壳聚糖纳米粒介导的HSV-tk基因对前列腺癌的体外杀伤作用

目的：考察壳聚糖纳米粒作为自杀基因载体进行前列腺自杀基因治疗的可行性，为前列腺癌的基因治疗寻找新的基因载体。方法：将壳聚糖纳米粒包裹的含报告基因的质粒pEGFP—C1分别转染前列腺癌细胞PC-3和22Rvl，并观察EGFP的表达情况。随后将含HSV-tk自杀基因的真核高效表达质粒pcDNA3-tk经壳聚糖纳米粒包裹后转染上述2种前列腺癌细胞，转染48h后，以RT-PCR检测HSV-tk基因的表达情况。最后对转染的2种细胞给予不同浓度的前体药更昔洛韦（ganci—clovir，GCV）作用24h后，采用MTT比色法测定药物对细胞增殖的影响，并进行统计学分析。结果：壳聚糖纳米粒能够将质粒pEGFP-C1转入2种前列腺癌细胞，并表达EGFP，在PC-3细胞中壳聚糖纳米介导的转染效率高于脂质体。采用壳聚糖纳米粒转染pcDNA3-tk于2种前列腺癌细胞后，RT-PCR证明在癌细胞中有HSV-tk基因的表达。给予前体药物GCV后，实验组细胞生长抑制率与只给壳聚糖或裸质粒的对照组相比明显增高，说明HSV-tk自杀基因纳米粒在细胞中表达，从而使HSV-tk／GCV自杀基因系统发挥杀伤细胞的作用。结论：壳聚糖纳米粒能将质粒基因pcDNA3-tk转入前列腺癌细胞并表达胸苷激酶发挥作用，可做为前列腺癌自杀基因治疗的基因载体。

HSV-tk gene delivered by chitosan nanoparticles killed prostate cancer cells in vitro

Objective:To identify the possibility of using chitosan nanoparticles as gene vector to deliver HSV-tk suicide gene and try to find a new gene vector for prostate cancer gene therapy.Methods:Prostate cancer cells PC-3 and 22Rvl were transfect- ed with plasmid pEGFP-C1 coated with chitosan nanoparticles and the expression of EGFP was observed.Furthermore,PC-3 and 22Rvl cells were transfected with eukary-otic plasmid pcDNA3-tk via Lipofectamine and chitosan nanoparticles mediation. RT-PCR was used to detect expression of HSV-tk gene at 48 h after transfection.The substrate ganciclovir(GCV)was cultured with the transfected PC-3 and 22Rvl cells at different concentrations for 24 h.MTT assay was used to evaluate the effect of GCV on the proliferation of two types of cancer cells.Results:Chitosan nanoparticles delivered pEGFP-C1 into PC-3 and 22Rv1 cells and EGFP was successfully expressed in the two types of cell lines.The transfeetion efficacy of chitosan nanoparticles was supe- rior than Lipofectamine in PC-3 cells.RT-PCR demonstrated that HSV-tk mRNA was expressed in the two prostate cancer cells after being transfected with chitosan/pcDNA3-tk.The growth inhibition ratio induced by GCV was significantly higher in chi- tosan/pcDNA3-tk group than chitosan or plasmid group indicating that chitosan nanoparticles could deliver HSV-tk suicide gene into prostate cancer PC-3 and 22Rvl cells and induce its expression thus exerting cell-killing effect of HSV-tk/GCV suicide gene system.Conclusions:Chitosan nanoparticles could successfully deliver peDNA3-tk into prostate cancer cells and induce expres- sion of thymidine kinase.This study suggests that chitosan nanoparticles have a good prospect as suicide gene carriers for pros- tate cancer gene therapy.