摘要
目的:
从人大肠癌不同远近部位取得组织进行体外培养,探讨人大肠组织体外培养条件,以及在不同条件下,维持肠隐窝完整结构的时间,建立人大肠组织体外培养的平台。用小檗碱处理体外培养的肠组织,探讨小檗碱维持隐窝结构稳定的机制。
方法:
大肠癌手术时,从距瘤体旁边约2cm、≥10cm,以及两者的中间位(约距瘤体5-6cm)处取得手术标本,运用重复贴壁法进行贴壁培养。逐日观察组织块贴壁和生长情况,在培养3d、7d、14d、20d时,取组织块固定、做病理学观察,以及免疫组化分析。无血清培养条件下,用105μmol/L小檗碱处理培养组织3d、7d,然后对培养组织进行病理检测与免疫组化分析。
结果:
用含不同浓度FBS(0%、10%、20%)的培养基培养组织并对其进行形态学观察,结果发现:血清浓度越大,组织细胞的生长会越受到抑制,培养期间观察到的从组织中爬出的细胞越少,组织越早发黑死亡。在不含血清的培养液中,组织存活率反而高;最靠近瘤体的组织生长活性最高,中间位及最远端组织依次次之。在含血清的培养液中,对培养不同时间的组织进行病理切片观察,我们发现越靠近瘤体的组织隐窝结构变化越大,甚至消失,距离瘤体越远的组织,隐窝越呈现出规则的微结构。无论是无血清培养还是含血清培养,培养3d组织块隐窝状态没有太大变化,但培养7d后,隐窝内的细胞会逐渐减少,开始崩解。用105μmol/L小檗碱和400ng/mlDkk1处理组织块3d和7d,与无血清培养组织块相比,作免疫组化分析,发现用入小檗碱和Dkk1处理的组织块其隐窝内细胞膜、细胞质、细胞核染色程度与使用无血清培养的组织块无明显差异。
结论:
人大肠组织块的存活时间与血清浓度成负相关,隐窝完整结构的保持时间与血清浓度呈负相关。尚不能确定小檗碱是通过抑制β-catenin在细胞核中的过度表达,从而抑制隐窝的异常增殖而使组织块隐窝结构保持完整,从而达到防治大肠癌的作用。
Object ive
Culturing the organization form the different parts of colorectal carcinoma to investigate the culture conditions of human congress bowel tissue in vitro and the time of maintaining the complete crypt structure under the different conditions. Using the medium containing berberine to culturing the organizations form the different parts of colorectal carcinoma for the aim to investigate how to maintain the stability of the crypt and treatment and prevention of colorectal carcinoma.
Methods
Obtaining surgical specimens from the place about2cm,5cm or10cm next to tumors,and then training the specimens by the way of repeating stick wall.Observationing the growth of organization,to take a picture on the3rd, the7th,the14th and the20th day.removing tissues to cut into slices,stained by HE. Training organization biopsy of large intestine cancer different parts by using respectively the Serum-free medium and the serum-free medium including105μmol/L berberine in3days, removing tissues to cut into slices,stained by HE,IHC.
Results
By observing the growth situation about the large intestine cancer tissues in vitro,we find that the tissues have the highest survival rate,the closer the tumors,the growth activity is higher.In the medium containing serum,along with the serum concentration increases.the growth of the cell is restrained.From the observing of the stained by HE,we seen the Whether serum-free culture or contain serum training,the situation about the crypt of The tissues did not chang significantly in3days. But after7days, the cells would be gradually reduced in the crypts, and even apart. Training organization biopsy of large intestine cancer different parts by using respectively Dkkl medium and the serum-free medium including105U mol/L berberine in3days and7days, we found the color of the cytoplasm, cell membrane and nuclei is same,the dyed cells is not obviously different to by using the serum-free medium including105μmol/L berberine or by using Dkk1medium. This did not suggest that berberine could restrain the excessive expression of β-catenin in the nucleus.
Conclusions
The survival time of humen intestinal tissues are negative correlated with the serum concentrations.the time of maintaining the structure of the crypt are negative correlated with the serum concentrations. This did not suggest that berberine could restrain the excessive expression of β-catenin in the nucleus and by this it can inhibition of the abnormal proliferation of the crypt,make the structure of the crypt maintain intact and treatment and prevention of colorectal carcinoma.
引文
[1]Browning TH, Trier JS. Organ culture of mucosal biopsies of human small intestine[J]. Clin Invest.1969;48:1423-1432.
[2]Kararli TT. Comparison of the gastrointestinal anatomy,physiology, and biochemistry of humans and commonly used laboratory animals [J].Biopharm Drug Dispos.1995;16:351-380.
[3]Young B, Heath JW. Wheaters functional histology.4th ed. London:Churchill Livingstone; 2000:249-273.
