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目的:探讨叶酸修饰的第五代聚酰胺-胺型树枝状聚合物(G5-PAMAM-D-fol)作为基因载体进行前列腺癌自杀基因治疗的可行性。方法:以GS-PAMAM-D-fol为基因载体将含有自杀基因HSV-TK的重组质粒pcDNA3-tk转染至前列腺癌细胞系PC-3和LNCaP,24h后以RT-PCR法检测HSV-TK基因在两种前列腺癌细胞系中的mRNA表达;再次转染后24h,对转染的两种细胞分别给予不同浓度的前体药物更昔洛韦(GCV),48h后采用四甲基偶氮唑盐比色法(MTT法)检测细胞抑制率。结果:RT-PCR结果证明G5-PAMAM-D-fol可将重组质粒pcDNA3-tk转染2种前列腺癌细胞并转录HSV-TK基因。MTT实验反映出A组(G5-PAMAM-D-fol/pcDNA3-tk)对PC-3和LNCaP细胞有明显的杀伤作用,并且比B组(G5-PAMAM-D/pcDNA3-tk)的细胞抑制率更高。C组(G5-PAMAM-D-fol)和D组(GS-PAMAM-D)随着GCV浓度的增加细胞生长并未受到明显的抑制,而且前者并未表现出比后者更明显的细胞杀伤作用。结论:G5-PAMAM-D-fol能够在体外将自杀基因重组质粒成功转入前列腺癌细胞并表达HSV-TK基因,其靶向性强且细胞毒性低。
Objective: To investigate the feasibility of using folic acid-modified fifth-generation polyamide-amine dendrimer (G5-PAMAM-D-fol) as a gene carrier for suicide gene therapy of prostate cancer. Methods: The recombinant plasmid pcDNA3-tk containing suicide gene HSV-TK was transfected into prostate cancer cell lines PC-3 and LNCaP with GS-PAMAM-D-fol as a vector and the expression of HSV-TK Gene expression in two prostate cancer cell lines mRNA expression; 24 hours after transfection, the two transfected cells were given different concentrations of the precursor drug ganciclovir (GCV), 48h after the use of tetramethylazo Azolium salt colorimetry (MTT method) to detect cell inhibition rate. Results: The results of RT-PCR showed that G5-PAMAM-D-fol transfected two kinds of prostate cancer cells with recombinant plasmid pcDNA3-tk and transcribed HSV-TK gene. MTT assay showed that group A (G5-PAMAM-D-fol / pcDNA3-tk) had a significant killing effect on PC-3 and LNCaP cells and was more cytotoxic than group B (G5-PAMAM-D / pcDNA3-tk) Higher rates. The cell growth of G5-PAMAM-D-fol group and GS-PAMAM-D group was not significantly inhibited with the increase of GCV concentration, and the former did not show more obvious cell killing than the latter effect. CONCLUSION: G5-PAMAM-D-fol can successfully transfer suicide gene recombinant plasmid into prostate cancer cells and express HSV-TK gene in vitro with strong targeting and low cytotoxicity.