RNAi沉寂肝癌细胞CDK2基因表达及树舌多糖GF辅助作用的研究
摘要
目的:构建针对CDK2的siRNA真核表达载体,转染肝癌细胞株SMMC7721,研究其对肝癌CDK2基因及细胞周期相关基因(RB、CyclinE、E2F1)表达的影响,以及树舌多糖GF对其的辅助作用。
方法:用基因克隆技术构建针对CDK2基因的siRNA真核表达载体,阳离子脂质体试剂法转染肝癌细胞株SMMC7721,实时荧光定量PCR与Western blot技术检测对CDK2基因的抑制效果并筛选出有效siRNA序列片段;并用实时荧光定量PCR技术研究RNAi抑制CDK2对细胞周期相关基因RB、CyclinE、E2F1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;并采用实时荧光定量PCR与Western blot技术研究树舌多糖GF对肝癌SMMC7721细胞CDK2、RB、CyclinE、E2F1基因表达的影响并探讨该多糖对RNAi抑制CDK2基因的辅助作用。
结果:
1.DNA测序证明成功构建针对CDK2基因的siRNA真核表达载体。
2.阳离子脂质体转染法成功将外源表达载体转入SMMC7721细胞,转染效率达90%以上。
3.肝癌细胞CDK2基因的有效siRNA干扰片段为190:GGAGCTTAACCATCCTAATAT与191:GACCCTAACAAGCGGATTTCG。
4.siRNA真核表达载体可以在mRNA与蛋白水平特异地阻断SMMC7721细胞CDK2基因的表达,其中190、191对CDK2基因mRNA水平的抑制率分别为72%、56%,对CDK2基因蛋白水平的抑制率分别为91.6%、79.5%。
5.RNAi抑制CDK2对细胞周期相关基因表达有影响,190片段使RB基因mRNA表达上调56%,191片段可使CyclinE与E2F1基因mRNA表达分别下调36%、38%。
6.树舌多糖GF 2.5μg·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%。
7.树舌多糖GF2.5μg·mL~(-1)、10μg·mL~(-1)可使CDK2、CyclinE、E2F1基因mRNA表达水平下调,使RB基因mRNA表达水平上调。
8.树舌多糖GF10μg·mL~(-1)与190序列合用使CDK2蛋白表达比单纯190抑制提高47.1%;树舌多糖GF2.5μg·mL~(-1)与191序列合用使CDK2mRNA表达比单纯191抑制提高3%,蛋白表达比单纯191抑制提高13.3%。
结论:
1.经DNA测序证明,成功构建6对以CDK2为靶基因的siRNA真核表达载体。
2.通过RNAi能特异地沉寂肝癌细胞中CDK2基因,表明CDK2基因可能成为肝癌基因治疗中的一个新的靶点。
3.肝癌细胞中RB、CyclinE、E2F1基因的mRNA表达对CDK2基因的表达有明显的依赖性,表明对细胞周期调控网络中CDK2基因的干扰抑制是肝癌基因治疗的有效手段之一。
4.树舌多糖GF对体外肝癌细胞株SMMC7721的增殖有明显抑制作用,机制可能是通过下调CDK2、CyclinE与E2F1基因表达,上调RB基因表达实现的。
5.树舌多糖GF对RNAi沉寂CDK2基因表达有一定辅助作用,表明树舌多糖GF可以成为肝癌基因治疗中的一个新的辅助药物。
Purpose:To Construct the 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 CDK2 gene and the related RB,Cyclin E and E2F1 genes in the cell cycle and the assistant action of GAPSGF[Polysaccharides component GF extracted by Ganoderma applanatum(Per.ex Gray)].
Methods:The siRNA eukaryotic expression plasmids of CDK2 gene were constructed and transfected into SMMC7721 cells with a lipofection method.The inhibitive effect of CDK2 gene and effective sequence of siRNA was identified with Real-time PCR and Western blot methods. Real-time PCR method was also utilized to detect the mRNA expression of RB,Cyclin E and E2F1 that are related to CDK2 gene.MTT was used to detect the inhibition of GAPSGF in cell proliferation of SMMC7221 and to search the effective dose GAPSGF for anti-tumor in vitro.It was studied that the the effect of GAPSGF on CDK2,RB,Cyclin E and E2F1 mRNA and protein expression of SMMC7721 cell with Real-time PCR and Western blot methods.The assistant effect of GAPSGF was investigated on the inhibition action of RNAi in CDK2 gene.
Results:
1.The siRNA eukaryotic expression plasmids of CDK2 gene have been successfully constructed that was proved by DNA sequence determination.
2.The siRNA eukaryotic expression plasmids of CDK2 gene have been successfully transfected into SMMC7721 cells with a lipofection method. The transfection rate was over 90%.
3.Effective siRNA sequence of CDK2 gene were 190: GGAGCTTAACCATCCTAATAT and 191:GACCCTAACAAGCGGATTTCG in SMMC7721 cells.
4.The siRNA eukaryotic expression plasmids of CDK2 gene can specificly inhibit mRNA and protein expression in SMMC7721,and the inhibition ratio of siRNA segment 190 and 191 in mRNA expression of CDK2 were 72%and 56%,respectively;and in protein expression of CDK2 were 91.6%and 79.5%,respectively.
5.The inhibition of RNAi in CDK2 could influence the related genes expression in cell cycle,The expression of RB gene is raised 56%by SiRNA segment 190,the expression of Cyclin E and E2F1 were reduced 36%and 38%by siRNA segment 191,respectively.
6.GAPSGF could obviously inhibit the cell proliferation in SMMC7721 at the effective doses of 2.5μg·mL~(-1),5μg·mL~(-1)and 10μg·mL~(-1) The inhibition ratio were 20.01%,20.47%and 24.44%.
7.GAPSGF could reduced the mRNA expression of CDK2,CyclinE and E2F1,raised the mRNA expression of RB at the effective doses of 2.5μg·mL~(-1)and 10μg·mL~(-1).
8.Comparing with application of siRNA segment 190 alone,the combining use of 10μg·mL~(-1)GAPSGF and siRNA segment 190 can raise the inhibition ratio of CDK2 protein expression,The inhibition ratio can increase 47.1%;Comparing with application of siRNA segment 191 alone, the combining use of 2.5μg·mL~(-1)GAPSGF and siRNA segment 190 can raise the inhibition ratio of CDK2 mRNA and protein expression,The inhibition ratio can increase 3%and 13.3%,respectively.