[4]Brennan PC, McCullough JS, Carr KE. Variations in cell and structure populations along the length of murine small intestine. Cells Tissues Organs.1999; 164:221-226.
[5]Snippert HJ, van der Flier LG, Sato T, van Es JH, van den BornM, Kroon-Veenboer C, et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell.2010; 142:134-144.
[6]Cheng H, Leblond CP. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am J Anat.1974; 141:461-479.
[7]Cheng H, Bjerknes M. Whole population cell kinetics of mouse duodenal, jejunal, ileal, and colonic epithelia as determined by radioautography and flow cytometry. Anat Rec. 1982;203:251-264.
[8]Porter EM, Bevins CL, Ghosh D, Ganz T. The multifaceted Paneth cell. Cell Mol Life Sci.2002;59:156-170.
[9]Ouellette AJ. Paneth cells and innate mucosal immunity. Curr Opin Gastroenterol. 2010;26:546-553.
[10]Goyal RK, Hirano I. The enteric nervous system. New Engl J Med.1996;334:1106-110 5.
[11]Thomson D. Some further researches on the cultivation of tissues in vitro. Proc R Soc Med.1914;7:21-46.
[12]Fischer A. A pure strain of cartilage cell in vitro. JEM.1922;36:379-386.
[13]Maximow A. Tissue cultures of young mammalian embryos. Contributions to Embryology.1925; 16:47-133.
[14]Strange ways TSP, Fell HB. Experimental studies on the differentiation of embryonic tissues growing in vivo and in vitro. I. The development of the undifferentiated limbbud (a) when subcutaneously grafted into the postembryonicchick and (b) when cultivated in vitro. Proc R Soc Lond B.1926;99:340-366.
[15]Trowell OA. The culture of mature organs in a syntheticmedium. Exp Cell Res. 1959;16:118-147.
[16]Danielsen EM, Sjostrom H, Noren O, Bro B, Dabelsteen E.Biosynthesis of intestinal microvillar proteins. Characterization of intestinal explants in organ culture and evidence for the existence of proforms of the microvillar enzymes.Biochem[J]. 1982;202:647-654.
[17]Wheeler EE, Challacombe DN. The trophic action of growth hormone, insulin-like growth factor-I, and insulin on human duodenal mucosa cultured in vitro. Gut.1997;40:57-60.
[18]Tham DM, Whitin JC, Kim KK, Zhu SX, Cohen HJ. Expression of extracellular glutathione peroxidase in human and mouse gastrointestinal tract. Am J Physiol.1998; 275:G1463-1471.
[19]Mitchell JD, Mitchell J, Peters TJ. Enzyme changes in human small bowel mucosa during culture in vitro. Gut.1974;15:805-811.
[20]Zachrisson K, Neopikhanov V, Wretlind B, Uribe A. Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa. Cytokine.2001;15:148-155.
[21]Losonsky GA, Fantry GT, Reymann M, Lim Y. Validation of a gastrointestinal explant system for measurement of mucosal antibody production. Clin Diagn Lab Immunol.199 9;6:803-807.
[22]Autrup H, Stoner GD, Jackson F, Harris CC, Shamsuddin AK,Barrett LA, et al. Explant culture of rat colon:a model system for studying metabolism of chemical carcinogens. In Vitro.1978b; 14:868-877.
[23]Stoppini L, Buchs PA, Muller D. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods.1991;37:173-182.
[24]Fletcher PS, Elliott J, Grivel JC, Margolis L, Anton P,McGowan I, et al. Ex vivo culture of human colorectal tissue for the evaluation of candidate microbicides. AIDS.2006;20:1237-1245.
[25]Jacobs JJ, Lehe C, Cammans KD, Das PK, Elliott GR. An in vitro model for detecting skin irritants:methyl greenpyronine staining of human skin explant cultures. Toxicol in Vitro.2002;16:581-588.
[26]Dionne S, Laberge S, Deslandres C, Seidman EG. Modulation of cytokine release from colonic explants by bacterial antigens in inflammatory bowel disease. Clin Exp Immunol.2003; 133:108-114.
[27]Jarry A, Bossard C, Bou-Hanna C, Masson D, Espaze E, Denis MG, et al. Mucosal IL-10 and tgf-beta play crucial roles in preventing lps-driven, ifn-gamma-mediated epithelial damage in human colon explants. J Clin Invest.2008;118:1132-1142.
[28]Lehe CL, Jacobs JJ, Hua CM, Courtellemont P, Elliott GR, Das PK. Subtoxic concentrations of allergenic haptens induce LC migration and maturation in a human organotypic skin explant culture model:a novel method for identifying potential contact allergens. Exp Dermatol.2006; 15:421-431.
[29]Zimmermann GS, Neurohr C, Villena-Hermoza H, Hatz R,Behr J. Anti-inflammatory effects of antibacterials on human bronchial epithelial cells. Respir Res.2009;29:10-8 9.
