| 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.
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