Conclusion:
1.Six pairs of siRNA eukaryotic expression plasmids of CDK2 gene have been successfully constructed by DNA sequence determination.
2.CDK2 gene can be specifically silenced by RNAi technique.It is indicated that CDK2 gene may be a new target for treating hepatoma in gene therapy.
3.The mRNA expression of RB,Cyclin E and E2F1 genes related to CDK2 gene have obviously dependence on CDK2 gene.Therefore,it indicates that the interference of CDK2 gene is a an effective means for treating hepatoma in cell regulation net.
4.GAPSGF can obviously inhibit cell proliferation of SMMC7721 in vitro.The mechanism may be produced by the down-regulation of CDK2, Cyclin E and E2F1 genes expression,and the up-regulation of RB gene expression.
5.GAPSGF has certain assistant action for the RNA interfere expression of CDK2.It is demonstrated that GAPSGF could become a new assistant medicine for treating hepatoma in gene therapy.
方法:用基因克隆技术构建针对CDK2基因的siRNA真核表达载体,阳离子脂质体试剂法转染肝癌细胞株SMMC7721,实时荧光定量PCR与Western blot技术检测对CDK2基因的抑制效果并筛选出有效siRNA序列片段;并用实时荧光定量PCR技术研究RNAi抑制CDK2对细胞周期相关基因RB、CyclinE、E2F1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;并采用实时荧光定量PCR与Western blot技术研究树舌多糖GF对肝癌SMMC7721细胞CDK2、RB、CyclinE、E2F1基因表达的影响并探讨该多糖对RNAi抑制CDK2基因的辅助作用。
结果:
1.DNA测序证明成功构建针对CDK2基因的siRNA真核表达载体。
2.阳离子脂质体转染法成功将外源表达载体转入SMMC7721细胞,转染效率达90%以上。
3.肝癌细胞CDK2基因的有效siRNA干扰片段为190:GGAGCTTAACCATCCTAATAT与191:GACCCTAACAAGCGGATTTCG。
4.siRNA真核表达载体可以在mRNA与蛋白水平特异地阻断SMMC7721细胞CDK2基因的表达,其中190、191对CDK2基因mRNA水平的抑制率分别为72%、56%,对CDK2基因蛋白水平的抑制率分别为91.6%、79.5%。
5.RNAi抑制CDK2对细胞周期相关基因表达有影响,190片段使RB基因mRNA表达上调56%,191片段可使CyclinE与E2F1基因mRNA表达分别下调36%、38%。
6.树舌多糖GF 2.5μg·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%。
7.树舌多糖GF2.5μg·mL~(-1)、10μg·mL~(-1)可使CDK2、CyclinE、E2F1基因mRNA表达水平下调,使RB基因mRNA表达水平上调。
8.树舌多糖GF10μg·mL~(-1)与190序列合用使CDK2蛋白表达比单纯190抑制提高47.1%;树舌多糖GF2.5μg·mL~(-1)与191序列合用使CDK2mRNA表达比单纯191抑制提高3%,蛋白表达比单纯191抑制提高13.3%。
结论:
1.经DNA测序证明,成功构建6对以CDK2为靶基因的siRNA真核表达载体。
2.通过RNAi能特异地沉寂肝癌细胞中CDK2基因,表明CDK2基因可能成为肝癌基因治疗中的一个新的靶点。
3.肝癌细胞中RB、CyclinE、E2F1基因的mRNA表达对CDK2基因的表达有明显的依赖性,表明对细胞周期调控网络中CDK2基因的干扰抑制是肝癌基因治疗的有效手段之一。
4.树舌多糖GF对体外肝癌细胞株SMMC7721的增殖有明显抑制作用,机制可能是通过下调CDK2、CyclinE与E2F1基因表达,上调RB基因表达实现的。
5.树舌多糖GF对RNAi沉寂CDK2基因表达有一定辅助作用,表明树舌多糖GF可以成为肝癌基因治疗中的一个新的辅助药物。
Purpose:To Construct the 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 CDK2 gene and the related RB,Cyclin E and E2F1 genes in the cell cycle and the assistant action of GAPSGF[Polysaccharides component GF extracted by Ganoderma applanatum(Per.ex Gray)].
Methods:The siRNA eukaryotic expression plasmids of CDK2 gene were constructed and transfected into SMMC7721 cells with a lipofection method.The inhibitive effect of CDK2 gene and effective sequence of siRNA was identified with Real-time PCR and Western blot methods. Real-time PCR method was also utilized to detect the mRNA expression of RB,Cyclin E and E2F1 that are related to CDK2 gene.MTT was used to detect the inhibition of GAPSGF in cell proliferation of SMMC7221 and to search the effective dose GAPSGF for anti-tumor in vitro.It was studied that the the effect of GAPSGF on CDK2,RB,Cyclin E and E2F1 mRNA and protein expression of SMMC7721 cell with Real-time PCR and Western blot methods.The assistant effect of GAPSGF was investigated on the inhibition action of RNAi in CDK2 gene.
Results:
1.The siRNA eukaryotic expression plasmids of CDK2 gene have been successfully constructed that was proved by DNA sequence determination.
2.The siRNA eukaryotic expression plasmids of CDK2 gene have been successfully transfected into SMMC7721 cells with a lipofection method. The transfection rate was over 90%.
3.Effective siRNA sequence of CDK2 gene were 190: GGAGCTTAACCATCCTAATAT and 191:GACCCTAACAAGCGGATTTCG in SMMC7721 cells.
4.The siRNA eukaryotic expression plasmids of CDK2 gene can specificly inhibit mRNA and protein expression in SMMC7721,and the inhibition ratio of siRNA segment 190 and 191 in mRNA expression of CDK2 were 72%and 56%,respectively;and in protein expression of CDK2 were 91.6%and 79.5%,respectively.
5.The inhibition of RNAi in CDK2 could influence the related genes expression in cell cycle,The expression of RB gene is raised 56%by SiRNA segment 190,the expression of Cyclin E and E2F1 were reduced 36%and 38%by siRNA segment 191,respectively.
6.GAPSGF could obviously inhibit the cell proliferation in SMMC7721 at the effective doses of 2.5μg·mL~(-1),5μg·mL~(-1)and 10μg·mL~(-1) The inhibition ratio were 20.01%,20.47%and 24.44%.
7.GAPSGF could reduced the mRNA expression of CDK2,CyclinE and E2F1,raised the mRNA expression of RB at the effective doses of 2.5μg·mL~(-1)and 10μg·mL~(-1).
