colon Rectum 1991;34(5):424-425.
6.Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the international collaborative group on HNPCC[J]. Gastroenterology 1999;116(6):1453-1456.
7. U.S. Preventive Services Task Force. Screening for colorectal cancer:U.S. Preventive Services Task Force recommendation statement[J]. Ann Intern Med.2008;149(9):627-637.
8. Baglietto L, Lindor NM, Dowty JG, et al. Risks of Lynch syndrome cancers for MSH6 mutation carriers[J]. J Natl Cancer Inst.2010; 102(3):193-201.
9. Stoffel E, Mukherjee B, Raymond VM, et al. Calculation of risk of colorectal and endometrial cancer among patients with Lynch syndrome[J]. Gastroenterology.2009; 137(5):1621-1627.
10.Wagner A,Tops C. Genetic testing in hereditary nonpolyposis colorectal cancer families with a MSH2,MLHl,or MSH6 mutation[J]. J Med Genet 2002,39(11):833-837.
11. Lynch HT. dela CA. Hereditary colorectal cancer [J]. N Engl J Med 2003,348 (10):919-932.
12.崔龙.遗传性非息肉性大肠癌的诊治进展[J].中国实用外科杂志,2005,25(3)：189-192.
13.杨柏林.微卫星不稳定性结直肠癌分了病理特征及hMLH1基因甲基化[J].大肠肛门病外科杂志2005；11(4)：312-314.
14. Fidalgo P, Almeida MR, West S. Detection of mutations in mismatch repair genes in Portuguese families with hereditary nonpolyposis colorectal cancer by a multimethod approach[J].Eur J Hum Genet 2000; 8:49-53.
15. Rijcken FM, Hollema H, Kleibeuker JH. Proximal adenomas in hereditary non-polyposis colorectal cancer are prone to rapid malignant transformation [J]. Gut 2002;50:382-386.
16. Rijcken FE, Koornstra JJ, van der Sluis T, Boersma-van EW, el al. Early carcinogenic events in HNPCC adenomas:differences with sporadic adenomas[J]. Dig Dis Sci 2008; 53(6):1660-1668
17. Vasen HF, Nagengast FM, Khan PM. Interval cancers in hereditary non-polyposis colorectal cancer (Lynch syndrome) [J]. Lancet 1995; 345:1183-1184.
18.deJong AE, Morreau H, vanPuijenb-roek M, et al.The role of mismatch repair gene defects in the development of adenomas in patients with HNPCC[J]. Gastroenterology 2004; 126:42-48.
19. Boland CR, ThibodeauSN, HamiltonSR, et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition:development of international criteria for the determination of microsatellite instability in colorectal cancer[J]. Cancer Res,1998;58:5248-5257.
20. Aaltonen LA, Salovaara R, Kristo P, et al. Incidence ot hereditary nonpolyposis colorectal cancer and the fleasibility of molecular screening for the disease [J]. N Engl J Med 1998;338:1481-1487.
21. Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer(Lynch syndrome) and microsatellite instability[J]. J Natl Cancer Inst 2004; 96:261-268.
22.金黑鹰,丁义江,刘秀芳,等.修改Bethseda标准筛选遗传性非息肉病性结直肠癌患者的队列研究[J].中华医学杂志2007；87(21)：1445-1447.
23. Pino V, Castells A, Andreu M, et al. Accuracy of revised Bethesda guidelines, microsatellite instability, and immunohistochemistry for the identification of patients with hereditary nonpolyposis colorectal cancer[J]. JAMA 2005; 293:1986-1994.
24. Mueller J, Gazzoli L, Garber JE. Comprehensive molecular analysis of mismatch rpair gene defects in suspected Lynch syndrome cases[J]. Cancer Res 2009,69(17):7053-7061.
25. Coolbaugh-Murphy MI, Xu JP, Ramagli LS, et al. Microsatellite instability in the peripheral blood leukocytes of HNPCC patients [J]. Hum Mutat 2010;31(3):317-324.
26. Hendriks Y, Franken P, Dierssen JW, et al. Conventional and tissue micro-array immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumours[J].Am J Pathol 2003; 162:469-477.
27. Engel C, Forberg C, Holinski-Feder E, et al. Novel strategy for optimal sequential application of clinical criteria, immunohistochemistry and microsatellite analysis in the diagnosis of hereditary nonpolyposis colorectal cancer[J]. Int J Cancer 2006; 118:115-122.
28.deJong AE, van Puijen broek M, Hend-riks Y, et al. Microsatellite instability, immunohistochemistry,and additional PMS2 staining insuspected hereditary nonpolyposis colorectal cancer[J]. Clin Cancer Res 2004; 10:972-980.
29. Shia J, Klimstra DS, Nafa K, et al. Value of immunohistochemical detection of DNA mismatch repair proteins in predicting germline mutation in hereditary colorectal neoplasms[J]. Am J Surg Pathol. 2005;29:96-104.
