壳聚糖纳米粒介导的CD基因对前列腺癌的体外杀伤作用

Killing effect of CD gene induced by chitosan nanoparticles on prostate cancer in vitro

目的考察壳聚糖(Chitosan)纳米粒作为自杀基因载体进行前列腺癌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含报告基因质粒pEGFP-C1转染至前列腺癌细胞系PC-3和22Rvl,观察EGFP的表达情况,再将含胞嘧啶脱氨酶(Cytosinedeamiase,CD)自杀基因的真核表达质粒pcDNA3-CD转染至该2种前列腺癌细胞,转染48h后,以RT-PCR检测CD基因的表达。对转染的两种细胞给予不同浓度的前体药5-氟胞嘧啶(5-fluorocytosine,5-FC)24h后,采用MTT比色法测定药物对细胞增殖的影响,并进行统计学处理。结果表明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细胞其转染效率高于脂质体转染试剂。采用壳聚糖纳米粒转染pcDNA3-CD于2种前列腺癌细胞后,RT-PCR证明在癌细胞中有CD基因的表达。给予前体药物5-FC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,说明壳聚糖纳米粒可将CD自杀基因递送至前列腺癌细胞中,并在细胞中进行表达,从而使CD/5-FC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转入前列腺癌细胞并表达,可做为前列腺癌自杀基因治疗的基因载体,有良好的应用前景。

Objective To detemine possibility of chitosan as gene vecter for cytosine deamiase （CD） suicide genc therapy in prostate cancer. Methods Prostate cancer cells PC- 3, 22Rvl were transfected by plasmid pEGFP- C1 coated with chitosan nanoparticles and obeserve the expression of EGFP. Furthermore, lipofectamine and chitosan nanoparticles were used as gene vecters to deliver plasmid pcDNA3 - CD which expresses CD in eukaryocytes into PC- 3 and 22Rvl. 48 hours after transfection, RT- PCR was used to detect expression of CD gene. The substrate 5 - fluorocytosine （5 - FC） was added to the transfected PC - 3,22Rvl cells at different concentrations. 24 hour later, MTT was used to evaluate the effect of 5 - FC on the proliferation of two cancer cells. Results EGFP was successfully expressed in prostate cancer PC- 3 and 22Rvl. CD mRNA existed in 2 prostate cancer cells which were transfected by chitosan/pcDNA3 - CD. The number of live cells in chitosan nanoparticles group with CD is much less than that of control group after 5 - FC was added, significant difference was found between them. Condu^on Chitosan nanoparticles can deliver CD gene to prostate cancer cells effectively; it is promising gene vecter for suicide gene therapy in prostate cancer.