8.Comparing with application of siRNA segment 190 alone,the combining use of 10μg·mL~(-1)GAPSGF and siRNA segment 190 can raise the inhibition ratio of CDK2 protein expression,The inhibition ratio can increase 47.1%;Comparing with application of siRNA segment 191 alone, the combining use of 2.5μg·mL~(-1)GAPSGF and siRNA segment 190 can raise the inhibition ratio of CDK2 mRNA and protein expression,The inhibition ratio can increase 3%and 13.3%,respectively.
Conclusion:
1.Six pairs of siRNA eukaryotic expression plasmids of CDK2 gene have been successfully constructed by DNA sequence determination.
2.CDK2 gene can be specifically silenced by RNAi technique.It is indicated that CDK2 gene may be a new target for treating hepatoma in gene therapy.
3.The mRNA expression of RB,Cyclin E and E2F1 genes related to CDK2 gene have obviously dependence on CDK2 gene.Therefore,it indicates that the interference of CDK2 gene is a an effective means for treating hepatoma in cell regulation net.
4.GAPSGF can obviously inhibit cell proliferation of SMMC7721 in vitro.The mechanism may be produced by the down-regulation of CDK2, Cyclin E and E2F1 genes expression,and the up-regulation of RB gene expression.
5.GAPSGF has certain assistant action for the RNA interfere expression of CDK2.It is demonstrated that GAPSGF could become a new assistant medicine for treating hepatoma in gene therapy.
引文
[1]林毅,陈书长.肝癌的治疗现状与展望[J].癌症进展杂志,2007,5(1):79-84
[2]任凤梅,张晓春.中医药治疗肝癌的实验研究进展[J].医药导报,2007,26(3):273-275
[3]孙昀,耿小平.肝癌的基因治疗现状与问题[J].肝胆外科杂志,2006,14(4):315-317
[4]任明欣,宋于刚,陈学清.RNAi研究进展[J].世界华人消化杂志,2004,12(3):748-750
[5]干慧珠,张桂珍.RNA干扰技术与肿瘤研究进展[J].国外医学内科学分册,2004,31(12):511-515
[6]Jorgensen R.Altered gene expression in plants due to trans interactions between homologous genes[J].Trends Biotechnol,1990,8:343-344
[7]胡亚华,陈维进,候晓华.RNAi及其抗肿瘤应用研究进展[J].现代肿瘤医学,2007,(15)1:98-101
[8]Guo S,Kemphues KJ.par-1,a gene required for establishing polarity in C,elegans embryos,encodes a putative Ser/Thr kinase that is asymrnetrically distributed[J].Cell,1995,81:611-620
[9]安立峰,董震.RNA干扰--肿瘤研究的新工具[J].中华肿瘤杂志2005,27(7):385-388
[10]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811
[11]Hunter CP.Shrinking the Black Box of RNAi[J].Current Biology,2000,(10):137-140
[12]方华舟.核糖核酸干扰(RNAi)研究进展及其在植物学中的应用[J].内蒙古农业科技,2007,5(5):70-74
[13]Tuschl T.Functiona I genomics:RNA sets the standard[J].Nature,2003,421(6920):220-221
[14]Bernstein E,Caudy AA,Hammond SM,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409:363-366
[15]Nagy P,Arndt Jovin DJ,Jovin TM.Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor(erbB1)and induce apoptosis in erbB1-overexpressing ceils[J].Exp Cell Res,2003,285(1):39-49
[16]符生苗.RNA干扰及其技术在临床医学中的应用前景[J].中国热带医学,2006,6(7):1271-1273
[17]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].Nature,2001,411(6836):494-498
[18]Bertrand JR,Pottier M,Vekris A,et ai.Comparison of antisense oligonu cleotides and siRNAs in cell culture and in vivo[J].Biochem Biophys Res Commun,2002,296(4):1000-1004
[19]Gil J,Esteban M.Induction of apoptosis by the dsRNA-dependent protein kinase(PKR):mechanism of action[J].Apoptosis,2000,5:107-114
[20]Fraser AG.Fuctional genomic analysis of cell division in C elegans chrosome 1by systematic RNAi[J].Nature,2000,408:325-330
[21]Gonczy P,Echeverri G,Oegema K,et al.Functional genomic analysis of cell division in Celegans using RNAi of genes on chromosome Ⅲ[J].Nature,2000,408:331-336
[22]Prins M.RNA-mediated virus resistence in transgenic plants[J].Anch Virology,1996,141:2259-2276
[23]Kapadia SB,Brideau-Andersen A,Chisari FV.Interference of hepatitis C virus RNA replication by short interfering RNAs[J].Proc Natl Acad Sci USA,2003,100(4):2014-2018
[24]Capodici J,Kariko K,Weissman D.Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference[J].J Immunol,2002,169(9):5196-5201
[25]Wilda M,FucKs U,Wosmarm W,et al.Killing of leukemic cellswith a BCR-ABL fusion gene by RNA interference(RNAi)[J].Onco-gene,2002,21(37):5716-5724
[26]Scherr M,Battmer K,Winkler T,et al.Specific inhibition of bcr-abl gene expression by small interfering RNA[J].Blood,2003,101(4):1566-1569
[27]Brummelkamp TR,Bernards R,Agami R.Stable suppression oftumorigenicity byvirus-med iated RNA interference[J].Cancer Cell.2002,2(3):243-247
[28]Lin SL,Chuong CM,Ying SY.A novel mRNA-cDNA interfere-ence phenomenon for silencing bcl-2 expression in human L CaPcells[J].Biochem Biophys Res Commun,2001,281(3):639-643
[29]ElbashirSM,Harborth J,Weber K,et al.Analysis of gene function insomatic mammalian cells using small interfering RNAs[J].Methods,2002,(26):199-213
[30]杨连君,曹雪涛,于益芝.细胞周期蛋白及其与肿瘤等疾病的关系[J].中国肿瘤生物治疗杂志,2003,10(3):223-225
[31]李峰,陈临溪.细胞周期蛋白D与细胞周期调控研究进展[J].国外医学·生理、病理科学与临床分册,2005,25(3):270-273
[32]Murray AW,Marks D.Can sequencing shed light on cell cycling[J].Nature,2001,409:844-846
[33]Mihara M,ShintaniS,Nakahara Y,et al.Overexpression of CDK2 is aprognostic indicator of oral cancer progression[J].Jpn J Cancer Res,2001,92(3):352-360
[34]文川,万伍卿.Cyclin/CDK复制前复合物途径对细胞周期的调控[J].医学综述,2007,13(18):1373-1375
[35]Evan GI,Vouaden KH.Proliferation,cell cycle and apoptosis in cancer[J].Nature,2001,411(6835):342-348
[36]邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1):70-73
[37]张玉霞,喻伦银,刘铭球.细胞周期调控研究进展[J].国外医学遗传学分册,2001,24(5):262-266
[38]陈济,宋方洲.CyclinE-CDK2分子活性调节机制[J].医学分子生物学杂志,2006,3(3):220-222
[39]张素丽,郑红兵.CyclinE、CDK2在子宫平滑肌肿瘤中的表达[J].肿瘤防治研究,2006,33(7):531-532
[40]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].J BiolChem,2003,278(41):3978-3993
[41]Lee MH,Yang HY.Negative regulations of cyclin dependent kinases and their roles in cancers[J].Cell Mol Life Sci,2001,58(12-13):1907-1922
[42]Knudson AG.Mutation and cancer:statistical study of retinoblastoma[J].Proc NatlAcad Sci USA,1971,18:820-823.