30. Shia J, Ellis NA, Klimstra DS. The utility of immunohistochemical detection of DNA mismatch repair gene proteins [J]. Virchows Arch.2004;445:431-441.
31. Lynch PM. The hMSH2 and hMLHl genes in hereditary nonpolyposis colorectal cancer[J]. Surg Oncol Clin N Am 2009; 18(4):611-624.
32.杨柏霖,谷云飞,赖仁胜,等.散发性结直肠癌微卫星不稳定性及其临床病理生物学的关系[J].世界华人消化杂志,2007,15(10)1160-
33. Boland CR, Koi M, Chang DK, et al. The biochemical basis of microsatellite instability and abnormal immunohistochemistry and clinical behavior in Lynch syndrome:from bench to bedside [J]. Fam Cancer. 2008;7:41-52.
34. Shia J,Tang LH, Vakiani E,et al. Immunohistochemistry as First-line Screening for Detecting Colorectal Cancer Patients at Risk for Hereditary Nonpolyposis Colorectal Cancer Syndrome[J]. Am J Surg Pathol 2009;33:1639-1645.
35. Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group. Recommendations from the EGAPP Working Group:genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives[J]. Genet Med.2009;11(1):35-41.
36. Palomaki GE, McClain MR, Melillo S, Hampel HL, Thibodeau SN. EGAPP supplementary evidence review:DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome[J]. Genet Med.2009;11(1):42-65.
37. Peltomaki P, Gao X, Mecklin JP. Genotype and phenotype in hereditary nonpolyposis colon cancer:a study of families with different vs. shared predisposing mutations[J]. Fam Cancer 2001; 1:9-15.
38. Wang Y, Friedl W, Lamberti C, et al. Hereditary nonpolyposis colorectal cancer:frequent occurrence of largegenomic deletions in MSH2 and MLH1 genes[J]. Int J Cancer 2003; 103:636-641.
39. Okkels H, Lindorff-Larsen, Thorlasius-Ussing O, et al. MSH6 Mutations are Frequent in Hereditary Nonpolyposis Colorectal Cancer Families With Normal pMSH6 Expression as Detected by Immunohistochemistry[J]. Appl Immunohistochem Mol Morphol 2012 Oct;20(5):470-477.
40. Nilbert M, Wikman FP, Hansen TVO, et al. Major contribution from recurrent alterations and MSH6 mutations in Danish Lynch syndrome population[J]. Fam Cancer.2009;8:75-83.
41. Wijnen J, de Leeuw W, Vasen HF, et al. Familial endometrial cancer in female carriers of MSH6 germline mutations[J]. Nature Genet.1999;23:142-144.
42. Schweizer P, Moisio A-L, Kuismanen SA, et al. Absence of MSH2 and MSH6 characterizes endometrial but not colon carcinomas in hereditary nonpolyposis colorectal cancer[J]. Cancer Res.2001;61:2813-2815.2001 Apr 1;61(7):2813-2815.
43. Suchy J, Kurzawski G, Jakubowska A, et al. Ovarian cancer of endometrioid type as part of the MSH6 gene mutation phenotype[J]. J Hum Genet.2002;47:529-531.
44. Goodfellow PJ, Buttin BM, Herzog TJ, et al. Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers[J]. PNAS.2003;100:5908-5913.
45. Devlin LA, Graham CA, Price JH, et al. Germline mutations are more prevalent in endometrial cancer patient cohorts thanHereditary Non Polyposis Colorectal Cancer cohorts[J]. Ulster Med J.2007;77:25-30.
46. Patrick M, Lynch D. The hMSH2 and hMLH1 Genes in Hereditary Nonpolyposis Colorectal Cancer[J]. Surg Oncol Clin N Am 2009; 18:611-624.
47. Morak M, Massdorf T, Sykora H, et al. First evidence for digenic inheritance in hereditary colorectal cancer by mutations in the base excision repair genes[J]. Eur J Cancer.2011;47(7):1046-1055
48. Wei W, Liu F, Liu L, et al. Distinct mutations in MLH1 and MSH2 genes in Hereditary Nonpolyposis Colorectal Cancer (HNPCC) families from China [J.]BMB reports 2011; 44(5):317-322.
49. Sheng JQ, Zhang H, Ji M, et al. Genetic diagnosis strategy of hereditary non-polyposis colorectal cancer[J]. World J Gastroenterol 2009;15(8):983-989
50.倪敏,金黑鹰,丁义江,等.微卫星不稳定结直肠癌hMLH1和hMSH2及hMSH6种系突变与hMLH1启动子甲基化检测[J].中华胃肠外科杂志2008；11(4)：358-361
51. Goel A, Xicola RM, Nguyen TP, et al. Aberrant DNA Methylation in Hereditary Nonpolyposis Colorectal Cancer Without Mismatch Repair Deficiency[J]. GASTROENTEROLOGY 2010;138:1854-1862.
