RNAi阻断CDK2基因表达对RB基因表达的影响及树舌多糖GF辅助作用的研究
|
摘要
目的：通过将己构建成功的针对CDK2基因的siRNA真核表达载体，转染至人肝癌细胞株SMMC7721中，研究其对细胞周期调控网络中RB基因表达的影响，以及中药树舌多糖GF对其的辅助作用。
方法：将已构建成功的针对CDK2基因的siRNA真核表达载体通过阳离子脂质体试剂法转染肝癌细胞株SMMC7721，利用实时荧光定量PCR技术研究RNAi抑制CDK2基因后对细胞周期调控网络中相关基因RB的mRNA水平的影响；MTT法检测树舌多糖GF体外对SMMC7721细胞增殖抑制情况并筛选出体外抗瘤的有效树舌多糖GF剂量；并采用荧光定量PCR技术研究树舌多糖对肝癌SMMC7721细胞中RB基因表达的影响并探讨该多糖对其的辅助作用。
结果：
1．树舌多糖GF2．5μg·mL~(-1)、5μg·mL~(-1)、10μg·mL~(-1)对肝癌细胞增殖有明显抑制作用，抑制率分别为20．01％、20．47％、24．44％。
2．树舌多糖GF2．5μg·mL~(-1)、10μg·mL~(-1)可使RB基因mRNA表达水平上调。与SMMC7721相比，树舌多糖GF2．5μg·mL~(-1)组与树舌多糖GF10μg·mL~(-1)组RB基因mRNA水平分别上调63％、64％。
3．RNAi抑制CDK2基因表达后对RB基因mRNA表达有影响，siRNA190组中RB基因上调56％，siRNA19．1组中RB基因的mRNA表达上调11％。
4．与单纯siRNA191组相比，树舌多糖GF2．5μg·mL~(-1_、10μg·mL~(-1)与siRNA191组合用可分别使肝癌细胞中RB基因mRNA水平上调5％、4％；与单纯siRNA190组相比，树舌多糖GF2．5μg·mL~(-1)、10μg·mL~(-1)与siRNA190组合用均未能使肝癌细胞中RB基因mRNA水平上调。
结论：
1．肝癌SMMC7721细胞的过度增殖与RB基因缺失性表达关系密切。
2．树舌多糖GF对体外肝癌细胞株SMMC7721的增殖有明显抑制作用，其机制可能是通过上调RB基因表达实现的。
3．肝癌细胞中RB基因的mRNA表达与CDK2基因的表达有明显的关联性，RNAi特异地沉寂肝癌细胞中CDK2基因后，RB基因表达上调。
4．树舌多糖GF对RNAi特异沉寂肝癌细胞CDK2基因表达后上调RB基因表达有一定的辅助作用，表明树舌多糖GF可能成为肝癌基因治疗中的一个新型辅助药物。
Purpose: To select the effective siRNA eukaryotic expression plasmids (pGPU6/GFP/Neo) of CDK2 gene, then to transfect in the human hepatocellular carcinoma cells line(SMMC7721). For studying its effect on the RB gene in the cell cycle and the assistant action of GAPSGF [Polysaccharides component GF extracted by Ganoderma applanatum (Per. ex Gray) ].
Methods: The effective siRNA eukaryotic expression plasmids of CDK2 gene were selected and transfected into SMMC7721 cells with a lipofection method. Real-time PCR method was also utilized to detect the mRNA expression of RB.MTT was used to detect the inhibition of GAPSGF on SMMC7221 and to search the effective dose GAPSGF for anti-tumor in vitro. It was studied that the the effect of GAPSGF on RB mRNA expression of SMMC7721 cell with Real-time PCR method. The assistant effect of GAPSGF was investigated on the enhancement action of RB gene after RNA interference .
Results :
1. GAPSGF could obviously inhibit the cell proliferation in SMMC7721 at the effective doses of 2.5μ·g·mL~(-1), 5μg·mL~(-1) and 10μg·mL~(-1). The inhibition ratio were 20.01%, 20.47% and 24.44%.
2. GAPSGF could raised the mRNA expression of RB at the effective doses of 2.5μg·mL~(-1) and 10μg·mL~(-1). The mRNA expression of RB are raised 63% and 64% by 2.5μg·mL~(-1) and 10μg·mL~(-1) GAPSGF, respectively.
3. The inhibition of RNAi in CDK2 could influence RB gene expression in cell cycle.The mRNA expression of RB gene are raised 56% and 11% by siRNA segment 190and siRNA segment 191, respectively.