[43]Friend SH,R Bernards,S Rogelj,et al.A human DNA segment with properties of the gene that predisposes to retino-blastoma and osteosareoma[J].Nature,1986,323:643-646
[44]陈主初.病理生理学[M].北京:人民卫生出版社,2001:245
[45]Rovesdi L,Rcielei R,Novins J.Identification of a cellular transcription factor involved ElA trans-activation[J].Cell,1986;45:219-228
[46]王懿娜,方红.E2F因子与表皮细胞的增殖和凋亡[J].肿瘤,2003,23(2):158-159
[47]Zhang Y,Cheilappan SP.Cloning and characterization of human DP2,anovel dimerization partner of E2F[J].Oncogene,1995,10:2085-2093
[48]刘柏合,吴坤.E2F转录因子在细胞周期调控中作用的研究进展[J].卫生毒理学杂志,2000,14(1):53-55
[49]刘辰,钱其军,吴孟超,E2F转录因子与肿瘤基因治疗[J].国外医学肿瘤学分册,2004,3(7):499-502
[50]Mundle SD,Saberwal G.Evolving intricacies and im plications of E2F1regulation[J].Fasebj J,2003,17(6):569-574
[51]中华病理学杂志委员会.全国端粒酶p53和细胞凋亡的研究应用[J].中华病理学杂志,2000,29(1):9
[52]吴艳萍,李立平,张颖,等.细胞周期调控分子与肿瘤关系的研究进展[J].军医进修学院学报,2006,27(4):306-308
[53]Mitselou A,Loachim E,Zagorianakou N,et al.Expression of the cell-cycle-regulatory proteins(cyclinD and E)in endometrial carcinomas:correlations with hormone receptor status,proliferating indices,tumor suppressor gene products(p53,pRb),and clinicopathological parameters[J].Eur J Gynaecol Oncol,2004,25(6):719-724
[54]Martinsson HS,Starborg M,Erlandsson F.Single cell analysis of G(1)check points-the relationship between the restriction point and phosphorylation of pRb[J].Expcell Res,2005,305(2):383-391
[55]Yu M,Zhan Q,Finn OJ.Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by nonfunctional p53[J].Mol Immunol,2002,38(12-13):981-987
[56]YasmeenA,BerdelWE,ServeH,et al.E and A type cyclins as markers for cancer diagnosis and prognosis[J].Expert Rev mol Diagn,2003,3(5):617-33
[57]Szeps M,Erickson S,Cruber A,et al.Effects of interferon alpha on cell cydle regulatory proteins in lerkemic cell[J].Leuk lymphoma,2003,44(6):1019-1025
[58]林福地,谢庆祥,张闽峰,等.CyclinE和CDK2在肺癌中表达及其临床意义[J].肿瘤防治杂志,2001,8(3):259-260
[59]李丽,齐凤英,左连富,等.CyclinE、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义[J].肿瘤,2005,25(2):158-162
[60]桑建利,王永潮,周清源.利用原位杂交分析几种cyclin和CDK基因在人乳腺癌中的异常表达[J].解剖学报,1999,30(3):225-226
[61]谢庆祥,林福地,韩聪祥,等.CDK2和CyclinE在肾细胞癌中表达的意义[J].肿瘤防治研究,2005,32(2):91-93
[62]郑敏,王亚.中药多糖抗肿瘤的药理学研究进展[J].国外医学中医中药分册,2000,(22)5:259-263
[63]乔红梅,贺新怀,席孝贤.中药抗肿瘤机制研究浅析[J].陕西中医,2003,(24)5:454-456
[64]周金黄,刘干中.免疫药理学进展--基础与临床[M].北京:中国科学基础出版社,1993:367
[65]尹龙,徐亮,胡格,等.抗肿瘤中药及其有效成分的作用研究现状[J].动物医学进展,2006,27(1):39-42
[66]余上才,张育正.牛膝多糖抗肿瘤作用及免疫机制实验研究[J].中华肿瘤杂志,1995,17(4):275-278
[67]赵永娟,王蕾,侯琳,等.半夏多糖抗肿瘤作用研究[J].中国药理学通报,2006,22(3):368-371
[68]庞战军,陈瑗,周玫,等.云芝多糖对巨噬细胞GPX基因表达的影响[J].生物化学与生物物理学报,1999,31(3):284-288
[69]刁波,唐瑛,王晓琨.中药多糖研究新进展[J].中国医药导报,2008,(5)3:21-22
[70]万军梅,张永忠,吴巍,等.抑瘤扶正丸提高肿瘤化疗疗效的实验研究[J].中成药,2000,22(10):712-714
[71]韩鸿彬.华蟾素抗肿瘤作用及其机制的研究进展[J].中国肿瘤生物治疗杂志,2005,12(2):160-162
[72]袁庆欣,张艳,宋婷婷,等.抗肿瘤中药成分的研究探微[J].中华中医药学刊,2007,25(3):617-618
[73]张贵君,于英君,田中俊弘.树舌的生药鉴定[J].中药材,1996,19(5):226-228
[74]焦连庆,于敏.树舌多糖组分的高效液相色谱分析[J].中国医院药学杂志,2005,79(12):121
[75]李玉芳,王杰.中药多糖抗肿瘤机制研究进展[J].实用癌症杂志,2007,22(5):546-550
[76]孙巧红,刘正红,刘丽波.树舌多糖对HepA瘤小鼠的抑瘤作用及血清LSA 含量的影响[J].中国中医药科技,1996,3(6):24
[77]王玉,潘洪明,刘枫.树舌多糖对HepA瘤小鼠骨髓细胞染色体SCE影响的研究[J].中国优生与遗传杂志,2002,10(3):42-44
[78]于英君,杜家忠.树舌多糖对小鼠HepA瘤细胞3H-TdR和[6-3H]-Glucose掺入的影响[J].中医药信息,1997,(12)3:46-47
[79]于英君,孙玺媛.树舌多糖对HepA瘤小鼠癌细胞凋亡影响的初探[J].中医药学报,2003,5:34-35
[80]王玉,徐广有.树舌多糖对癌细胞AgNOR表达的影响[J].齐齐哈尔医学院学报,2001,22(10):14-17