[30]Seeber JW, Zorn-Kruppa M, Lombardi-Borgia S, Scholz H, et al. Characterisation of human corneal epithelial cell cultures maintained under serumfree conditions. Altern Lab Anim.2008;36:569-583.
[31]Yeh TH, Tsai CH, Chen YS, Hsu WC, Cheng CH, Hsu CJ, et al.Increased communication among nasal epithelial cells in air-liquid interface culture. Laryngoscope.2007;117:1439-1444.
[32]Clayton RF, Rinaldi A, Kandyba EE, Edward M, Willberg C,Klenerman P, et al. Liver cell lines for the study of hepatocyte functions and immunological response. Liver Int. 2005;25:389-402.
[33]Preynat-Seauve O, Suter DM, Tirefort D, Turchi L, Virolle T,Chneiweiss H, et al. Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture. Stem Cells.2009;27:509-520.
[34]Ootani A, Toda S, Fujimoto K, Sugihara H. An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture. Biochem Biophys Res Commun.2000;271:741746.
[35]Randall KJ, Foster JR. The demonstration of immunohistochemical biomarkers in methylmethacrylate embedded plucked human hair follicles.Toxicolathol.2007; 35:952-957.
[36]Bareiss PM, Metzger M, Sohn K, Rupp S, Frick JS, Autenrieth IB, et al. Organotypical tissue cultures from adult murine colon as an in vitro model of intestinal mucosa. Histochem Cell Biol.2008; 129:795-804.
[37]Schiff LJ. Organ cultures of rat and hamster colon. In Vitro.1975;11:46-49.
[38]Autrup H, Barrett LA, Jackson FE, Jesudason ML, Stoner G,Phelps P, et al. Explant culture of human colon. Gastroenterology.1978a;74:1248-1257.
[39]Shamsuddin AK, Barrett LA, Autrup H, Harris CC, Trump BF.Long-term organ culture of adult rat colon. Pathol ResPract.1978;163:362-372.
[40]Finney KJ, Ince P, Appleton DR, Sunter JP, Watson AJ. A comparison of crypt-cell proliferation in rat colonic mucosa in vivo and in vitro. J Anat.1986; 149:177-188.
[41]Howdle PD. Organ culture of gastrointestinal mucosa—a technique for the study of the human gastrointestinal tract. Postgrad Med J.1984;60:645-652.
[42]Mazzarella G, Stefanile R, Camarca A, Giliberti P, Cosentini E,Marano C, et al. Gliadin activates HLA class I-restricted CD8+ T cells in celiac disease intestinal mucosa and induces the enterocyte apoptosis. Gastroenterology.2008;134:1017-1027.
[43]Dionne S, D Agata ID, Hiscott J, Vanounou T, Seidman EG.Colonic explant production of IL-land its receptor antagonist is imbalanced in inflammatory bowel disease (IBD). Clin Exp Immunol.1998;112:435-442.
[44]Di Sabatino A, Pickard KM, Rampton D, Kruidenier L, Rovedatti L, Leakey NA, et al. Blockade of transforming growth factor beta upregulates T-box transcription factor T-bet, and increases T helper cell type 1 cytokine and matrix metalloproteinase-3 production in the human gut mucosa. Gut.2008;57:605-612.
[45]Lindley RM, Hawcutt DB, Cornell MG, Faussone-Pellegrini MS, et al. Human and mouse enteric nervous system neurosphere transplants regulate the function of aganglionic embryonic distal colon. Gastroenterology.2008;135:205-216.
[46]Metzger M, Bareiss PM, Danker T, Wagner S, Hennenlotter J,Guenther E, et al. Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system. Gastroenterology.2009;137:2063-2073.
[47]Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med.2009; 15:701-706.
[48]Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N,Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature.2009;459:262-265.
[49]Brusko TM, Hulme MA, Myhr CB, Haller MJ, Atkinson MA. Assessing the in vitro suppressive capacity of regulatory T cells. Immunol Invest.2007;36:607-628.
[50]Nigsch F, Klaffke W, Miret S. In vitro models for processes involved in intestinal absorption. Expert Opin Drug Metab Toxicol.2007;3:545-556.
[51]Buck V. Who will start the 3Rs ball rolling for animal welfare? Nature. 2007;446:856-856.
[52]De Kanter R, Monshouwer M, Draaisma AL, De Jager MH, De Graaf IAM, Proost JH, et al. Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon. Xenobiotica. 2004;34:229-241.
[53]De Kanter R, Monshouwer M, Meijer DKF, Groothuis GMM. Precision cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to nonhepatic tissues. Curr Drug Metab.2002a;3:39-59.
[54]De Kanter R, De Jager MH, Draaisma AL, Jurva JU, Olinga P, Meijer DKF, et al. Drug-metabolizing activity of human and rat liver, lung, kidney and intestine slices. Xenobiotica.2002b;32:349-62.