4. Comparing with application of siRNA segment 191 alone, the combining use of GAPSGF and siRNA segment 191 can raise the mRNA expression of RB. The mRNA expression of RB are raised 5% and 4% by2.5μg·mL-l and 10μg·mL-lGAPSGF,respectively.Comparing with application of siRNA segment 190 alone, the combining use of GAPSGF and siRNA segment 190 can not raise the mRNA expression of RB.
Conclusion:
1. Comparing with the normal hepatocyte,the mRNA expression of RB gene in the SMMC7721 cell have obviously down-regulation.
2. GAPSGF can obviously inhibit cell proliferation of SMMC7721 in vitro. The mechanism may be produced by the up-regulation of RB gene expression.
3. The mRNA expression of RB gene have obviously relevance on CDK2 gene. Silencing specifically the CDK2 gene by RNAi technique can raise the mRNA expression of RB gene.
4. GAPSGF has certain assistant action for the mRNA expression of RB gene after RNA interference. It is demonstrated that GAPSGF could become a new assistant medicine for treating hepatoma in gene therapy.
方法：将已构建成功的针对CDK2基因的siRNA真核表达载体通过阳离子脂质体试剂法转染肝癌细胞株SMMC7721，利用实时荧光定量PCR技术研究RNAi抑制CDK2基因后对细胞周期调控网络中相关基因RB的mRNA水平的影响；MTT法检测树舌多糖GF体外对SMMC7721细胞增殖抑制情况并筛选出体外抗瘤的有效树舌多糖GF剂量；并采用荧光定量PCR技术研究树舌多糖对肝癌SMMC7721细胞中RB基因表达的影响并探讨该多糖对其的辅助作用。
结果：
1．树舌多糖GF2．5μg·mL~(-1)、5μg·mL~(-1)、10μg·mL~(-1)对肝癌细胞增殖有明显抑制作用，抑制率分别为20．01％、20．47％、24．44％。
2．树舌多糖GF2．5μg·mL~(-1)、10μg·mL~(-1)可使RB基因mRNA表达水平上调。与SMMC7721相比，树舌多糖GF2．5μg·mL~(-1)组与树舌多糖GF10μg·mL~(-1)组RB基因mRNA水平分别上调63％、64％。
3．RNAi抑制CDK2基因表达后对RB基因mRNA表达有影响，siRNA190组中RB基因上调56％，siRNA19．1组中RB基因的mRNA表达上调11％。
4．与单纯siRNA191组相比，树舌多糖GF2．5μg·mL~(-1_、10μg·mL~(-1)与siRNA191组合用可分别使肝癌细胞中RB基因mRNA水平上调5％、4％；与单纯siRNA190组相比，树舌多糖GF2．5μg·mL~(-1)、10μg·mL~(-1)与siRNA190组合用均未能使肝癌细胞中RB基因mRNA水平上调。
结论：
1．肝癌SMMC7721细胞的过度增殖与RB基因缺失性表达关系密切。
2．树舌多糖GF对体外肝癌细胞株SMMC7721的增殖有明显抑制作用，其机制可能是通过上调RB基因表达实现的。
3．肝癌细胞中RB基因的mRNA表达与CDK2基因的表达有明显的关联性，RNAi特异地沉寂肝癌细胞中CDK2基因后，RB基因表达上调。
4．树舌多糖GF对RNAi特异沉寂肝癌细胞CDK2基因表达后上调RB基因表达有一定的辅助作用，表明树舌多糖GF可能成为肝癌基因治疗中的一个新型辅助药物。
Purpose: To select the effective siRNA eukaryotic expression plasmids (pGPU6/GFP/Neo) of CDK2 gene, then to transfect in the human hepatocellular carcinoma cells line(SMMC7721). For studying its effect on the RB gene in the cell cycle and the assistant action of GAPSGF [Polysaccharides component GF extracted by Ganoderma applanatum (Per. ex Gray) ].
Methods: The effective siRNA eukaryotic expression plasmids of CDK2 gene were selected and transfected into SMMC7721 cells with a lipofection method. Real-time PCR method was also utilized to detect the mRNA expression of RB.MTT was used to detect the inhibition of GAPSGF on SMMC7221 and to search the effective dose GAPSGF for anti-tumor in vitro. It was studied that the the effect of GAPSGF on RB mRNA expression of SMMC7721 cell with Real-time PCR method. The assistant effect of GAPSGF was investigated on the enhancement action of RB gene after RNA interference .
Results :
1. GAPSGF could obviously inhibit the cell proliferation in SMMC7721 at the effective doses of 2.5μ·g·mL~(-1), 5μg·mL~(-1) and 10μg·mL~(-1). The inhibition ratio were 20.01%, 20.47% and 24.44%.
2. GAPSGF could raised the mRNA expression of RB at the effective doses of 2.5μg·mL~(-1) and 10μg·mL~(-1). The mRNA expression of RB are raised 63% and 64% by 2.5μg·mL~(-1) and 10μg·mL~(-1) GAPSGF, respectively.
