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目的探讨超声联合微泡造影剂介导野生型p53(wtp53)基因转染宫颈癌HeLa细胞的可行性、效率性及作用效果。方法以前期实验所筛选的超声辐照参数(300kHz,0.5W/cm2,30s)为实验条件,将HeLa细胞分为质粒组(A组)、质粒+超声组(B组)、质粒+超声+微泡组(C组)、质粒+脂质体组(D组)、空白对照组(E组),分别进行处理。转染24～48h后,收集各组细胞,荧光显微镜观察细胞基因转染效率、RT-PCR检测p53mRNA表达,流式细胞仪检测细胞周期,MTT检测细胞增殖抑制情况。结果荧光显微镜下见C、D组均有较多绿色荧光蛋白表达的细胞(转染率分别为14.15%和10.86%),B组仅有极少的荧光细胞(0.81%);A组和E组无荧光表达;RT-PCR结果显示,C组和D组均可见特异性p53电泳条带,C组的电泳条带的光密度比值高于D组(P<0.05);流式细胞仪测细胞周期显示,转染野生型p53基因能使细胞周期出现明显的G1期阻滞,C、D组与E组相比差异有显著性(P<0.05);MTT测定结果显示C组细胞生长明显受抑,且随时间的延长,抑制程度逐渐增强。结论适当浓度的微泡和优化的超声辐照条件可增强基因转染效率,野生型p53基因能使HeLa细胞产生G1期阻滞,抑制宫颈癌细胞生长。
Objective To investigate the feasibility, efficiency and effect of ultrasound combined with microbubble contrast-mediated transfection of wild-type p53 (wtp53) gene into cervical cancer HeLa cells. Methods The ultrasound irradiation parameters (300kHz, 0.5W / cm2, 30s) screened in the previous experiment were used as the experimental conditions. HeLa cells were divided into three groups: group A, plasmid + ultrasound group B, plasmid + ultrasound + Micro-bubble group (C group), plasmid + liposome group (D group) and blank control group (E group) were treated respectively. The cells were collected 24-48 hours after transfection. The transfection efficiency of cells was observed by fluorescence microscopy. The expression of p53 mRNA was detected by RT-PCR, the cell cycle was detected by flow cytometry, and the inhibition of cell proliferation by MTT assay. Results In the fluorescence microscope, cells with more green fluorescent protein were observed in C and D groups (transfection rates were 14.15% and 10.86%, respectively), while there were only very few fluorescent cells in group B (0.81%). Groups A and E The results of RT-PCR showed that there was a specific band of p53 in both groups C and D. The optical density of electrophoresis bands in group C was higher than that in group D (P <0.05). Flow cytometry Cell cycle showed that transfected with wild-type p53 gene could significantly arrest the cell cycle in G1 phase. There was significant difference between C and D groups and E group (P <0.05). MTT assay showed that the cell growth of group C was obvious Suppressed, and with the extension of time, the degree of inhibition gradually increased. Conclusion Appropriate concentration of microbubbles and optimized ultrasound irradiation conditions can enhance the gene transfection efficiency. Wild-type p53 gene can cause G1 phase arrest in HeLa cells and inhibit the growth of cervical cancer cells.