[55]Hanahan D, Weinberg RA.The hallmarks of cancer.Cell 2000; 100(1):57-70.
[56]Kopper L, Hajdu M.Tumor stem cells.Pathol Oncol Res,2004;10(2):69-73.
[57]Shih I M. Top-down morphogenesis of colorectal tumors.Proc Natl Acad Sci USA, 2001; 98(5):2640-2645.
[58]Preston SL.Bottom-up histogenesis of colorectal adenomas:origin in the monocryptal adenoma and initial expansion by crypt fission.Cancer Res,2003;63(13):3819-3825.
[59]Pearson H. Surviving a knockout blow. Nature,2002;415(6867):8-9.
[60]Calabrese P, Tavare S, Shibata D.Pretumor Progression:clonal evolution of human stem cell populations.Am J pathol,2004;164(4):1337-1346.
[61]Sell S,Pierce CB.Maturation arrest stem cell differentiation is a common pathway for the cellular portion of carcinomas and epithelial cancers.Lab Invest,1994;70(1):6-22
[62]Renan M J. How many mutations are required for tumorigenesis:Implications from human cancer data.Mol Carcinogensis,1993;7(3):139-146
[63]Trosko JE,Chang CC.Stem cell theory of carcinogenesis.Toxicol Lett,1989;49(2-3): 283-295
[64]HEASLEY LE, HAN SY.JNK regulation of oncogenesis [J].Mof Cells,2006;21(2): 167-167.
[65]Luo W, Zou H, Jin L, Lin S, LI Q, Ye Z, Rui H, Lin SC.Axin contains three separable domains that confer intramolecular, homodimeric, and heterodimeric interactions involved indistinct functions.J Biol Chem 2005;280:5054-5060.
[66]Krieghoff E, Behrens J, Mayr B.Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention.J Cell Sci 2006;119:1453-1463.
[67]Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways. Science,2004; 303(5663):1483-1487.
[68]Vinean E.Frizzled/WNT signalling:the insidious promoter of tumour growth and progression.Front Biosci,2004;9:1023-1034
[69]Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature,2005; 434(7035): 843-850.
[70]Cadigan KM, Liu Yl.wnt signaling:complexity at the surface.J Cell Sci,2006;119:395- 402.
[71]Takahashi M, Tsunoda T, Seiki M, Nakamura Y, Furukawa Y.Identification of membrane-type matrix metalloproteinase-1 as atarget of the beta-catenin/Tcf4 complex inhuman colorectal cancers.Oncogene,2002;21:5861-5867
[72]Gordon MD, Nusse R. Wnt Signaling:multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem,2006;281 (32):22429-22433.
[73]KRIEGHOFE, BEHRENS J,MAYR B.Nucleo-cytoplasmic distribu-tion of β-caentin is regulated by retention [J].Cell Sci,2006; 10:119-119.
[74]KUPHAL F, BEHREN S J.E-cadherin modulates Wnt-dependent transcription in colorectal cancer cells butdoes not alter Wnt-inde-pendent gene expression in fibroblasts [J].ExpCellRes,2006; 312(4):457.
[75]Ilyas M.wnt signaling and the meehanistic basis of tumour development[J]. Pathol, 2005;205:130-144.
[76]McMillan M, Kahn M. Investigating wnt signaling:achemogenomic safari. Drug Discov Today,2005;10:1467-1474.
[77]Katoh Y, Katoh M.Hedgehog signaling pathway and gastric caneer.Cancer Biol Ther, 2005;4:1050-1054.
[78]Webster MT, RoZyeka M, Sara E, Davis E, Smalley M, Young N, DaleTC, Wooster R.Sequenee variants of the axin gene in breast, colon, and other caneers:ananalysis of mutations that interfere with GSK-3 binding. Genes Chromosomes Cance, 2000;28:443-453.
[79]Fukushima H,Yamamoto H,Itoh F,Horiuchi S,Min Y,Iku S,Imai K.Frequent alterations of the beta-catenin and TCF-4 genes,but not of the APC gene,in colon cancers with high-frequency microsatellite instability [J]. Exp Clin Cancer Res,2001;20:553-559.
[80]Lustig B, Behrens J.The wnt signaling Pathway and its role in tumor development[J]. Cacer Res Clin Oncol,2003;129:199-221.
[81]Roh H, Green DW, Boswell CB, PIPPin JA, Drebin JA Suppression of beta-catenin in hibits the neoplastic growth of APC-mutant colon caneer cells.Cancer Res, 2001;61:6563-6568.
[82]Brantjes H, Barker N, van Es J, et al. TCF:lady justice casting the final verdict on the outcome of Wnt signalling [J]. Biol Chem,2002;383(2):255-261.