3. The inhibition of RNAi in CDK2 could influence RB gene expression in cell cycle.The mRNA expression of RB gene are raised 56% and 11% by siRNA segment 190and siRNA segment 191, respectively.
4. Comparing with application of siRNA segment 191 alone, the combining use of GAPSGF and siRNA segment 191 can raise the mRNA expression of RB. The mRNA expression of RB are raised 5% and 4% by2.5μg·mL-l and 10μg·mL-lGAPSGF,respectively.Comparing with application of siRNA segment 190 alone, the combining use of GAPSGF and siRNA segment 190 can not raise the mRNA expression of RB.
Conclusion:
1. Comparing with the normal hepatocyte,the mRNA expression of RB gene in the SMMC7721 cell have obviously down-regulation.
2. GAPSGF can obviously inhibit cell proliferation of SMMC7721 in vitro. The mechanism may be produced by the up-regulation of RB gene expression.
3. The mRNA expression of RB gene have obviously relevance on CDK2 gene. Silencing specifically the CDK2 gene by RNAi technique can raise the mRNA expression of RB gene.
4. GAPSGF has certain assistant action for the mRNA expression of RB gene after RNA interference. It is demonstrated that GAPSGF could become a new assistant medicine for treating hepatoma in gene therapy.
引文
[1]杨广顺，吴志全，吴孟超．原发性肝癌的规范化综合治疗[J]．中华外科杂志，2001，39(10)：742-744
[2]SherrCJ. Cancer cell cydes[J]. Science, 1996, 274: 1672-1677
[3]韦伟，龚建平，裘法祖．细胞周期调控与肿瘤的发生发展[J]．癌症，1999，18(1)：952-972
[4]Kamb A. Cell-cycle regulators and cancer[J]. Trends Genet, 1995, 11: 136-140
[5]Hunter T, Pines J. Cyclins and cancer II: cyclinD and CDK inhibitors come ofage [J]. Cell, 1994, 79: 573-582
[6]Johnson DG, Walker CL. Cyclins and cycle checkpoints [J]. Annu Rev PharmacolToxicol, 1999, 39: 295-312
[7]Hartwell LH, Kastan NiB. Cell cycle control and cancer [J]. Science, 1994, 266:1821-1828
[8]ParkinDM, BrayF, FerlayJ, etal. Estimation the world cancer burden:globocan2000 Int[J]. Cancer, 2001, 94(1): 153-156
[9]蔡琳，Binh Yang，DonaldM，等．亚太若干地区恶性肿瘤流行趋势分析[J]．肿瘤，2004，24(5)：422-426
[10]佚名．八大癌症威胁国人健康[J]．医药与保健，2003，(5)：53
[11]徐宁志，董志伟．中国肿瘤流行状况与防治对策述评[J]．肿瘤防治杂志，2003，10(1)： 15-18
[12]周宜强，王黎军．中医防治肿瘤的现状及肿瘤学科的发展思路[J]．世界中医药，2007， 2(5)：259
[13]陈锐深，曹洋．中医药治疗恶性肿瘤的特色和优势[J]．中医药学刊，2006，24(1)： 12-13
[14]何赛萍．中药抗肿瘤研究思路[J]．浙江中医学院学报，2001，25(4)4：61-62
[15]李循，孔繁智，朱婉萍．中药多糖抗肿瘤作用研究进展[J]．中药多糖抗肿瘤作用研究进展，2006，41(2)：113-116
[16]刁波，唐英，王晓琨，等．中药多糖研究新进展[J]．中国医药导报，2008，5(3)： 21-22
[17]王学宏，李明春．中药多糖的免疫及抗肿瘤作用研究进展[J]．齐鲁医学杂志，??2000，15(9)：230-231
[18]邵树军，刘彩玉，刘雄伯，等．牛膝多糖对小鼠免疫功能影响的研究[J]．肿瘤防治杂志，2002，9(1)：57
[19]田国燕，史亦谦．中药多糖的免疫调节作用研究进展[J]．医学研究杂志，2007，36(4)： 26-28
[20]何彦丽，苏俊芳．中药多糖抗肿瘤免疫药理研究的新思路——对树突状细胞的影响[J]．中国中西医结合杂志，2003，23(1)：73
[21]刘杰麟，费樱．戴氏虫草和粉被虫草多糖对巨咙细胞等活性的影响[J]．免疫学杂志，2001，17(3)：189-191
[22]潘洪明，于英君．中药多糖抗肿瘤研究进展[J]．齐齐哈尔医学院学报，2001，22(12)： 1439
[23]郑应馨，徐恒卫．具有抗肿瘤活性的多搪及其作用机理研究概况[J]．中国药师，2003，6(6)：368-369
[24]杜景卫，侯晓青，夏作里．中药多糖抗肿瘤作用机制及其影响因素[J]．中国药师，2006，9(9)：852-854
[25]宋为民，法京．人参和绞股蓝的抗突变作用[J]．中草药，1992，23(3)：136
[26]丁健．抗肿瘤药物的研究新进展[J]．中国新药杂志，2000，17(3)：150
[27]魏小龙，茹祥斌．低分子质量地黄多糖体外对LEWIS肿瘤细胞P53基因表达的影响[J]．中国药理学通报，1998，14(3)：245-248
[28]吕晓英，曾令福，王双印，等．红毛五加多糖对体外人胃癌细胞的抑制作用及其作用机制研究[J]．实用癌症杂志，2000，16(1)：6-8
[29]沈先荣，贾福星。血管生成抑制因子在肿瘤防治中的研究进展[J]．国外医学药学分册，1994，21(4)：215
[30]李家琦，夏英．中药诱导干扰素作用的探索[J]．上海中医药杂志，1994，28(1)：34
[31]于英君，杜家忠，刘丽波，等．树舌多糖对小鼠HepA癌细胞3H-TdR和(6-3H)-Glucose掺入的影响[J]．中医药信息，1997，14(3)：46