[81]张庆梅,潘洪明,于英君.树舌多糖GF对小鼠HepA瘤TNF-α含量的影响[J].中医药学刊,2003,21(6):913
[82]宋高臣,孙玺媛,于英君.树舌多糖对小鼠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
[83]李杰,张楚菁,中药多糖的免疫调节及抗肿瘤近年研究概述[J].天津药学, 2003,15(6):62-64
[84]管宇,于英君.树舌多糖GF注射液对HepA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].药物研究,2005,32(3):31-32
[85]于英君,刘丽波,何维.树舌多糖GF免疫调节作用研究[J].中医药信息,1999,2:64
[86]杨晶凡,刘丽波,于英君.树舌多糖对HepA瘤细胞Ras蛋白表达的影响[J].中国药学刊,2003,21(11):1831-1832
[87]孙力,杨晶凡,于英君.树舌多糖对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
[88]于英君,张庆梅,郭丽新,等.树舌多糖GF对HepA癌细胞p53基因表达的影响[J].中医药学报,2002,30(2):56-57
[89]于英君,潘洪明,张庆梅.树舌多糖GF对HepA瘤细胞p16基因表达的影响[J].中医药学刊,2002,2(20):160-162
[90]张庆梅,刘丽波,潘洪明.树舌多糖GF对小鼠HepA癌细胞Rb基因表达的影响[J].中医药学刊,2002,20(4):431-443
[91]于英君,李丽阳.树舌多糖GF注射液对环磷酰胺增效减毒作用的实验研究[J].齐齐哈尔医学院学报,2004,25(10):25-27
[92]宋高臣,于英君.树舌多糖GF注射液与环磷酰胺联合抗肿瘤作用的实验研究[J].中医药信息,2004,21(6):49-50
[93]宋高臣,于英君.树舌多糖GF注射液对化疗药物增效减毒作用及其机制研究[J].中国老年学杂志,2005,13(4):102-104
[94]曹艳菲,宋高臣,于英君,等.树舌多糖GF抗肿瘤的研究进展[J].牡丹江医学院学报,2007,28(3):69-72
[95]Guangchao S,Christina S,Bachir A,et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells[J].PNAS,2002,99(8):5515-5520
[96]Jinyan D,Hans R,Martin A,et al.Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF[J].Cancer Cell,2004,12(6):565-576
[97]MeihongC,Lishu Z,Hongyan Z,et al.Auniversalplasmid library encoding all permutations of small interfering RNA[J].PNAS,2005,102(7):2356-2361
[98]LeBlanc HN,Ashkenazi A.Apo2L/TRAIL and its death and decoy receptors[J].Cell Death Differ,2003,10(1):66-75
[99]Tuschl T.Expanding small RNA interference[J].Nat Biotechnol,2002,20(5):446-448
[100]Paddison PJ,Caudy AA,Bernstein E,et al.Short hairpin RNAs(shRNAs)induce sequence-specific silencing in mammalian cells[J].GenesDev,2002,16(8):948-958
[101]McCaffrey AP,Meuse L,Pham TT,et al.RNAinterference in adult mice[J].Nature,2002,418(6893):38-39
[102]Xia DY,Sanders A,Shah M,et al.Rela-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in ailograft acceptor mice[J].Transplantation,2001,72:907-914
[103]Borkhardt A.Blocking oncogenes in ma lignan t cells by RNA inter-ference-new hope for a highly specific can cer treatment[J].CancerCell,2002,2(3):167-168.
[104]J.萨姆布鲁克,D.W.拉塞尔著,黄培堂译.分子克隆实验指南[M].第3版,北京:科学出版社,2002:1276-1298
[105]Charles J,Sherr.The pezcoller lecture:cancer cell cycles Revisited[J].Cancer Research,2000,60:3389-3695
[106]Murray A W,Marks D.Can sequencing shed light on cell Cycling[J].Nature,2001,409:844-846
[107]DμMan-Scheel M,Weng L,Xin S,et al.Hedgehog regulates cell growth and proliferation by inducing Cyclin D and Cyclin E[J].Nature,2002,417(6886):299-304
[108]郭德生.细胞周期调控与细胞癌变[J].国外医学生理病理科学与临床分册,1997,17(1):1-3
[109]Hwang HC,Clurman BE.Cyclin E in normal and neoplastic cell cycles [J].Oncogene,2005,24(17):2776-2786
[110]Musgrove EA,Sarcevic B,Sutherland R.Inducible expression of CyclinD1 in T_47D hμMan breast cancer cells is sufficient for cdk2 activation and pRb hy perphorylation[J].Journal of Cellular Biochemistry,1996,60:363-378
[111]桑建利,王永潮.CDK4反义RNA对乳腺癌细胞增殖及CyclinD1,CyclinE 和CDK2表达的影响[J].科学通报,1999,44(2):184-188
[112]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
[113]Francoi D,Lang R.Rapid colormeric assay for cell groth and survival[J].J Immunol Meth,1986;89:271-280
[114]Porter LA,Dellinger RW,Tynan JA,et al.A novel cell cycle regulator that enhances proliferation through activation of CDK2[J].J Cell Biol,2002,157:357.