1. Jemal A, Bray F, Center MM, et al. Global Cancer Statistics. CA Cancer J Clin 2011;61 (2):69-90.
2. Suo T,Mahteme H,Qin XY. Hyperthermic intraperitoneal chemotherapy for gastric and colorectal cancer in Mainland China[J]. World J Gastroenterol.2011; 17(8):1071-5.
3.孙燕,石远凯.临床肿瘤内科手册[M]第5版北京：人民卫生出版社2009：491-492.
4.王强,郑海涛,丁德祥.结直肠癌的流行病学和筛查进展[J].中国现代医生杂志2008,46(18)；103-104
5.万德森.结直肠癌流行病学与预防[J].中国中西医结合外科杂志2011,17(1)；3-6
6. 孟可,许斌.中医中药治疗大肠癌概况[J].辽宁中医药大学学报2012,4(5)141-142
7. 王辉,孙桂芝,花宝金.中医药防治肿瘤的源流、现状和发展趋势[J].中华中医药学刊2012,30(9)；1973-1975
8.余莉芳,李勇.上海名老中医治疗消化病经验精粹[M]. 北京：中国中医药出版社,2007；75-90.
9.任建琳,谢正强.辨证治疗结肠癌32例[J]. 陕西中医2012；33(6)；690-691
10.张恩欣周岱翰论治大肠癌经验[J]. 实用中医药杂志,2006；22(9)；562
11.白建平,邓宏,张海波,等.刘伟胜教授治疗大肠癌经验简介[J].新中医,2010；42(11)：132-133
12.郁仁存.中医肿瘤学[M].北京：科学出版社,1983；255
13.丁金芳,黄云胜,李明花. 施志明治疗大肠癌经验举要[J].上海中医药杂志,2007；41(5)；43-44
14.王晓戎,袁孝兵,李平,等.大肠癌中医证候分布临床调查研究[J].长春中医药大学学报,2011；27(3)；377-379
15.赵海燕,屠德敬,夏溪,等.151例辅助治疗期大肠癌患者中医证候分布研究[J].中华中医药杂志,2011；26(4)：861-864
16.武迎梅,时水治,潘媛,等.扶正解毒化瘀法治疗大肠癌初探[J].北京中医,2006；25(8)；473-475
17.贾小强,邱辉忠,黄乃健,等.大肠癌辨证分型与肿瘤浸润转移相关性的前瞻性研究[J].中华中医药杂志,2005；20(6)；344-346
18.贾小强,杜永宏,张丽娟.中西医结合治疗大肠癌疗效分析[J].辽宁中医杂志2005；32(7)；703-704
19.司富春,岳静宇.近30年大肠癌中医证型和用药规律分析[J].中华中医药杂志(原中国医药学报)2012；27(7)；1929-1931
20.赵桂侠,郑坚,顾缨,等.大肠癌中医证型的文献分析[J].辽宁中医药大学学报,2009；11(6)；72-74
21.邓志红,孙珏.大肠癌中医辨证文献研究[J].云南中医学院学报,2011；34(2)；54-56
22.李克桑,李琦.中医药防治大肠癌临床研究进展[J].辽宁中医杂志2012；39(5)：953-955
23.曹洋,刘展华,陈志坚.陈锐深教授治疗大肠癌的经验[J].中医药学刊,2005；23(10)；1750-1751
24.张新,孙华,李亚东.孙桂芝治疗大肠癌经验[J]. 山东中医杂志,1998；7(4)：173
25.陶丽,朱莹杰,吕仙梅等.中医辨证治疗对老年大肠癌术后Ⅱ、Ⅲ期患者生存期影响的临床研究[J].中西医结合学报2010；8(12)：1159-1164
26.朱旭东.辨证分型治疗中晚期大肠癌76例[J].江苏中医1998；11(3)：28-29
27.曾小粤.中西医结合治疗中晚期大肠癌86例[J].中医研究2006；19(4)：37-38
28.林岩,许尤琪,丁蓉.肠安康合剂联合FOLFOX4方案治疗晚期结直肠癌临床疗效观察[J].实用 临床医药杂志2010；14(23)：52-54
29.高海德,柳仲林,陈宏生.贞芪扶正胶囊在大肠癌术后化疗中的应用[J].医师进修2005；28(6)：39-40
30.张雨,杨勇,耿昌海,等.化疗联合中药灌肠治疗晚期结直肠癌[J].湖北中医杂志2003；25(9)：34
31.郑兴斌,孙素红. 复方苦参注射液在直肠癌术后化疗中的临床观察[J].中国临床医生2005；33(6)：27-28
32.王鸿礼.中医药治疗晚期直肠癌20例临床分析[J].中医药研究1994；(4)：27