[32]苗本春，李静，耿美玉，等．海洋硫酸多糖聚古罗糖醋(912)抗肿瘤作用机理探讨[J]．中国海洋药物杂志，2003，3：11-15
[33]Lee WH, Bookstein R, Hong FD, et al. Human retinoblastoma susceptibility gene cloning identification and sequence[J]. Science, 1987, 235: 1394-1402
[34]Knudson AG. Mutation and cancer: statistical study ofretinoblastoma[J]. Proc Natl Acad Sci USA, 1971, 18: 820-823
[35]Friend SH, R Bernards, S Rogelj, et al. A human DNA segment with properties of the gene that predisposes to retino-blastoma and osteosarcoma[J]. Nature, 1986, 323: 643-646
[36]Fung YK, Murphree AL, T'Ang A, et al. Structural evid-ence for the authenticity of the human retinoblastoma gene[J]. Science, 1987, 236: 1657-1661
[37]Lee WH, R Bookstein, F Hong, et al. Human retino-blastoma susceptibility gene: cloning, identification, and sequ-ence[J]. Science, 1987, 235: 1394-1399
[38]Ekholm-Reed S, Mendez J, Tedesco D, et al. Deregulation of cyclin E in human cells interferes with prereplication complexassembly[J]. Cell Biol. 2004, 165(6): 789-800
[39]Deeds L, Teodorescu S, Chu M, et al. A p53-independent G1 cell cycle checkpoint induced by the suppression of protein kinase C alpha and theta isoforms[J]. Biol Chem, 2003, 278(41): 39782-93
[40]Lee MH, Yang HY. Negative regulations of cyclin- de-pendent kinases and their roles in cancers[J]. Cell Mol Life Sci, 2001, 58(12-13): 1907-1922
[41]Nielsen SJ, Schneider R, Bauer UM, et al. Rb targets histone H3 methylation and HP1 to promoters[J]. Nature, 2001, 412(6846): 561-565
[42]Berezuts K E, Yu B, Morozov A, et al. Differential regulation of the pocket domain of the retinoblastoma family proteins by the HPV16 E7 oncoprotein[J]. Cell Growth Differ, 1997, 8(12): 1277-1286
[43]Wang NP, To H, Lee WH, etal. Tumorsuppressor activity of Rb and p53 Gene in human breast carcinoma cells[J]. Oncogene, 1993, 8: 219-224
[44]Xu HJ, Kuzumaki N, Kawakami Y, etal. Altered retinoblastoma protein expression in nonsmall cell lung cancer: its synergistic effects with altered as and p53 protein status on prognosis[J]. Cancer, 1997, 79: 1329-1337
[45]Marchetti A, Doglionic C, Barbareschi V, et al. Cyclin D1 and retinoblastoma susceptibity gene alterations in non-small-cell lung cancer , Int[J]. Cancer, 1998,??75(2)： 187-192
[46]冀文茹，乌恩，施琳等，胰腺癌胰腺炎CD44V6，RB，C myc基因检测分析[J]．内蒙古医学院学报，2001，23(3)：167-169
[47]刘清源，施学忠，李来生，等．Rb对HepG2肝癌细胞系增殖的影响[J]．医药论坛杂志2006，27(22)：10-11
[48]刘凯，何煦，赵连三，等．利用组织芯片研究Rb基因在原发性肝细胞肝癌中的表达[J]．中国普外基础与临床杂志，2002，9(3)：177-179
[49]Marc F, Hansen. Molecuar genetic consideration in osteosarcoma[J]. Clinthop,1991, 270(5): 273-81
[50]Guo S, Kemphues K[J]. Cell, 1995, 81(4): 611-620
[51]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interferenceby double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669):744-745
[52]Hannon GJ. RNA interference[J]. Nature, 2002, 418(6894): 244-251
[53]Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled RNA(Val)promoter significantly induce RNAi mediated gene silencing in cytoplasm of humancell[J]. Nucleic Acids Res, 2003, 31(2): 700-707
[54]Caplen NJ . Gene therapy progress and prospects. Downregulating geneexpression: the impact of RNA interference[J]. Gene Ther, 2004 ,11(16): 1241-1248
[55]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interferenceby double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669):806-811.
