高强度聚焦超声联合脂质体介导p53基因转染胰腺癌细胞的实验研究

Experimental study on p53 plasmid transfected into pancreatic cancer cells by high intensity focused ultrasound combined with liposome

目的探讨高强度聚焦超声(HIFU)联合脂质体介导p53基因转染胰腺癌细胞的可行性以及转染后对细胞生物学行为的影响。方法实验分为5组:Ⅰ组为单纯细胞组,Ⅱ组为HIFU+质粒组,Ⅲ组为HIFU+脂质体+质粒组,Ⅳ组为脂质体+质粒组,Ⅴ组为单纯质粒组。每组予以相应处理后,采用反转录聚合酶链反应(RT-PCR)法检测各组细胞p53mRNA表达情况,四甲基偶氮唑盐(MTT)法及流式细胞术检测细胞增殖、凋亡的情况。结果荧光显微镜下见Ⅲ组有较多绿色荧光蛋白表达的细胞(转染率为10.6%),Ⅱ组及Ⅳ组有少量的荧光细胞(转染率分别为1.2%,4.8%);Ⅴ组仅有极少的荧光细胞(0.3%);Ⅰ组无荧光表达;Ⅲ组和Ⅳ组经RT-PCR法均检测到p53mRNA表达,且2组灰度值差异有统计学意义(P<0.05);MTT结果显示Ⅱ、Ⅲ、Ⅳ组细胞均有明显的生长抑制作用,而其中Ⅲ组的增殖抑制作用最为明显,与其他各组比较差异具有统计学意义(P<0.05);Ⅲ组细胞凋亡率为(14.5±1.6)%,与其他4组比较,凋亡率明显增高(P<0.05)。结论 HIFU联合脂质体可提高p53质粒转染率;p53质粒转染后促凋亡作用增强,肿瘤细胞生长进一步受到抑制。

Objective To explore the feasibility,efficiency and effect of p53 gene transfected into pancreatic cancer cells by high intensity focused ultrasound combined with liposome.Methods Pancreatic cancer cells were divided into 5groups：simple cells group（Ⅰ）,HIFU-plasmid group（Ⅱ）,HIFU-liposome-plasmid group（Ⅲ）,liposome-plasmid group（Ⅳ）and simple cells group（V）.At 24~48hafter transfection,the transfection efficiency was detected by fluorescence microscope,the mRNA expression of p53 was detected by RT-PCR,the apoptosis of the tumor cells was detected by flow cytometry,and the inhibition rate of cells growth was detected by MTT assay.Results Fluorescence microscopy showed that the expression of green fluorescent protein in group Ⅲ was higher than that in group Ⅱ and group Ⅳ（P〈0.05）,and there were no green fluorescent cells in group I.RTPCR confirmed that the specific p53 electrophoresis strips were seen in groupⅢand groupⅣ.The optical density of the strip from group Ⅲ was higher than that from groupⅣ（P〈0.05）.The results of MTT showed that the significant cell growth inhibition exists in group Ⅱ,group Ⅲ and group Ⅳ.The cell inhibitory rate in group Ⅲwas significantly higher than those in other groups（P〈0.05）.The cell inhibitory rate in groupⅢ was（14.2±1.55）%.The result was significantly different with other four groups（P〈0.05）.Conclusion The transfection efficiency of p53 gene into pancreatic cells can be enhanced by optimum HIFU intensity and liposome.P53 gene can inhibit the growth of pancreatic cancer cells.