Tf-PEG-PEI载细胞特异性干扰质粒靶向沉默前列腺癌核干因子的体外实验研究

Silencing effect of cell-specific RNA interference plasmid pPSMAe/p-shNS-ploy （A）loaded by transgenic vector Tf-PEG-PEI targeting nucleostemin on prostate cancer cells in vitro

目的探讨非病毒基因转移载体转铁蛋白一聚乙二醇一聚乙烯亚胺（Tf-PEG-PEI）载前列腺特异性膜抗原增强子／启动子（PSMAe／p）为驱动序列的干扰质粒的细胞特异性沉默效果。方法应用聚乙二醇（PEG）修饰聚乙烯亚胺（PEI），偶联转铁蛋白（Tf）合成靶向载体Tf-PEG-PEI。以Tf-PEG-PEI为载体将细胞特异性干扰质粒pPSMAe／p-shNS-ploy（A）转入前列腺癌LNCap和PC-3细胞中，观察细胞形态变化，检测核干因子（NS）基因和蛋白水平、细胞增殖情况以及细胞周期的变化。结果成功修饰靶向载体Tf-PEG-PEI，细胞转染后，表达前列腺特异性膜抗原的LNCap细胞中NS表达水平显著降低，细胞膜边缘突起增多，更趋向于分化，细胞增殖速度减慢。在LNCaP细胞中，干扰组S期细胞的百分率[（14．17-4-0．32）％]明显低于载体组[（19．65±0．64）％]和对照组[（20．614-0．80）％，均P〈0．01）]；干扰组G，期细胞的百分率[（74．69±1．01）％]高于载体组[（71．06±0．38）％]和对照组[（70．17±0．32）％，均P〈0．01]。而在各组PC-3细胞中，干扰组、载体组和对照组处于S期的百分率分别为（28．86±1．13）％、（29．47±0．47）％和（29．864-0．36）％，差异无统计学意义（P〉0．05）；处于G1期的百分率分别为（63．34±0．81）％、（62．56±0．62）％和（61．71±1．07）％，差异无统计学意义（P〉0．05）。结论非病毒载体Tf-PEG-PEI具有高效基因转移的能力，PSMAe／p驱动shRNA靶向干扰NS基因具有细胞特异性，使用细胞特异性启动子是实现前列腺癌靶向基因治疗的有效策略。

Objective To explore the transgenic etticiency of non-viral vector Tf-PEG-PEI and the cell specific silencing effect of plasmid pPSMAe/p-shNS-ploy（A） on prostate cancer cells. Methods Polyethyleneimine （PEI） was modified by using polyethylene glycol and transferrin to synthesize the non-viral vector Tf-PEG- PEI. NS-specific plasmids pPSMAe/p-shNS-ploy（A） and Tf-PEG-PEI were used to transfect prostate cancer LNCap and PC-3 cells. The changes of cell morphology, proliferation ability and cell cycle were studied after down-regulating the NS gene level. Results Tf-PEG-PEI was successfully modified. After transfection, the PSMA-expressing LNCaP cells became larger and showed more pseudopodia, having a tendency to differentiate. Their cell proliferation ability was reduced, and the detection of cell cycle showed a decrease of S phase and an increase of G1 phase after knocking down NS gene. These targets were not changed in non- PSMA-expresing PC-3 cells. Conclusions The non-viral vector Tf-PEG-PEI has a high ability to transfer targeted gene into target cells. The cellular specificity of short-hairpin RNA transcription driven by PSMAe/p is confirmed by silencing NS gene. The use of cell specific promoter may be an effective strategy of gene theranv for orostate cancer.