[83]Kohno H, Suzuki R, Sugie S, et al. Beta-catenin mutations in a mouse model of inflammation-related colon carcinogenesis induced by 1,2-dimethylhydrazine and dextran sodium sulfate [J]. Cancer Sci,2005; 96(2):69-76.
[84]Valizadeh A, Karayiannakis AJ, El Hariry I, et al. Am [J]. Pathol,1997; 150:1977- 1984
[85]Kobayashi M, Honma T, Matsuda Y, et al. Br [J]. Cancer,2000; 82:1689-1693
[86]Polette M, Mestdagt M, Bindels S, et al. Beta-catenin and ZO-1:shuttle olecules involved in tumor invasion-associated epithelialmesenchymal transition processes [J]. Cells Tissues Organs,2007; 185(1-3):61-65.
[87]Powell DM,Mifflin RC,Valentich JD,etal.Myofibroblasts Ⅱ intestinal subepithelial myofibroblasts. Am J Physiol,1999; 277(2ptl), C183-C201
[88]Watt FM,Hogan BL out of Eden:stem cells and their niches. Scienee,2000; 287(5457):1427-1430
[89]Walters Julian RF.Cell and molecular biology of the smallintestine:new insights into differentiation,growth and repair.Gastroenterol,2004;20(2):70-76
[90]苏金玲,孙保存.大肠隐窝干细胞及其与大肠癌的发生[J].国外医学肿瘤学分册,2005;32(6):454-456
[91]Preston SL.Bottom-up histogenesis of colorectal adenomas:origin in themonocryptal adenoma and initial expansion by cryptfission.Cancer Res,2003;63(13):3819-3825
[92]Pinto D, Clevers H. Wnt, stem cells and cancer in the intestine.Biol Cell,2005;97(3):18 5-196
[93]Radtke F,Clevers H.Self-renew a land cancer of the gut:two sides of a coin.Science,2 005;307(5717):1904-1909
[94]Gregorieff A.Expressionpattern of Wnt signaling components in the adultintestine. Gastroenterology,2005; 129(2):626-638
[95]Leedham SJ, Brittan M, McDonald S A C, et al. Intestinal stemcells. J Cell Mol Med,2005;9(1):11-24
[96]van Es J H, Clevers H. Notch and Wnt inhibitors as potential new drugs for intestinal neoplastic disease.Trends Mol Med,2005; 11(11):496-502
[97]Katoh Y, Katoh M. Hedgehog signaling pathway and gastrointestinal stem cell signaling network.Int J Mol Med,2006;18(6):1019-1023
[98]van Es J H, van Gijn M E, Riccio O. Notch/y-secretase inhibition turn sproliferative cells in intestinal crypts and adenomas into goblecells.Nature,2005;435(7044):95 9-963
[99]Pinto D,GregorierTA,BegthelH,etal.Canonical Wntsig-nalsarees sential for homeostasis of the intestinal epithelium.Genes Dev,2003; 17(14):1709-1713
[100]文彬,陈蔚文.隐窝干细胞和结肠癌的发生[J].生命科学,2007;19(3):321-325
[101]Watt FM, Hogan BL.Out of Eden:stem cells and their niches.Science,2000;287(545 7):1427-1430.
[102]Bach SP, Renehan AG, Potten CS.Stem cells:the intestinal stem cell as a paradigm.Carcinogenesis,2000; 21(3):469-476.
[103]Booth C, Potten CS.Gut instincts:thoughts on intestinal epithelial stem cells.J Clin Invest,2000; 105(11):1493-1499.
[104]Walters Julian RE.Cell and molecular biology of the small intestine:new in sights into differentiation,growth and repair.Gastroenterol.2004;20(2):70-76
[105]Potten CS,Booth C,Pritchard DM.The intestinal epithelial stem cell:the mucosal governor.Int J Path,1997;78(4):219-243
[106]Kim KM,Shibata D,Methylation reveals a niche:stem cell succession in human coloncrypts.Oncogene,2002;21(35):5441-5449.
[107]Booth C,Potten CS.Gut instincts:thoughts on intestinal epithelial Stem cells. J Clin Invest,2000;105(11):1493-1499.
[108]Marshman E,Booth C,Potten CS.The intestinal epithelial stem cell. Bioessays, 2002;24(1):91-98
[109]Yatabe Y, Tavare S, Shibata D. Investigating stem cells in human colon By using methylation patterns. Proc Nat Acad Sci USA,2001;98(19):10839-10844.
[110]Bach SP,Renehan AG,Potten CS. Stem cells:the intestinal stem cell As a paradigm.Carcinogenesis,2000;21(3):469-476.
[111]Houchen CW,George RJ,Sturmoski MA,etal.FGF-2 enhances intes-Tinal stem cell survival and its expression is induced after radiation in-jury.Am J Physiol,1999; 276(1):249-258
[112]Glinsky GV,Beregovska O,Glinskn AB.Microarray analy sisidentifies a death from cancer signature predicting therapy failure in patients with multiple types of cancer.J lin Invest,2005; 115(6):1503-1521.
