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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 study the transfection of human thyroid peroxidase (TPO) gene mediated by lipofectamine 2000 into lung cancer cells in vitro and to detect the expression of TPO protein in infected cells by using gene transfection technique. Theoretical and experimental basis. Methods: The plasmid pcDAN3.1-hTPO containing TPO gene was amplified, purified, identified by restriction enzyme and DNA sequencing. A549 lung cancer cells in vitro resuscitation and culture and divided into two groups: transfected plasmid pcDAN3.1-hTPO as the experimental group transfected with empty plasmid pcDAN3.1 as the control group. Lipofectamine 2000 was used as a vector to mediate TPO gene transfection into lung cancer cells. Western blotting and immunohistochemistry were used to detect the expression of TPO protein in lung cancer cells. Results: ① Enzyme digestion and DNA sequencing showed that the inserted gene of plasmid pc-DAN3.1-hTPO was hTPO gene, whose fragment size and orientation were correct. ② The lung cancer cells cultured in vitro had normal viability and quantity, 96% cell viability, and 1 × 106 / ml cell growth density to meet the experimental requirements. ③ After transfection of A549 cells with plasmid, western blotting showed that TPO protein was expressed in lung cancer A549 cells but not in control cells. ④ The results of immunohistochemistry showed that TPO protein was positive in lung cancer A549 cells and mainly distributed on the cell membrane in the experimental group, the positive expression rate was up to 75%, while the TPO protein expression was negative in the control group Significant (P = 0.000). Conclusion: ① The nucleotide sequence of hTPO gene fragment obtained is exactly the same as reported by GeneBank. ② Lipofectamine2000-mediated, TPO gene can be effectively transfected into lung cancer cells. ③ Lung cancer cells transfected with human thyroid peroxidase gene can successfully express TPO protein in vitro.