[115]Farley J,Smith L M,Dary K M,et al.Cyclin E expression is a significant predictor of survival in advanced,suboptimally debulked ovarian epithelial cancer:A Gynecologic Ontology Group study[J].Cancer Res,2003,63(6):1235-1241
[116]Ben-Porath I,Weinberg R.The signals and pathways activating cellular senescence[J].Int J Biochem Cell Biol,2005,37(5):961-976
[117]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
[118]李兆忠,张玲,王芸,等.C-myc反义基因对人高转移肺癌细胞增殖和黏附活性的抑制作用[J].中华肿瘤生物治疗杂志,2002,9(1):19-22
[119]陈孝银,沈卓群.琼玉膏对化疗实验肺癌小鼠nm23及PCNA基因蛋白表达的影响[J].安徽中医学院学报,2000,19(2):47-48
[120]王建平,魏品康,许玲.消痰散结方对MKN-45人胃癌细胞ICMA-1表达的影响[J].中医药研究,2002,18(1):38-39
[121]王昌俊.中医药与自杀基因疗法结合提高肝癌疗效的思路[J].中医药学刊,2006,24(11):2015-2018
[2]任凤梅,张晓春.中医药治疗肝癌的实验研究进展[J].医药导报,2007,26(3):273-275
[3]孙昀,耿小平.肝癌的基因治疗现状与问题[J].肝胆外科杂志,2006,14(4):315-317
[4]任明欣,宋于刚,陈学清.RNAi研究进展[J].世界华人消化杂志,2004,12(3):748-750
[5]干慧珠,张桂珍.RNA干扰技术与肿瘤研究进展[J].国外医学内科学分册,2004,31(12):511-515
[6]Jorgensen R.Altered gene expression in plants due to trans interactions between homologous genes[J].Trends Biotechnol,1990,8:343-344
[7]胡亚华,陈维进,候晓华.RNAi及其抗肿瘤应用研究进展[J].现代肿瘤医学,2007,(15)1:98-101
[8]Guo S,Kemphues KJ.par-1,a gene required for establishing polarity in C,elegans embryos,encodes a putative Ser/Thr kinase that is asymrnetrically distributed[J].Cell,1995,81:611-620
[9]安立峰,董震.RNA干扰--肿瘤研究的新工具[J].中华肿瘤杂志2005,27(7):385-388
[10]Fire A,Xu S,Montgomery MK,et al.Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J].Nature,1998,391(6669):806-811
[11]Hunter CP.Shrinking the Black Box of RNAi[J].Current Biology,2000,(10):137-140
[12]方华舟.核糖核酸干扰(RNAi)研究进展及其在植物学中的应用[J].内蒙古农业科技,2007,5(5):70-74
[13]Tuschl T.Functiona I genomics:RNA sets the standard[J].Nature,2003,421(6920):220-221
[14]Bernstein E,Caudy AA,Hammond SM,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409:363-366
[15]Nagy P,Arndt Jovin DJ,Jovin TM.Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor(erbB1)and induce apoptosis in erbB1-overexpressing ceils[J].Exp Cell Res,2003,285(1):39-49
[16]符生苗.RNA干扰及其技术在临床医学中的应用前景[J].中国热带医学,2006,6(7):1271-1273
[17]Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].Nature,2001,411(6836):494-498
[18]Bertrand JR,Pottier M,Vekris A,et ai.Comparison of antisense oligonu cleotides and siRNAs in cell culture and in vivo[J].Biochem Biophys Res Commun,2002,296(4):1000-1004
[19]Gil J,Esteban M.Induction of apoptosis by the dsRNA-dependent protein kinase(PKR):mechanism of action[J].Apoptosis,2000,5:107-114
[20]Fraser AG.Fuctional genomic analysis of cell division in C elegans chrosome 1by systematic RNAi[J].Nature,2000,408:325-330
[21]Gonczy P,Echeverri G,Oegema K,et al.Functional genomic analysis of cell division in Celegans using RNAi of genes on chromosome Ⅲ[J].Nature,2000,408:331-336
[22]Prins M.RNA-mediated virus resistence in transgenic plants[J].Anch Virology,1996,141:2259-2276
[23]Kapadia SB,Brideau-Andersen A,Chisari FV.Interference of hepatitis C virus RNA replication by short interfering RNAs[J].Proc Natl Acad Sci USA,2003,100(4):2014-2018
[24]Capodici J,Kariko K,Weissman D.Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference[J].J Immunol,2002,169(9):5196-5201
[25]Wilda M,FucKs U,Wosmarm W,et al.Killing of leukemic cellswith a BCR-ABL fusion gene by RNA interference(RNAi)[J].Onco-gene,2002,21(37):5716-5724
[26]Scherr M,Battmer K,Winkler T,et al.Specific inhibition of bcr-abl gene expression by small interfering RNA[J].Blood,2003,101(4):1566-1569
[27]Brummelkamp TR,Bernards R,Agami R.Stable suppression oftumorigenicity byvirus-med iated RNA interference[J].Cancer Cell.2002,2(3):243-247
[28]Lin SL,Chuong CM,Ying SY.A novel mRNA-cDNA interfere-ence phenomenon for silencing bcl-2 expression in human L CaPcells[J].Biochem Biophys Res Commun,2001,281(3):639-643
[29]ElbashirSM,Harborth J,Weber K,et al.Analysis of gene function insomatic mammalian cells using small interfering RNAs[J].Methods,2002,(26):199-213
[30]杨连君,曹雪涛,于益芝.细胞周期蛋白及其与肿瘤等疾病的关系[J].中国肿瘤生物治疗杂志,2003,10(3):223-225
[31]李峰,陈临溪.细胞周期蛋白D与细胞周期调控研究进展[J].国外医学·生理、病理科学与临床分册,2005,25(3):270-273
[32]Murray AW,Marks D.Can sequencing shed light on cell cycling[J].Nature,2001,409:844-846
[33]Mihara M,ShintaniS,Nakahara Y,et al.Overexpression of CDK2 is aprognostic indicator of oral cancer progression[J].Jpn J Cancer Res,2001,92(3):352-360
[34]文川,万伍卿.Cyclin/CDK复制前复合物途径对细胞周期的调控[J].医学综述,2007,13(18):1373-1375
[35]Evan GI,Vouaden KH.Proliferation,cell cycle and apoptosis in cancer[J].Nature,2001,411(6835):342-348
[36]邹向阳,李连宏.细胞周期调控与肿瘤[J].国际遗传学杂志,2006,29(1):70-73
[37]张玉霞,喻伦银,刘铭球.细胞周期调控研究进展[J].国外医学遗传学分册,2001,24(5):262-266
[38]陈济,宋方洲.CyclinE-CDK2分子活性调节机制[J].医学分子生物学杂志,2006,3(3):220-222
[39]张素丽,郑红兵.CyclinE、CDK2在子宫平滑肌肿瘤中的表达[J].肿瘤防治研究,2006,33(7):531-532
[40]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].J BiolChem,2003,278(41):3978-3993
[41]Lee MH,Yang HY.Negative regulations of cyclin dependent kinases and their roles in cancers[J].Cell Mol Life Sci,2001,58(12-13):1907-1922
[42]Knudson AG.Mutation and cancer:statistical study of retinoblastoma[J].Proc NatlAcad Sci USA,1971,18:820-823.