[56]Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411(6836): 494-498
[57]Borkhardt A. Blocking oncogenes in malignant cells by RNA interference-newhope for a highly specific cancer treatment[J]. Cancer Cell, 2002, 2(3): 167-168
[58]Ellen D, Brusselmans K,Heyns W, et al. RNA interference-mediated silencingof the fatty acid synthase gene attenuates growth and induces morphologicalchanges and apoptosis of LNCap prostate cancer cells. Cancer Res, 2003, 63:??3799-3804
[59]Li KY, Shiaw YL, Charles F, et al. Use of RNA interference to target cyclinE-overexpressing hepatocellular[J]. Carcinoma, 2003, 63: 3593-3597
[60]Chen Y, Stamatoyannopoulos G, Song CZ, etal. Down regulation of CXCR4by inducible small interfering RNA inhibits breast cancer cell inva-sion invitro[J]. Cancer Res, 2003, 63(16): 4801-4804
[61]Scherr M, Battmer K, Winkler T, et al. Specific inhibition of bcr-abl geneexpression by small interfering RNA[J]. Blood, 2003, 101(4): 1566-1569
[62]Krishnamachary B, Berg-Dixon S, Kelly B, et al. Regulation of colon carcinomacell invasion by hypoxia-inducible factor 1 [J]. Cancer Res, 2003, 63(5): 1138-43
[63]于英君，孙玺嫒．树舌多糖GF对小鼠HepA癌细胞凋亡影响的初探[J]．中 医药学报，2003，5： 34
[64]于英君，刘丽波，孙巧红，等． 树舌多糖注射液抗肿瘤作用的研究[A]．国 际传统医药大会论文摘要汇编[C]，2000，3 56
[65]管宇，于英君．树舌多糖GF注射液对荷瘤鼠T细胞亚群、白介素-2及巨噬 细胞吞噬功能的影响[J]．中国药业，2005，1 4(3)：31-33
[66]于英君，张庆梅，郭丽新，等．树舌多糖G F对HepA瘤细胞p53基因表达的 影响[J]．中医药学报，2002，30(2)：56-57
[67]于英君，潘洪明，张庆梅，等．树舌多糖GF对HepA瘤细胞p16基因表达的 影响[J]．Chinese Archives of Traditional Chinese Medicine，2002，20(2)：160-162
[68]张庆梅，刘丽波，潘洪明，等．树舌多糖GF对小鼠HepA瘤细胞Rb基因表 达的影响[J]．中医药学刊，2002，20(4)：431-443
[69]潘洪明，于英君．树舌多糖、猪苓多糖对小鼠HepA癌细胞TNF-α表达的影 响[J]．中国基层医药，2002，9(6)：486-487
[70]杨晶凡，刘丽波，于英君．树舌多糖GF对HepA瘤细胞Ras蛋白表达的影响 [J]．中医药学刊，2003，21(11)：1831-1832
[71]孙力，杨晶凡，于英君．树舌多糖GF对HepA瘤细胞C-myc蛋白表达的影响 [J]．中国中医药科技，2004，11(3)：137
[72]宋高臣，孙玺嫒，于英君．树舌多糖GF对小鼠HepA瘤基因组DNA甲基化 影响的实验研究[J]．中国优生与遗传杂志，2005，13(1)：51-52
[73]于赫，于英君．树舌多糖GF对HepA瘤细胞MDM-2基因表达的影响研究[J]．．中医药学刊，2005，23(2)：283-284
[74]宋高臣，于英君，管宇，等．树舌多糖GF注射液对化疗药物的增效减毒作用及其机制研究[J]．中国老年学杂志，2005，25(4)：424-425
[75]司徒镇强，吴军正，主编．细胞培养[M]．第1版，北京：世界图书出版公司，2004：250-252
[76]Xia DY, Sanders A, Shah M, et al. Rela-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in allograft acceptor mice[J]. Transplantation, 2001, 72: 907-914
[77]全成实，郭晓峰．喉鳞癌组织中Rb基因突变的分析[J]．吉林大学学报，医学版，2002，28(4)：383-385
[78]Maddison Lisette A, Sutherland Brent W, Barrios Roberto J, et al. Conditiondeletion of Rb causes early stage prostate cancer[J]. Cancer Research, 2004, 64(17):6018-6025
[79]CHOUDHARY J, GRANT S. Proteomics in postgenomic neuro-science: theending of the beginning [J]. Nat Neurosci, 2004, 7(3): 440-445
[80]李育，王明艳．中药抗肿瘤的分子机理[J]．中国中医药信息杂志，2005，12(1)：95-96
