目的 利用分枝状聚乙烯亚胺(BPEI)、树枝状聚合物聚酰胺-胺(PAMAM)和线型的聚乙烯亚胺(LPEI)包载具有抑制前列腺癌细胞LNCaP增殖、侵袭和转移的miRNA-15a和miRNA-16-1构建3种聚阳离子纳米复合物,对其抑制LNCaP细胞作用进行考察。方法 利用粒径分析仪测定3种纳米复合物的粒径Zeta电位,利用凝胶电泳实验测定载体材料包裹miRNA的稳定性; 利用荧光显微镜和流式细胞仪考察3种复合物对LNCaP细胞的转染效率;CCK8 法测定抑制LNCaP细胞增殖效果;Western blot法测定BCL-2、Cyclin D1和Wnt3a蛋白表达效果。结果 分枝状聚乙烯亚胺,树枝状聚合物聚酰胺-胺和线型的聚乙烯亚胺3种材料包裹miRNA可形成稳定的纳米复合物,在N/P=5时,分枝状聚乙烯亚胺/miRNA-CY3对LNCaP的转染效率高于树枝状聚合物聚酰胺-胺/miRNA-CY3和线型的聚乙烯亚胺/miRNA-CY3,具有统计学意义(P<0.01) 。分枝状聚乙烯亚胺、树枝状聚合物聚酰胺-胺和线型的聚乙烯亚胺都可以携带miRNA-15a和miRNA-16-1进入LNCaP细胞,并且阻滞LNCaP细胞中BCL-2、Cyclin D1和Wnt3a蛋白的表达。结论 分枝状聚乙烯亚胺、树枝状聚合物聚酰胺-胺和线型的聚乙烯亚胺3种材料包裹miRNA-15a和miRNA-16-1可形成稳定,并且对前列腺癌细胞LNCaP细胞具有高转染效率的纳米系统。并可以有效阻滞LNCaP细胞中BCL-2、CCDN1和Wnt3a蛋白的表达,有望成为治疗前列腺癌的基因给药系统。

OBJECTIVE To identify the possibility of branched polyethyleneimine (BPEI), polyamidoamine dendrimers (PAMAM) and linear polyethylenimine (LPEI) as gene vector for miRNA-15a and miRNA-16-1 gene therapy in prostate cancer. METHODS The diameter and Zeta potential of complex were measured by Zetasizer Nano-ZS9, and its stability was also observed. The efficiency of transfection in vitro was detected by flow cytometer and the positively transfected cells were detected by fluorescence microscope. The inhibition of complex on LNCaP cell was determined by CCK8 assay. The expression of protein BCL-2, CCDN1 and Wnt3a were detected by Western blot method. RESULTS BPEI, PAMAM and LPEI with miRNA formed nano-sized particles. The transfection rate of BPEI/miRNA-CY3 was significantly higher than that of PAMAM/miRNA-CY3 and LPEI/miRNA-CY3 at N/P ratio (P<0.01). The protein level of BCL-2, CCDN1 and Wnt3a in BPEI, PAMAM and LPEI were all lower than the control group in LNCaP cells. CONCLUSION With good stability and transfection rate, BPEI/miRNA, PAMAM/miRNA and LPEI/miRNA can be a promising nano-vector of miRNA transfer system. The complex can inhibit the expression of BCL-2, CCDN1 and Wnt3a gene specifically and efficiently, which may be used for prostate cancer treament.