[43]Friend SH,R Bernards,S Rogelj,et al.A human DNA segment with properties of the gene that predisposes to retino-blastoma and osteosareoma[J].Nature,1986,323:643-646
[44]陈主初.病理生理学[M].北京:人民卫生出版社,2001:245
[45]Rovesdi L,Rcielei R,Novins J.Identification of a cellular transcription factor involved ElA trans-activation[J].Cell,1986;45:219-228
[46]王懿娜,方红.E2F因子与表皮细胞的增殖和凋亡[J].肿瘤,2003,23(2):158-159
[47]Zhang Y,Cheilappan SP.Cloning and characterization of human DP2,anovel dimerization partner of E2F[J].Oncogene,1995,10:2085-2093
[48]刘柏合,吴坤.E2F转录因子在细胞周期调控中作用的研究进展[J].卫生毒理学杂志,2000,14(1):53-55
[49]刘辰,钱其军,吴孟超,E2F转录因子与肿瘤基因治疗[J].国外医学肿瘤学分册,2004,3(7):499-502
[50]Mundle SD,Saberwal G.Evolving intricacies and im plications of E2F1regulation[J].Fasebj J,2003,17(6):569-574
[51]中华病理学杂志委员会.全国端粒酶p53和细胞凋亡的研究应用[J].中华病理学杂志,2000,29(1):9
[52]吴艳萍,李立平,张颖,等.细胞周期调控分子与肿瘤关系的研究进展[J].军医进修学院学报,2006,27(4):306-308
[53]Mitselou A,Loachim E,Zagorianakou N,et al.Expression of the cell-cycle-regulatory proteins(cyclinD and E)in endometrial carcinomas:correlations with hormone receptor status,proliferating indices,tumor suppressor gene products(p53,pRb),and clinicopathological parameters[J].Eur J Gynaecol Oncol,2004,25(6):719-724
[54]Martinsson HS,Starborg M,Erlandsson F.Single cell analysis of G(1)check points-the relationship between the restriction point and phosphorylation of pRb[J].Expcell Res,2005,305(2):383-391
[55]Yu M,Zhan Q,Finn OJ.Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by nonfunctional p53[J].Mol Immunol,2002,38(12-13):981-987
[56]YasmeenA,BerdelWE,ServeH,et al.E and A type cyclins as markers for cancer diagnosis and prognosis[J].Expert Rev mol Diagn,2003,3(5):617-33
[57]Szeps M,Erickson S,Cruber A,et al.Effects of interferon alpha on cell cydle regulatory proteins in lerkemic cell[J].Leuk lymphoma,2003,44(6):1019-1025
[58]林福地,谢庆祥,张闽峰,等.CyclinE和CDK2在肺癌中表达及其临床意义[J].肿瘤防治杂志,2001,8(3):259-260
[59]李丽,齐凤英,左连富,等.CyclinE、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义[J].肿瘤,2005,25(2):158-162
[60]桑建利,王永潮,周清源.利用原位杂交分析几种cyclin和CDK基因在人乳腺癌中的异常表达[J].解剖学报,1999,30(3):225-226
[61]谢庆祥,林福地,韩聪祥,等.CDK2和CyclinE在肾细胞癌中表达的意义[J].肿瘤防治研究,2005,32(2):91-93
[62]郑敏,王亚.中药多糖抗肿瘤的药理学研究进展[J].国外医学中医中药分册,2000,(22)5:259-263
[63]乔红梅,贺新怀,席孝贤.中药抗肿瘤机制研究浅析[J].陕西中医,2003,(24)5:454-456
[64]周金黄,刘干中.免疫药理学进展--基础与临床[M].北京:中国科学基础出版社,1993:367
[65]尹龙,徐亮,胡格,等.抗肿瘤中药及其有效成分的作用研究现状[J].动物医学进展,2006,27(1):39-42
[66]余上才,张育正.牛膝多糖抗肿瘤作用及免疫机制实验研究[J].中华肿瘤杂志,1995,17(4):275-278
[67]赵永娟,王蕾,侯琳,等.半夏多糖抗肿瘤作用研究[J].中国药理学通报,2006,22(3):368-371
[68]庞战军,陈瑗,周玫,等.云芝多糖对巨噬细胞GPX基因表达的影响[J].生物化学与生物物理学报,1999,31(3):284-288
[69]刁波,唐瑛,王晓琨.中药多糖研究新进展[J].中国医药导报,2008,(5)3:21-22
[70]万军梅,张永忠,吴巍,等.抑瘤扶正丸提高肿瘤化疗疗效的实验研究[J].中成药,2000,22(10):712-714
[71]韩鸿彬.华蟾素抗肿瘤作用及其机制的研究进展[J].中国肿瘤生物治疗杂志,2005,12(2):160-162
[72]袁庆欣,张艳,宋婷婷,等.抗肿瘤中药成分的研究探微[J].中华中医药学刊,2007,25(3):617-618
[73]张贵君,于英君,田中俊弘.树舌的生药鉴定[J].中药材,1996,19(5):226-228
[74]焦连庆,于敏.树舌多糖组分的高效液相色谱分析[J].中国医院药学杂志,2005,79(12):121
[75]李玉芳,王杰.中药多糖抗肿瘤机制研究进展[J].实用癌症杂志,2007,22(5):546-550
[76]孙巧红,刘正红,刘丽波.树舌多糖对HepA瘤小鼠的抑瘤作用及血清LSA 含量的影响[J].中国中医药科技,1996,3(6):24
[77]王玉,潘洪明,刘枫.树舌多糖对HepA瘤小鼠骨髓细胞染色体SCE影响的研究[J].中国优生与遗传杂志,2002,10(3):42-44
[78]于英君,杜家忠.树舌多糖对小鼠HepA瘤细胞3H-TdR和[6-3H]-Glucose掺入的影响[J].中医药信息,1997,(12)3:46-47
[79]于英君,孙玺媛.树舌多糖对HepA瘤小鼠癌细胞凋亡影响的初探[J].中医药学报,2003,5:34-35
[80]王玉,徐广有.树舌多糖对癌细胞AgNOR表达的影响[J].齐齐哈尔医学院学报,2001,22(10):14-17
[81]张庆梅,潘洪明,于英君.树舌多糖GF对小鼠HepA瘤TNF-α含量的影响[J].中医药学刊,2003,21(6):913
[82]宋高臣,孙玺媛,于英君.树舌多糖对小鼠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
[83]李杰,张楚菁,中药多糖的免疫调节及抗肿瘤近年研究概述[J].天津药学, 2003,15(6):62-64
[84]管宇,于英君.树舌多糖GF注射液对HepA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].药物研究,2005,32(3):31-32
[85]于英君,刘丽波,何维.树舌多糖GF免疫调节作用研究[J].中医药信息,1999,2:64