[2]SherrCJ. Cancer cell cydes[J]. Science, 1996, 274: 1672-1677
[3]韦伟，龚建平，裘法祖．细胞周期调控与肿瘤的发生发展[J]．癌症，1999，18(1)：952-972
[4]Kamb A. Cell-cycle regulators and cancer[J]. Trends Genet, 1995, 11: 136-140
[5]Hunter T, Pines J. Cyclins and cancer II: cyclinD and CDK inhibitors come ofage [J]. Cell, 1994, 79: 573-582
[6]Johnson DG, Walker CL. Cyclins and cycle checkpoints [J]. Annu Rev PharmacolToxicol, 1999, 39: 295-312
[7]Hartwell LH, Kastan NiB. Cell cycle control and cancer [J]. Science, 1994, 266:1821-1828
[8]ParkinDM, BrayF, FerlayJ, etal. Estimation the world cancer burden:globocan2000 Int[J]. Cancer, 2001, 94(1): 153-156
[9]蔡琳，Binh Yang，DonaldM，等．亚太若干地区恶性肿瘤流行趋势分析[J]．肿瘤，2004，24(5)：422-426
[10]佚名．八大癌症威胁国人健康[J]．医药与保健，2003，(5)：53
[11]徐宁志，董志伟．中国肿瘤流行状况与防治对策述评[J]．肿瘤防治杂志，2003，10(1)： 15-18
[12]周宜强，王黎军．中医防治肿瘤的现状及肿瘤学科的发展思路[J]．世界中医药，2007， 2(5)：259
[13]陈锐深，曹洋．中医药治疗恶性肿瘤的特色和优势[J]．中医药学刊，2006，24(1)： 12-13
[14]何赛萍．中药抗肿瘤研究思路[J]．浙江中医学院学报，2001，25(4)4：61-62
[15]李循，孔繁智，朱婉萍．中药多糖抗肿瘤作用研究进展[J]．中药多糖抗肿瘤作用研究进展，2006，41(2)：113-116
[16]刁波，唐英，王晓琨，等．中药多糖研究新进展[J]．中国医药导报，2008，5(3)： 21-22
[17]王学宏，李明春．中药多糖的免疫及抗肿瘤作用研究进展[J]．齐鲁医学杂志，??2000，15(9)：230-231
[18]邵树军，刘彩玉，刘雄伯，等．牛膝多糖对小鼠免疫功能影响的研究[J]．肿瘤防治杂志，2002，9(1)：57
[19]田国燕，史亦谦．中药多糖的免疫调节作用研究进展[J]．医学研究杂志，2007，36(4)： 26-28
[20]何彦丽，苏俊芳．中药多糖抗肿瘤免疫药理研究的新思路——对树突状细胞的影响[J]．中国中西医结合杂志，2003，23(1)：73
[21]刘杰麟，费樱．戴氏虫草和粉被虫草多糖对巨咙细胞等活性的影响[J]．免疫学杂志，2001，17(3)：189-191
[22]潘洪明，于英君．中药多糖抗肿瘤研究进展[J]．齐齐哈尔医学院学报，2001，22(12)： 1439
[23]郑应馨，徐恒卫．具有抗肿瘤活性的多搪及其作用机理研究概况[J]．中国药师，2003，6(6)：368-369
[24]杜景卫，侯晓青，夏作里．中药多糖抗肿瘤作用机制及其影响因素[J]．中国药师，2006，9(9)：852-854
[25]宋为民，法京．人参和绞股蓝的抗突变作用[J]．中草药，1992，23(3)：136
[26]丁健．抗肿瘤药物的研究新进展[J]．中国新药杂志，2000，17(3)：150
[27]魏小龙，茹祥斌．低分子质量地黄多糖体外对LEWIS肿瘤细胞P53基因表达的影响[J]．中国药理学通报，1998，14(3)：245-248
[28]吕晓英，曾令福，王双印，等．红毛五加多糖对体外人胃癌细胞的抑制作用及其作用机制研究[J]．实用癌症杂志，2000，16(1)：6-8
[29]沈先荣，贾福星。血管生成抑制因子在肿瘤防治中的研究进展[J]．国外医学药学分册，1994，21(4)：215
[30]李家琦，夏英．中药诱导干扰素作用的探索[J]．上海中医药杂志，1994，28(1)：34
[31]于英君，杜家忠，刘丽波，等．树舌多糖对小鼠HepA癌细胞3H-TdR和(6-3H)-Glucose掺入的影响[J]．中医药信息，1997，14(3)：46
[32]苗本春，李静，耿美玉，等．海洋硫酸多糖聚古罗糖醋(912)抗肿瘤作用机理探讨[J]．中国海洋药物杂志，2003，3：11-15
[33]Lee WH, Bookstein R, Hong FD, et al. Human retinoblastoma susceptibility gene cloning identification and sequence[J]. Science, 1987, 235: 1394-1402
[34]Knudson AG. Mutation and cancer: statistical study ofretinoblastoma[J]. Proc Natl Acad Sci USA, 1971, 18: 820-823
[35]Friend SH, R Bernards, S Rogelj, et al. A human DNA segment with properties of the gene that predisposes to retino-blastoma and osteosarcoma[J]. Nature, 1986, 323: 643-646
[36]Fung YK, Murphree AL, T'Ang A, et al. Structural evid-ence for the authenticity of the human retinoblastoma gene[J]. Science, 1987, 236: 1657-1661
