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目的:探讨前列腺特异性膜抗原增强子/启动子(PSMAe/p)驱动短发夹RNA(shRNA)靶向干扰前列腺癌细胞的特异性。方法:运用RNA干扰技术,以转铁蛋白-聚乙二醇-聚乙烯亚胺(Tf-PEG-PEI)作为基因转移载体,以前列腺癌LNCap细胞为研究对象,并以前列腺癌PC-3细胞、膀胱癌T24细胞及人胚肾HEK293细胞做为对照,将外源增强绿色荧光蛋白(EGFP)基因及以PSMAe/p为启动序列靶向EGFP的干扰质粒转入培养细胞,以实时定量PCR(qRT-PCR)及Western blot分别检测EGFP基因及蛋白在4种细胞各组中的表达情况,研究PSMAe/p驱动shRNA靶向干扰前列腺癌细胞的特异性。结果:在前列腺癌LNCap细胞组中,与单独转入EGFP基因(pEGFP-C1)的细胞组(A组)及转入EGFP基因(pEGFP-C1)+空载体质粒组(pPSMAe/p-UPRT)(C组)相比,转入EGFP基因(pEGFP-C1)及干扰质粒[pPSMAe/p-shEGFP-poIy(A)]组(B组),EGFP基因表达及蛋白表达水平下调,分别与对照组(A组、C组)相比差异具有统计学差异(P<0.05),而转入EGFP基因(pEGFP-C1)的细胞组(A组)和转入EGFP基因(pEGFP-C1)+空载体质粒组pPSMAe/p-UPRT)(C组)间比较差异无统计学意义(P>0.05);而在前列腺癌PC-3细胞、膀胱癌T24细胞及人胚肾HEK293细胞中EGFP基因及蛋白表达水平在实验组与对照组、对照组间比较差异无统计学意义(p>0.05)。结论:PSMAe/p驱动shRNA具有细胞特异性,其可驱动特异基因片段在前列腺癌LNCap细胞中表达,从而实现基因治疗的细胞特异性,可作为前列腺癌基因治疗的靶向策略之一。
Objective: To investigate the specificity of prostatic membrane-specific membrane antigen enhancer / promoter (PSMAe / p) driven short hairpin RNA (shRNA) targeting interference with prostate cancer cells. METHODS: Tc-PEG-PEI was used as a gene transfer vector and prostate cancer LNCap cells were treated with RNA interference technique. PC-3 cells , Bladder cancer T24 cells and human embryonic kidney HEK293 cells were used as control, and the exogenous enhanced green fluorescent protein (EGFP) gene and the interfering plasmid targeting EGFP with PSMAe / p as starting sequence were transferred into cultured cells. Real-time quantitative PCR qRT-PCR) and Western blot were used to detect the expression of EGFP gene and protein in each of the four cell groups, and to study the specificity of PSMAe / p-driven shRNA targeting interfering prostate cancer cells. Results: In the prostate cancer cell line LNCap, the cells transfected with EGFP gene (pEGFP-C1) (group A) and EGFP gene (pEGFP-C1) + empty vector plasmid group (pPSMAe / p-UPRT) Compared with control group (group C), EGFP gene expression and protein expression were down-regulated to EGFP gene (pEGFP-C1) and plasmid pPSMAe / p-shEGFP-poIy (Group A and group C), and the difference was statistically significant (P <0.05). The cells transfected with EGFP gene (group A) and EGFP gene (pEGFP-C1) + empty vector (P> 0.05). However, the expression of EGFP gene and protein in human prostate cancer PC-3 cells, bladder cancer T24 cells and human embryonic kidney HEK293 cells was not significantly different There was no significant difference between the experimental group and the control group and the control group (p> 0.05). CONCLUSIONS: The PSMAe / p-driven shRNA is cell-specific and can drive the expression of specific gene fragments in prostate cancer LNCap cells to achieve the cell specificity of gene therapy, which may serve as one of the targeted strategies for gene therapy of prostate cancer.