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目的探讨第5代阳离子聚酰胺-胺型树枝状聚合物(G5-PAMAM-D)作为小发卡RNA (shRNA)真核表达质粒的载体,进行前列腺癌RNA干扰基因治疗的可行性。方法根据增强型绿色荧光蛋白(EGFP)、信号转导子和转录激活子3(STAT3)cDNA序列结构,分别设计构建表达EGFP mRNA、STAT3 mRNA的特异shRNA真核表达质粒pSilencing 4.1-EGFP-shRNA和pSilencing 4.1- STAT3-shRNA。在体外以G5-PAMAM-D为载体,将质粒pEGFP-C1和pSilencing 4.1-EGFP-shRNA共转染前列腺癌细胞PC-3、22Rvl,通过观察EGFP表达检测干扰效果以及转染效率;然后,以同样的方式将pSilencing 4.1-STAT3-shRNA转入前列腺癌细胞系,四甲基偶氮唑盐(MTT)法检测其对细胞生长的抑制作用,丫啶橙染色观察细胞凋亡。制作前列腺癌小鼠模型,将G5-PAMAM-D与质粒pSilencing 4.1-STAT3-shRNA的混合物注射人荷瘤鼠肿瘤内,观察对肿瘤生长的抑制作用。结果体外实验表明,G5-PAMAM-D可将pSilencing 4.1.EGFP-shRNA转入前列腺癌细胞,明显沉默EGFP的表达,并且G5-PAMAM-D可将pSilencing 4.1-STAT3-shRNA有效转入前列腺癌细胞中,明显抑制肿瘤细胞的生长,增加细胞的凋亡。体内实验证明,G5-PAMAM-D/pSilencing 4.1-STAT3-shRNA可抑制荷瘤鼠前列腺癌的生长,而各对照组肿瘤生长无明显抑制。结论G5-PAMAM-D作为基因载体,可将shRNA的真核表达质粒在体内外转入前列腺癌组织细胞,并有效表达shRNA;G5-PAMAM-D可能成为应用前景广阔的小干扰RNA(siRNA)的载体。
Objective To investigate the feasibility of using the fifth generation cationic polyamide-amine dendrimer (G5-PAMAM-D) as a vector for eukaryotic expression plasmid of small hairpin RNA (shRNA) for RNA interference gene therapy of prostate cancer. Methods According to the sequence of enhanced green fluorescent protein (EGFP), signal transducer and activator of transcription 3 (STAT3) cDNA sequence, we designed and constructed the eukaryotic expression plasmid pSilencing 4.1-EGFP-shRNA of specific shRNA expressing EGFP mRNA and STAT3 mRNA, pSilencing 4.1- STAT3-shRNA. The recombinant plasmid pEGFP-C1 and pSilencing 4.1-EGFP-shRNA were co-transfected into prostate cancer cells PC-3 and 22Rv1 by G5-PAMAM-D in vitro. The interference effect and transfection efficiency were detected by observing the expression of EGFP. PSilencing 4.1-STAT3-shRNA was transfected into prostate cancer cell line in the same way. The inhibitory effect on cell growth was detected by MTT assay. Apoptosis was observed by acridine orange staining. A prostate cancer mouse model was made. The mixture of G5-PAMAM-D and plasmid pSilencing 4.1-STAT3-shRNA was injected into human tumor-bearing mice to observe the inhibitory effect on tumor growth. Results In vitro experiments showed that G5-PAMAM-D transfected pSilencing 4.1.EGFP-shRNA into prostate cancer cells significantly silenced EGFP expression, and G5-PAMAM-D pSTilencing 4.1-STAT3-shRNA effectively transfected into prostate cancer cells In the obvious inhibition of tumor cell growth, increase cell apoptosis. In vivo experiments show that, G5-PAMAM-D / pSilencing 4.1-STAT3-shRNA can inhibit the growth of tumor-bearing mouse prostate cancer, while the control group tumor growth was not significantly inhibited. Conclusion G5-PAMAM-D can be transfected into prostate cancer cells in vitro and in vivo with G5-PAMAM-D as a gene vector, and can efficiently express shRNA. G5-PAMAM-D may be used as a small interfering RNA (siRNA) Carrier.