[37]Lee WH, R Bookstein, F Hong, et al. Human retino-blastoma susceptibility gene: cloning, identification, and sequ-ence[J]. Science, 1987, 235: 1394-1399
[38]Ekholm-Reed S, Mendez J, Tedesco D, et al. Deregulation of cyclin E in human cells interferes with prereplication complexassembly[J]. Cell Biol. 2004, 165(6): 789-800
[39]Deeds L, Teodorescu S, Chu M, et al. A p53-independent G1 cell cycle checkpoint induced by the suppression of protein kinase C alpha and theta isoforms[J]. Biol Chem, 2003, 278(41): 39782-93
[40]Lee MH, Yang HY. Negative regulations of cyclin- de-pendent kinases and their roles in cancers[J]. Cell Mol Life Sci, 2001, 58(12-13): 1907-1922
[41]Nielsen SJ, Schneider R, Bauer UM, et al. Rb targets histone H3 methylation and HP1 to promoters[J]. Nature, 2001, 412(6846): 561-565
[42]Berezuts K E, Yu B, Morozov A, et al. Differential regulation of the pocket domain of the retinoblastoma family proteins by the HPV16 E7 oncoprotein[J]. Cell Growth Differ, 1997, 8(12): 1277-1286
[43]Wang NP, To H, Lee WH, etal. Tumorsuppressor activity of Rb and p53 Gene in human breast carcinoma cells[J]. Oncogene, 1993, 8: 219-224
[44]Xu HJ, Kuzumaki N, Kawakami Y, etal. Altered retinoblastoma protein expression in nonsmall cell lung cancer: its synergistic effects with altered as and p53 protein status on prognosis[J]. Cancer, 1997, 79: 1329-1337
[45]Marchetti A, Doglionic C, Barbareschi V, et al. Cyclin D1 and retinoblastoma susceptibity gene alterations in non-small-cell lung cancer , Int[J]. Cancer, 1998,??75(2)： 187-192
[46]冀文茹，乌恩，施琳等，胰腺癌胰腺炎CD44V6，RB，C myc基因检测分析[J]．内蒙古医学院学报，2001，23(3)：167-169
[47]刘清源，施学忠，李来生，等．Rb对HepG2肝癌细胞系增殖的影响[J]．医药论坛杂志2006，27(22)：10-11
[48]刘凯，何煦，赵连三，等．利用组织芯片研究Rb基因在原发性肝细胞肝癌中的表达[J]．中国普外基础与临床杂志，2002，9(3)：177-179
[49]Marc F, Hansen. Molecuar genetic consideration in osteosarcoma[J]. Clinthop,1991, 270(5): 273-81
[50]Guo S, Kemphues K[J]. Cell, 1995, 81(4): 611-620
[51]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interferenceby double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669):744-745
[52]Hannon GJ. RNA interference[J]. Nature, 2002, 418(6894): 244-251
[53]Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled RNA(Val)promoter significantly induce RNAi mediated gene silencing in cytoplasm of humancell[J]. Nucleic Acids Res, 2003, 31(2): 700-707
[54]Caplen NJ . Gene therapy progress and prospects. Downregulating geneexpression: the impact of RNA interference[J]. Gene Ther, 2004 ,11(16): 1241-1248
[55]Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interferenceby double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669):806-811.