33.雒琳,杨宇飞,李培红,等.中药扶正胶囊和祛邪胶囊减少Ⅱ、Ⅲ期大肠癌术后复发转移的队列研究[J].中国中西医结合杂志..2006；26：677-680
34.陆清昀.扶正抗癌方对大肠癌肿瘤患者免疫功能的影响[J]. 浙江中医药大学学报2010；34(5)：688-689
35.刘帆.自拟扶正消瘤汤治疗中晚期大肠癌42例疗效观察[J].现代实用肿瘤学2007；19(6)：461-463
36.秦伟夫,贺菊乔.益气复元汤对大肠癌术后患者化疗减毒增效的临床观察[J].辽宁中医杂志2011；38(2)：290-291
37.熊绍权,刘碧清.肠复康胶囊治疗大肠癌临床疗效探析[J].中药药理与临床2003；19(1)：45-46
38.陈培丰.单纯中医药治疗晚期直肠癌18例[J].陕西中医1995；16(1)：121
39.郑玉玲,王新杰.肠达顺灌肠液治疗湿热蕴结型大肠癌的临床研究[J].河南中医,2002,22(1)：12-14
40.张宇翔.张东岳教授治疗肛管直肠癌的学术经验[J].中医药管理杂志2006；14(2)：56-58
41.宋扬文,陈忻.中药黄芩药理作用的研究进展[J].中国中医药科技2010；17(4)：375-376
42.洪铁,杨振,绳娟,等.黄芩苷抗肿瘤作用及机制的研究[J].中国药理学通报2008；24(12)：1676-1678
43.郑海峰,段国辰.黄芩苷抑制肿瘤机制的研究进展[J].河北医科大学学报,2012；33(3；363-366
44.车庆明,黄新立,李艳梅,等.黄芩苷的药物代谢产物研究[J].中国中药杂志2001；6(11)：768
45.刘雄,高建德.黄芩研究进展[J].甘肃中医学院学报2007；24(2)：46-50.
46.文敏,李雪,付守廷.黄芩苷药理作用研究新进展[J].沈阳药科大学学报2008；25(2)：158
47.林红.黄芩苷抗肿瘤作用及机制研究进展[J].新医学学刊2008；5(3)：453
48.顾正勤,孙颖浩,许传亮.黄芩苷诱导前列腺癌细胞株DU145凋亡的体外研究[J].中国中药杂志2005；30：63-66.
49.李玉祥、樊华、张劲松等.中药抗癌的体外研究[J].中国药科大学学报1999；30：37-42
50.王英俊,付莉,华海婴.黄芩苷诱导结肠癌细胞株SW-1116凋亡的研究[J].中医研究2007；20(6)：22-23.
51.杨柏霖.结直肠癌微卫星不稳定研究及黄芩等中药对错配修复基因突变表型的影响[D].南京中医药大学博士论文2006.
52. Wang N,Tang LJ, Zhu GQ, et al. Apoptosis induced by baicalin involving up-regulation of p53 and bax in MCF-7 cells [J]. Asian Nat Prod Res,2008;10(11-12):1129-1135.
53. Gao J. Morgan WA, Sanchez-medina A, et al. Theethanol extract of scutellaria baicalensis and it active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells [J]. Toxicol Appl Pharmacol 2011;(3):221-228
54.唐菁,张玲,顾洪涛,等.淫羊藿苷、黄芩苷联合多柔比星抑制肝癌细胞AP RIL表达和逆转肿瘤免疫逃逸[J].中国肿瘤生物治疗杂志2007；14(6)：516-521
55.李宏捷,谢文利,朱江.黄芩苷的抗肿瘤作用及对肿瘤细胞端粒酶的影响[J].江苏医药2008；34(9)：931-933
56.郑静,胡建达,黄毅,等.黄芩苷对耐阿霉素白血病细胞株HL—60/ADR增殖、凋亡的影响[J].中国实验血液学杂志2009；17(5)：1198-1202
57.杨柏霖,刘飞,谷云飞,等.黄芩苷对错配修复基因功能缺失LoVo细胞的抑制作用及其机制[J].世界华人消化杂志2008；16(21)：2376-2380.
58.刘秀芳,金黑鹰,丁义江,等.抗突变中药对错配修复基因功能缺失结直肠癌细胞的抑制作用[J].世界华人消化杂志2007；15(20)：2201-2204.
59.杨柏霖,陈红锦,陈玉根,等.黄芩苷抑制错配修复基因功能缺失的结直肠癌裸鼠原位移植瘤的实验研究[J].中华外科杂志2012；50(9)：843-847.
60.洪怡,何伟,李丹,等.黄芩苷脂质体的制备及体外抗肿瘤作用[J].中国实验方剂学杂志2012；18(3)：29-31