纳米脂质体槲皮素联合8-Br-cAMP诱导人Rb44细胞恶性逆转化效应

Malignancy reverse of human Rb44 cells induced by nanoliposomal quercetin combined with 8-Br-cAMP

目的纳米脂质体槲皮素(nanoliposomal quercetin,nLQ)可抑制PI3K/AKt信号途径,8-Br-cAMP为PKAII型激动剂,探讨二者协同诱导人视网膜母细胞瘤(retinoblasto-ma,Rb)44细胞的恶性逆转化效应。方法将培养的Rb44细胞分为4组:(1)加纳米脂质体槲皮素(终浓度为40μmol.L-1)组(nLQ组);(2)加8-Br-cAMP(终浓度为20μmol.L-1)组(Br组);(3)联合药物组(nLQ+Br组);(4)不加药物培养的对照组。以MTT实验检测培养的各组Rb44细胞生长抑制率,采用免疫细胞化学和蛋白质免疫斑点印迹检测PTEN、cyclinD1、c-myc、p21waf1、MMP9及VEGF的表达,原位杂交技术检测p16、Rb抑癌基因的表达。结果 MTT实验显示联合药物组的细胞生长抑制率显著高于任何单个药物组。与对照组相比,nLQ和8-Br-cAMP组明显抑制培养的Rb44细胞的生长,并呈现协同抑制效应。免疫细胞化学及蛋白质免疫斑点印迹显示:与对照组相比,cyclinD1、c-myc、MMP9、VEGF的表达下调,PTEN、p21waf1表达上调。原位杂交显示:与对照组相比,药物组的p16及Rb抑癌基因的表达上调。结论 nLQ或8-Br-cAMP可诱导人Rb44细胞恶性逆转化,且呈协同效应。

Objective To investigate the malignancy reverse of human retinoblastoma 44（Rb44）cells induced by nanoliposomal quercetin（nLQ）inhibiting PI3K/AKt signal pathway and 8-Br-cAMP as PKAⅡexcitomotor.Methods The cultured Rb44 cells were divided into four groups,with nLQ（final concentration was 40 μmol·L-1,nLQ group）,with 8-Br-cAMP（final concentration was 20 μmol·L-1,Br group）,combined drug group（nLQ with Br group） and group without any drug（control group）.Growth inhibitive rate of cultured Rb44 cells in each group was detected by MTT assay.The expression of cyclin D1,c-myc,VEGF,PTEN,MMP9 and p21waf1 were detected by immunohistochemical method and protein immunodotting-blotting method;Expression of p16 and Rb tumor suppressor genes were measured by in situ hybridization.Results MTT assay showed growth inhibitive rate of nLQ with Br group was obviously higher than other single drug groups.Compared with control group,nLQ group or 8-Br-cAMP group obviously inhibited growth of cultured Rb44 cells with coordinate repression.Immunohistochemical method and protein immunodotting-blotting method showed expression of cyclin D1,c-myc,VEGF and MMP9 were with down regulation,while PTEN,p21waf1 were with up regulation compared with control group.In situ hybridization showed expression of p16 and Rb tumor suppressor genes in drug groups were with up regulation compared with control group.Conclusion nLQ or 8-Br-cAMP can induce malignancy reverse of human Rb44 cells with coordinate repression.