【作者】 李玲；

【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， 硕士

【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。更多还原

【Abstract】 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.更多还原