[86]杨晶凡,刘丽波,于英君.树舌多糖对HepA瘤细胞Ras蛋白表达的影响[J].中国药学刊,2003,21(11):1831-1832
[87]孙力,杨晶凡,于英君.树舌多糖对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
[88]于英君,张庆梅,郭丽新,等.树舌多糖GF对HepA癌细胞p53基因表达的影响[J].中医药学报,2002,30(2):56-57
[89]于英君,潘洪明,张庆梅.树舌多糖GF对HepA瘤细胞p16基因表达的影响[J].中医药学刊,2002,2(20):160-162
[90]张庆梅,刘丽波,潘洪明.树舌多糖GF对小鼠HepA癌细胞Rb基因表达的影响[J].中医药学刊,2002,20(4):431-443
[91]于英君,李丽阳.树舌多糖GF注射液对环磷酰胺增效减毒作用的实验研究[J].齐齐哈尔医学院学报,2004,25(10):25-27
[92]宋高臣,于英君.树舌多糖GF注射液与环磷酰胺联合抗肿瘤作用的实验研究[J].中医药信息,2004,21(6):49-50
[93]宋高臣,于英君.树舌多糖GF注射液对化疗药物增效减毒作用及其机制研究[J].中国老年学杂志,2005,13(4):102-104
[94]曹艳菲,宋高臣,于英君,等.树舌多糖GF抗肿瘤的研究进展[J].牡丹江医学院学报,2007,28(3):69-72
[95]Guangchao S,Christina S,Bachir A,et al.A DNA vector-based RNAi technology to suppress gene expression in mammalian cells[J].PNAS,2002,99(8):5515-5520
[96]Jinyan D,Hans R,Martin A,et al.Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF[J].Cancer Cell,2004,12(6):565-576
[97]MeihongC,Lishu Z,Hongyan Z,et al.Auniversalplasmid library encoding all permutations of small interfering RNA[J].PNAS,2005,102(7):2356-2361
[98]LeBlanc HN,Ashkenazi A.Apo2L/TRAIL and its death and decoy receptors[J].Cell Death Differ,2003,10(1):66-75
[99]Tuschl T.Expanding small RNA interference[J].Nat Biotechnol,2002,20(5):446-448
[100]Paddison PJ,Caudy AA,Bernstein E,et al.Short hairpin RNAs(shRNAs)induce sequence-specific silencing in mammalian cells[J].GenesDev,2002,16(8):948-958
[101]McCaffrey AP,Meuse L,Pham TT,et al.RNAinterference in adult mice[J].Nature,2002,418(6893):38-39
[102]Xia DY,Sanders A,Shah M,et al.Rela-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in ailograft acceptor mice[J].Transplantation,2001,72:907-914
[103]Borkhardt A.Blocking oncogenes in ma lignan t cells by RNA inter-ference-new hope for a highly specific can cer treatment[J].CancerCell,2002,2(3):167-168.
[104]J.萨姆布鲁克,D.W.拉塞尔著,黄培堂译.分子克隆实验指南[M].第3版,北京:科学出版社,2002:1276-1298
[105]Charles J,Sherr.The pezcoller lecture:cancer cell cycles Revisited[J].Cancer Research,2000,60:3389-3695
[106]Murray A W,Marks D.Can sequencing shed light on cell Cycling[J].Nature,2001,409:844-846
[107]DμMan-Scheel M,Weng L,Xin S,et al.Hedgehog regulates cell growth and proliferation by inducing Cyclin D and Cyclin E[J].Nature,2002,417(6886):299-304
[108]郭德生.细胞周期调控与细胞癌变[J].国外医学生理病理科学与临床分册,1997,17(1):1-3
[109]Hwang HC,Clurman BE.Cyclin E in normal and neoplastic cell cycles [J].Oncogene,2005,24(17):2776-2786
[110]Musgrove EA,Sarcevic B,Sutherland R.Inducible expression of CyclinD1 in T_47D hμMan breast cancer cells is sufficient for cdk2 activation and pRb hy perphorylation[J].Journal of Cellular Biochemistry,1996,60:363-378
[111]桑建利,王永潮.CDK4反义RNA对乳腺癌细胞增殖及CyclinD1,CyclinE 和CDK2表达的影响[J].科学通报,1999,44(2):184-188
[112]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
[113]Francoi D,Lang R.Rapid colormeric assay for cell groth and survival[J].J Immunol Meth,1986;89:271-280
[114]Porter LA,Dellinger RW,Tynan JA,et al.A novel cell cycle regulator that enhances proliferation through activation of CDK2[J].J Cell Biol,2002,157:357.
[115]Farley J,Smith L M,Dary K M,et al.Cyclin E expression is a significant predictor of survival in advanced,suboptimally debulked ovarian epithelial cancer:A Gynecologic Ontology Group study[J].Cancer Res,2003,63(6):1235-1241
[116]Ben-Porath I,Weinberg R.The signals and pathways activating cellular senescence[J].Int J Biochem Cell Biol,2005,37(5):961-976
[117]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
[118]李兆忠,张玲,王芸,等.C-myc反义基因对人高转移肺癌细胞增殖和黏附活性的抑制作用[J].中华肿瘤生物治疗杂志,2002,9(1):19-22
[119]陈孝银,沈卓群.琼玉膏对化疗实验肺癌小鼠nm23及PCNA基因蛋白表达的影响[J].安徽中医学院学报,2000,19(2):47-48
[120]王建平,魏品康,许玲.消痰散结方对MKN-45人胃癌细胞ICMA-1表达的影响[J].中医药研究,2002,18(1):38-39
[121]王昌俊.中医药与自杀基因疗法结合提高肝癌疗效的思路[J].中医药学刊,2006,24(11):2015-2018