[56]Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells[J]. Nature, 2001, 411(6836): 494-498
[57]Borkhardt A. Blocking oncogenes in malignant cells by RNA interference-newhope for a highly specific cancer treatment[J]. Cancer Cell, 2002, 2(3): 167-168
[58]Ellen D, Brusselmans K,Heyns W, et al. RNA interference-mediated silencingof the fatty acid synthase gene attenuates growth and induces morphologicalchanges and apoptosis of LNCap prostate cancer cells. Cancer Res, 2003, 63:??3799-3804
[59]Li KY, Shiaw YL, Charles F, et al. Use of RNA interference to target cyclinE-overexpressing hepatocellular[J]. Carcinoma, 2003, 63: 3593-3597
[60]Chen Y, Stamatoyannopoulos G, Song CZ, etal. Down regulation of CXCR4by inducible small interfering RNA inhibits breast cancer cell inva-sion invitro[J]. Cancer Res, 2003, 63(16): 4801-4804
[61]Scherr M, Battmer K, Winkler T, et al. Specific inhibition of bcr-abl geneexpression by small interfering RNA[J]. Blood, 2003, 101(4): 1566-1569
[62]Krishnamachary B, Berg-Dixon S, Kelly B, et al. Regulation of colon carcinomacell invasion by hypoxia-inducible factor 1 [J]. Cancer Res, 2003, 63(5): 1138-43
[63]于英君，孙玺嫒．树舌多糖GF对小鼠HepA癌细胞凋亡影响的初探[J]．中 医药学报，2003，5： 34
[64]于英君，刘丽波，孙巧红，等． 树舌多糖注射液抗肿瘤作用的研究[A]．国 际传统医药大会论文摘要汇编[C]，2000，3 56
[65]管宇，于英君．树舌多糖GF注射液对荷瘤鼠T细胞亚群、白介素-2及巨噬 细胞吞噬功能的影响[J]．中国药业，2005，1 4(3)：31-33
[66]于英君，张庆梅，郭丽新，等．树舌多糖G F对HepA瘤细胞p53基因表达的 影响[J]．中医药学报，2002，30(2)：56-57
[67]于英君，潘洪明，张庆梅，等．树舌多糖GF对HepA瘤细胞p16基因表达的 影响[J]．Chinese Archives of Traditional Chinese Medicine，2002，20(2)：160-162
[68]张庆梅，刘丽波，潘洪明，等．树舌多糖GF对小鼠HepA瘤细胞Rb基因表 达的影响[J]．中医药学刊，2002，20(4)：431-443
[69]潘洪明，于英君．树舌多糖、猪苓多糖对小鼠HepA癌细胞TNF-α表达的影 响[J]．中国基层医药，2002，9(6)：486-487
[70]杨晶凡，刘丽波，于英君．树舌多糖GF对HepA瘤细胞Ras蛋白表达的影响 [J]．中医药学刊，2003，21(11)：1831-1832
[71]孙力，杨晶凡，于英君．树舌多糖GF对HepA瘤细胞C-myc蛋白表达的影响 [J]．中国中医药科技，2004，11(3)：137
[72]宋高臣，孙玺嫒，于英君．树舌多糖GF对小鼠HepA瘤基因组DNA甲基化 影响的实验研究[J]．中国优生与遗传杂志，2005，13(1)：51-52
[73]于赫，于英君．树舌多糖GF对HepA瘤细胞MDM-2基因表达的影响研究[J]．．中医药学刊，2005，23(2)：283-284
[74]宋高臣，于英君，管宇，等．树舌多糖GF注射液对化疗药物的增效减毒作用及其机制研究[J]．中国老年学杂志，2005，25(4)：424-425
[75]司徒镇强，吴军正，主编．细胞培养[M]．第1版，北京：世界图书出版公司，2004：250-252
[76]Xia DY, Sanders A, Shah M, et al. Rela-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in allograft acceptor mice[J]. Transplantation, 2001, 72: 907-914
[77]全成实，郭晓峰．喉鳞癌组织中Rb基因突变的分析[J]．吉林大学学报，医学版，2002，28(4)：383-385
[78]Maddison Lisette A, Sutherland Brent W, Barrios Roberto J, et al. Conditiondeletion of Rb causes early stage prostate cancer[J]. Cancer Research, 2004, 64(17):6018-6025
[79]CHOUDHARY J, GRANT S. Proteomics in postgenomic neuro-science: theending of the beginning [J]. Nat Neurosci, 2004, 7(3): 440-445
[80]李育，王明艳．中药抗肿瘤的分子机理[J]．中国中医药信息杂志，2005，12(1)：95-96