BPEI／miRNA、PAMAM／miRNA和PEI／miRNA治疗前列腺癌作用研究

Effect of BPEI /miRNA,PAMAM/miRNA and PEI/miRNA Delivery System on the Treatment of Prostate Cancer

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LNCa P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LNCa P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LNCa P细胞的转染效率;CCK8法测定抑制LNCa P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LNCa P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LNCa P细胞,并且阻滞LNCa P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LNCa P细胞具有高转染效率的纳米系统。并可以有效阻滞LNCa P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 （ PAM- AM） and linear polyethylenimine （LPEI） as gene vector for miRNA-15a and miRNA-16-1 gene therapy in prostate cancer. METH- ODS The diameter and Zeta potential of complex were measured by Zetasizer Nano-ZS9, and its stability was also observed. The effi- ciency of transfection in vitro was detected by flow cytometer and the positively transfected cells were detected by fluorescence micro- scope. 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 transfec- tion 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 prom- ising 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.