[113]Yuspa S H, Morgan D L. Mouse skin cells resistant to terminal differentiation associated with initiation of carcinogenesis.Nature,1981;293(5827):72-74.
[114]Al-HajjM, W icha MS, Benito-Hernandez A, et al.Pro-spective identification of tumorigenic breast cancer cells.Proc NatlAcad SciU SA,2003; 100(7):3983-3988.
[115]Singh SK, Clarke ID, TerasakiM, et al.Identification of a cancer stem cell in human brain tumors.CancerRes,2003;63(18):5821-5828.
[116]Marx J.Cancer research-Mutant stem cellsmay seed cancer.Science,2003; 301(5638): 1308-1310.
[117]Passegue E, Jamieson CH, Ailles LE, et al.Normal and leukemic hematopoiesis, are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad Sci USA,2003; 100(Suppl1):11842-11849.
[118]Perez-Losada J, Balmain A.Stem-cell hierarchy in skin cancer.NatRev Cancer,2003; 3(6):434-443-
[119]Ricci-VitianiL, Lombardi DG, Pilozzi E, et al.Identification and expansion of human colon-cancer-initiating cells.Nature,2007; 445(7123):111-115.
[120]O'Brien CA, PollettA, Gallinger S, et al.A human colon cancer cell capable of initiating tumourgrowth in immunode-ficientmice.Nature,2007; 445(7123):106-110.
[121]Dalera P, Dylla SJ, Park IK, etal.Phenotypic characterization of human colorectal cancer stem cells.Proc Natl Acad Sci USA,2007; 104(24):10158-10163
[122]李波,陈维良,陈凯先.小檗碱及其衍生物的研究进展[J].药学学报,2008;43(8):773-787.
[123]陈冠容,周培恩.老药新用[M].北京:人民卫生出版社,1993:212-212.
[124]成都中医学院.中药学,第2版[M].上海:上海科学技术出版社,1981:62-62.
[125]王纪保,李敏,蒋永增.临床实用药物概要[M].西安:陕西科学技术出版社,1993:90-90.
[126]Han Y, Lee J H. Berberine synergy with amphotericin B against disseminated candidiasis in mice [J]. Biol Pharm Bull,2005;28 (3):541-544.
[127]Park K D, Lee J H, Kim S H, et al. Synthesis of 132(substituted benzyl) berberine and berberrubine derivatives as antifungal agents [J].Bioorg Med Chem Lett, 2006;16 (15):3913-3916.
[128]Sriwilaijareon N, Petmitr S, Mutirangura A, et al. Stage specificity of plasmodium falciparum telomerase and its inhibition by berberine [J]. Parasitol Int,2002; 51:99-103.
[129]Wu L T, Tsou M F, Ho C C, et al. Berberine inhibits arylamine N-acetyltransferase activity and gene expression in Salmonella typhi [J].Curr Microbiol,2005;51 (4):255-261.
[130]Hayashi K, Minoda K, Nagaoka Y, et al. Antiviral activity of berberine and related compounds against human cytomegalovirus [J]. Bioorg Med Chem Lett,2007;17 (6):1562-1564
[131]袁淑贞.双黄连体外抗解脲支原体试验及临床疗效观察[J].中华皮肤科杂志,1996;29(3):181-181.
[132]李建军,佟菊贞,李亿,等.黄连素治疗非淋菌性尿道炎疗效观察[J].中药材,1999;22(6):319.
[133]Wu JB. Preliminary study on antiviral activity of berberine derivate HB213 against herpes simp lex virus and itsmechanisms of action [C]. Beijing:Chinese Academy ofMedical Sciences and Peking Union Medical College,2006.
[134]Hayashi K, Minoda K, Nagaoka Y. Antiviral activity of berberine and related compounds against human cytome2 galovirus [J]. BioorgMed Chem Lett,2007;17: 1562-1564.
[135]Tan HM. Five cases of diabetes treated by hydrochlo ricberberine [J]. Her Med, 1996;15:297-297.
[136]Chen HF, Cai FN. Clinical investigation for 44 cases of gerontism type II diabetes treated by berberine [J].Jiangxi J Tradit ChinMed,1999; 30:19.
[137]华卫国,宋菊敏,廖菡,等.黄连素对糖尿病性神经病变大鼠神经传导速度的影响及激素的调节[J].标记免疫分析与临床,2001;8(4):212.
[138]Zhang M, Yu FH, Ding XP. Clinical investigation of type II diabetes treated by berberine [J]. Hubei J Tradit Chin Med,2001; 23:16-16.
[139]王国锋,何文艳,周静.黄连素治疗慢性肝病并发糖尿病33例临床分析[J].现代中西医结合杂志,2001;10(16):1512-1512.
