脂质体介导的人甲状腺过氧化物酶基因转染肺癌A549细胞及其蛋白表达

Transfection of Human Thyroperoxidase (TPO) Gene with Liposome and the Expression of TPO Protein in A549 Cell

目的:利用基因转染技术,研究以脂质体Lipofectamine2000为载体介导的人甲状腺过氧化物酶(TPO)基因体外转染肺癌细胞,检测感染细胞内TPO蛋白的表达,为放射性碘治疗肺癌提供理论和实验依据。方法:将获得的含TPO基因的质粒pcDAN3.1-hTPO进行扩增、纯化,并经酶切鉴定和DNA测序。将肺癌A549细胞在体外复苏与培养并分为两组:转染质粒pcDAN3.1-hTPO的为实验组,转染空质粒pcDAN3.1的为对照组。以脂质体Lipofectamine2000为载体,介导TPO基因转染肺癌细胞。采用WesternBlot免疫印迹法和免疫组化法分别检测肺癌细胞中TPO蛋白的表达。结果:①酶切鉴定和DNA测序结果表明质粒pc-DAN3.1-hTPO中插入的基因为hTPO基因,其片段大小和方向正确。②体外培养的肺癌细胞活力及数量正常,细胞活力为96%,细胞生长密度为1×106/ml,满足实验要求。③质粒转染A549细胞后,WesternBlot免疫印迹法显示:在实验组中,肺癌A549细胞有TPO蛋白的表达,而在对照组中无表达。④免疫组化染色结果显示:在实验组的肺癌A549细胞中,TPO蛋白表达阳性且主要分布于细胞膜上,阳性表达率可达75%,而在对照组中TPO蛋白表达阴性,两组比较差异有显著性(P=0.000)。结论:①获得的hTPO基因片段的核苷酸序列与GeneBank报道完全一致。②在脂质体Lipofectamine2000的介导下,TPO基因能够有效地转染肺癌细胞。③转染人甲状腺过氧化物酶基因的肺癌细胞能够在体外成功地表达TPO蛋白。

Objective： To research the expression of human Thyroperoxidase （TPO） gene transfected to lung carcinoma cells with Lipofectamine2000 by gene transfection technique. To explore the possibilities of TPO protein expression in carcinoma cells and provide further the academic evidence in radioiodide therapy for lung cancer. Methods： The presented plasmid vector encoding hTPO gene （pcD-NA3.1-hTPO from Dr. Basil Rapoport） was amplified and purified, then identified by restriction enzyme digestion and DNA sequencing. Lung carcinoma A549 cells were resuscitated and cultured in vitro and divided into two groups： the experimental group transfected by plasmid pcDNA3.1-hTPO and control group transfected by plasmid pcDNA3.1 .We used Western Blot and immunohistochemistry assay method to detect the expression products of TPO gene after the two groups were transfected in vitro respectively with plasmid pcDNA3.1-hTPO and pcDNA3.1 mediated by liposome Lipofectamine2000 vector. Results：①The result of restriction enzyme digestion and DNA sequencing showed the objective gene in plasmid pcDNA3-hTPO was hTPO gene, both the size and direction were correct.②Lung carcinoma A549 cells cultured in vitro were well cultured and able to reach the needs of this experiment. Live cell rate was up to 96% and cell density reached 1×10^6cells/ml.③The result of Western Blot showed that there was positive expression of TPO protein in experimental group and negative in control group.④Immunohistochemistry assay showed the expression of NIS protein was positive in experimental group and TPO proteins were detected in the cell membranes; the TPO protein expression rate was 75%. But the expression of TPO protein was negative in the cell membranes of control group. There was significant statistical difference between two groups （P=0.000）. Conclusion： ①The hTPO gene we got was consistent with the sequence reported by GeneBank.②Thyroperoxidase （TPO） gene can be transfected to lung carcinoma cells with Lipofectamine2000 effectively.③The lung carcinoma cells transfected by Thyroperoxidase （TPO） gene could express the TPO protein in vitro.