【作者】 宋高臣；

【导师】 于英君；

【作者基本信息】 黑龙江中医药大学 ， 中西医结合基础， 2008， 博士

【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。更多还原

【Abstract】 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^-1and 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^-1and 10μg·mL^-1.8.Comparing with application of siRNA segment 190 alone,the combining use of 10μg·mL^-1GAPSGF 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^-1GAPSGF 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.更多还原