[140]Huo Y, Cen DZ, Tu ZL. Clinical observation on berberine in reducing blood glucose [J]. HebeiMed J,2002;24:93-94.
[141]Wei J, Wu JD, J iang JD. Clinical study on imp rovement of type II diabetes mellitus comp Heated with fatty liver treated by berberine [J]. Chin J Integr Tradit West Med Liver Dis,2004; 14:334-336.
[142]陈莉,张玉.黄连素合六味地黄丸治疗2型糖尿病123例[J].国医论坛,2002;17(2):37-37.
[143]Wei J, J iang JD, Wu JD. Research on the effectiveimp rovement of hyperlip idemia by berberine [J]. Chin J Diabetes(中华糖尿病杂志),2005;13:49-51.
[144]Gou YH, Zong YX. Fifty-nine cases of hyperlipemia treated by berberine [J]. PLAMed(人民军医),1995;2:35-36.
[145]Kong W, Wei J, Abidi P, et al. Berberine is a novel cholesterol lowering drug working through a unique mechanism distinct from statins [J]. Nat Med,2004;10 (12):1344-1351.
[146]Cicero A F, Rovati L C, Setnikar I. Eulipidemic effects of berberine administered alone or in combination with other natural cholesterollowering agents. A single2blind clinical investigation [J]. Arzneimittel for schung,2007;57 (1):26-30.
[147]Tang L Q, Wei W, Chen L M, et al. Effects of berberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats [J]. JEthnopharmacol,2006;108 (1):109-115.
[148]姜长贵,匡永团.黄连素治疗心律失常32例疗效观察[J].中国中西医结合急救杂志,1998;5(2):402-402.
[149]郑金荣,王兵.黄连素与谷维素联合治疗心律失常38例报告[J].中国基层医药,2004;11(6):752-752.
[150]Zeng X H, Zeng X J, Li Y Y. Efficacy and safety of berberine forcongestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy [J]. Am J Cardiol, 2003;92 (2):173-176.
[151]智光,黄大显,杨兴生.黄连素治疗心功能衰竭的实验和临床观察[J].中华内科杂志,1991;30(9):581-581.
[152]康巧真,叶菲,杨顺裕.黄连素治疗充血性心力衰竭17例疗效观察[J].中国自然医学杂志,2002;4(2):83-83.
[153]黄伟民,徐尚忠,徐有秋.黄连素抗心律失常机理-电压钳制术观察延迟激活钾离子流的变化[J].中华心血管病杂志,1992;5:310-310.
[154]周祖玉,徐建国,蓝庭剑,等.黄连素对灌流心脏发生心衰的保护作用[J].华西医科大学学报,2001;32(3):417-417.
[155]周祖玉,孙爱民,徐建国,等.黄连素对离体灌流心脏的能量保存作用[J].华西医科大学学报,2002;33(3):431-431.
[156]ColomboML, Bugatti C, Mossa A et al. Cytotoxicity evaluation of natureal cop tisine and synthesis of cop tisine from Berberine [J].Farm aco,2001;56(527):403-409.
[157]Orfila L, RodriguezM, Colman T et al. Structuralmodification of berberine alkaloids in relation to cytotoxic activity in vitro[J].J Ethnopharm acol,2000;71(3):449-456.
[158]Iizuka N, Miyamoto K, Okita K et al. Inhibitory effect of Cop tidis Rhizoma and Berberine on the p roliferation of human esophageal cancer cell lines[J].Cancerlett, 2000;148(1):19-25.
[159]Li XK, Motwani M, TongW et al. Huanglian, achinese herbal extract, inhibits cell growth by supp ressing the exp ression of cyclin B1 and inhib iting CDC2 kinase activity in human cancer cells[J].Mol Pharm acol,2000;58(6):1287-1293.
[160]Lin HL, Liu TY, LuiWY et al. Up-regulation of multidrug resistance transporter expression by Berberine in human and murine hepatoma cells[J].Cancer,19 99;85(9):1937-1942.
[161]Lin HL, Liu TY, Wu CW et al. Berberinemodulates expression of mdrl gene product and the responses of digestive track cancer cells to paclitaxel.[J].Br J Cancer,1999;81(3):416-422.
[162]谭宇蕙,陈冠林,郭淑杰等.小檗碱对人胃癌MGC2803细胞生长抑制及诱导凋亡的作用[J].中国药理学通报,2001;17(1):40-43.
[163]台卫平,罗和生.黄连素抑制结肠癌细胞环氧合酶-2的作用[J].中国内科杂志,2003;42(8):558-558.
[164]Yount G, Qian Y, Moore D, et al. Berberine sensitizes human glioma cells, but not normal glial cells, to ionizing radiation in vitro [J]. J Exp Ther Oncol,2004; 4(2): 137-143.
