| Objective: To explore a new approach to the gene therapy ofβ-thalassemia by blocking DNA binding sequence(sequence between the gene γ andδ) of peptide nucleic acid and artificially activating the expression of γ-globin gene.Methods: 1. Design the peptide nucleic acids: The study designed three peptide nucleic acid altogether(1) PYR-PNA, The peptide nucleic acid sequence which complementary and binding to the PYR’s DNA for blocking the switch of α â†'β-globin.(2) β-PNA, The peptide nucleic acids which transcriptional initiation region of the oligonucleotide with β- globin gene and down-regulated β- globin gene expression on γ- globin gene.(3) Scr-PNA, The Scramble PNA as a control PNA.2.The transfection efficiency and cytotoxicity assay: With the cationic liposome lipofectamine 2000 used as carrier, PNAs marked by FITC fluorophore were transfected into K562 cells. Transfection efficiency was calculated according to the observation of cells under fluorescence microscope, and cytotoxicity was examined by Cell Counting Kit(CCK-8). In order to achieve the best transfection results, the dose and proportion of PNA and liposomes were optimized by orthogonal experiment,as well as the serum concentration in medium used as dilution was optimized. 3. The effect of the peptide nucleic acid on γ-globin gene expression: Firstly, for K562 cells,they were divided into three experimental groups and two control groups which the experimental groups including the PYR-PNA group, the β-PNA group, the PYR-PNA+ β-PNA group and the control groups including the Scr-PNA group and the blank control group. With the best transfection conditions, each group of K562 cells transfected with the PNA by 24 h,48h and 72 h,then detect the expression of γ- globin gene at the transcriptional and translational by the Real time PCR and the Western blotting to comparison the effects of each group on γ-globin gene expression.Results: 1.The optimal transfection conditions of cells: When the cells with the density of 2.5-3×105/mL were cultured, the optimized transfection conditions were as follow: each 100 ul medium added with 6.25 pmol PNA, the volume proportion of PNA to liposomes was 1:3.5, and the medium used as dilution had no fetal bovine serum, 50 ul fetal calf serum was added after transfection 5h and the effect of 48 h transfection is best. 2. The result of the cytotoxicity and transfection efficiency: After optimization the conditions, the best transfection results were achieved, with a transfection efficiency of 87.2% and a relative growth rate(RGR) of 94.1%. 3.The result of the Real Time-PCR: The PYR-PNA group which compared to the control group that the γ- globin gene’s relative expression was increased 1.702 times in 24 h,2.036 times in 48 h and 1.498 times in 72 h, by statiscal, all P < 0.01, significant difference;The β-PNA group which compared to the control group that the γ- globin gene’s relative expression was increased 1.374 times in 24 h, 1.436 times in 48 h and1.132 times in 72 h, in 24 h which compared to the control group by statiscal, P<0.05,significant difference; The PYR-PNA + β-PNA group which compared to the control group in 24 h, 48 h and 72 h that all were no significant with the P>0.05;Theβ-PNA group and the PYR-PNA + β-PNA group which compared to the PYR-PNA group by statiscal, all P < 0.01, significant difference. 4.The result of the western blotting: The PYR-PNA group which compared to the control group that theγ-globin’s relative expression was increased 1.608 times in 24 h, 2.488 times in 48 h and 1.575 times in 72 h, by statiscal, all P<0.01, significant difference. The β-PNA group which compared to the control group that the γ- globin relative expression was increased 1.330 times in 24 h and 1.422 times in 48 h and 1.259 times in 72 h,there have significant difference in 24h(P<0.01)and 48h(P<0.05); The PYR-PNA +β-PNA group which compared to the control group that the γ- globin relative expression was increased 1.334 times in 24 h and 1.784 times in 48 h and 1.177 times in 72 h, which compared to the control group, by statiscal, in 24 h and 48h(P<0.01),significant difference; The β-PNA group and the PYR-PNA + β-PNA group which compared to the PYR-PNA group by statiscal, all P<0.01, significant difference.Conclusion: 1.After optimization the conditions, PNA could be transfected into K562 cells mediated by cationic liposome. 2. The PYR-PNA group can increase the γ-globin gene’s expression in level of transcription and translation in K562 cells.3. The study provides a new research method to explored further more γ- globin gene’s expression mechanism and the β-thalassemia’s gene therapy. |
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