[165]Lin C C, Kao S T, Chen G W, et al. Apoptosis of human leukemia HL260 cells and murine leukemia WEHI23 cells induced by berberine through the activation of caspase23 [J]. Anticancer Res,2006; 26(1 A):227-242.
[166]Lin J P, Yang J S, Lee J H, et al. Berberine induces cell cycle arrest and apoptosis in human gastric carcinoma SNU25 cell line [J].World J Gastroenterol,2006;12 (1):21-28.
[167]Hwang J M, Kuo H C, Tseng T H, et al. Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells [J]. Arch Toxicol,2006;80 (2):62-73.
[168]Mantena S K, Sharma S D, Katiyar S K. Berberine inhibits growth,induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP [Jj.Carcinogenesis,2006;27 (10):2018-2027.
[169]Jantova S, Cipak L, Letasiova S. Berberine induces apoptosis through a mitochondrial /caspase pathway in human promonocytic U937 cells [J]. Toxicol In Vitro,2007;21 (1):25-31.
[170]Kang MR, Chung IK. Down-regulation of DNA topoisomerase Ilalpha in human colorectal carcinoma cells resistant to a protoBerberine alkaloid, berberrubine[J].Mol Pharm acol,2002;61(4):879-884.
[171]Naasani I, Seimiya h, Yamori T et al. A potent telemerase inhibitor identified by exploiting the disease-oriented screening p rogram with COMPARE analysis [J]. Cancer Res,1999;59(16):4004-4011.
[172]何百成、康全等.小檗碱抗肿瘤作用与Wnt/β-catenin信号转导关系[J].中国药理学通报,2005;21(9):1108-1111
[173]Jung DW, Yoo GS, Choi JK. Detection of DNA in agarose gels using Berberine and Mordant Yellow 3R[J]. Anal Biochem,1999;272 (2):254-256.
[174]Qing SH, Rao KY, Jiang HY, Wexner SD. Racial differences in theanatomical distribution of colorectal cancer:a study of differences between American and Chinese patients. World J Gastroenterol,2003;9:721-725
[175]李连弟,钱克勤,张思维,等.中国12市县1993-1997年肿瘤发病和死亡登记资料统计分析[J].中国肿瘤,2002;11(9):497-507
[176]徐晓初,沈国英,张霞,戴克俊,董淑丽,白雪林,刘海温.上海市长宁区 1997-2001年居民主要恶性肿瘤死亡动态分析[J].上海预防医学杂志,2003:15:86-87
[177][澳]R费雷纳弗雷谢尼主编.章静波等译.人肿瘤细胞培养[M].第1版,北京:化学工业出版社,2006:19-20.
[178]杨志明.组织工程[M].第1版,北京:化学工业出版社,2002:19-21.
[179]Nakamura A,Isoyama S,Watanabe T,et al.Heterogeneous smooth muscle cell population derived from small and larger arteries. Microvasc Res,1998;55 (1):14-28.
[180]Man GT,Feng ZH. Improvement in the techniques of culturing and purifying pig,human,and rat endothelial cells and smooth muscle cells.Chin J Histochem Cytochem;2001;10 (2):123-128.
[181]Challacombe DN, Wheeler EE. Trophic action of epidermal growth factor on human duodenal mucosa cultured in vitro. Gut,1991;32:991-993.
[182]Zimmermann GS, Neurohr C, Villena-Hermoza H, Hatz R,Behr J. Anti-inflammatory effects of antibacterials on human bronchial epithelial cells. Respir Res,2009; 29:10-89.
[183]Broadhead CL, Bottrill K. Strategies for replacing animals in biomedical research. MolMed Today,1997;3:483-487.
[184]王莉珍.黄连在治疗恶性肿瘤中应用经验[J].实用中医内科杂志,2003;17(5):373-374
[185]阴健主编.中药现代研究与临床应用(1)[M].北京:学苑出版社,1993;578-579.
[186]林菁.小檗碱对K562细胞生长的抑制作用[J].福建医学院学报,1996;30(4):309-311.
[187]Wu SN,Yu HS,Jan CR,et al.Inhibitory effects of berberine onvoltage and calcium--activated potassium currents in humanmyeloma cells[J].Life Sci,1998;62(25):228 3-2294.
[188]Chi CW,Chang YF,Chao TW,et al.Flowcytometric analysis of the effect of berberine on the expression of glucocorticoid receptors in human hepatoma HepG2 cells.[J].Life Sci,1994;54(26):2099-2107.
[189]AnisKV, RajeshkumarNV, KuttanR. Inhibition of chemical car-cinogenesis by berberine in rats andmice[J]. Pharmacol,2001; 53(5):763-768.
[190]NishinoH, KitagawaK, FujikiH, eta.l Berberine sulfate inhibitstumor-promoting activity of teleocidin in two-stage carcinogene-sis onmouse skin[J]. Oncology, 1986;43(2):131-134.