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该研究利用短发卡RNA(small hairpin RNA,shRNA)表达载体沉默HT9急性早幼粒白血病耐药细胞MDR1基因表达,以提高细胞对三尖杉酯碱、阿霉素的敏感性。通过设计合成编码shRNA的DNA模板序列,定向克隆到pSilencer 2.1-U6 neo质粒,成功构建1个P-gp蛋白基因特异的shRNA表达载体,稳定电转染HT9细胞,实时荧光定量PCR分析MDR1 mRNA表达,Western blot检测细胞P-gp蛋白表达,流式细胞术检测P-gp蛋白外排泵功能,MTT法检测细胞对药物敏感性。结果显示,成功构建了shRNA表达载体pSilencer 2.1-U6 neo-MDR1,转染HT9细胞后,PCR检测重组质粒整合到HT9/sh-2.1-1细胞基因组DNA,获得稳定遗传;HT9/sh-2.1-1细胞MDR1 mRNA表达降低了78.84%(P<0.01),P-gp蛋白表达降低了48.27%(P<0.05),细胞内Rho123相对荧光强度由(10.8±0.58)%升高至(73.56±1.37)%;转染细胞对三尖杉酯碱、阿霉素敏感性明显增强,IC50分别由(2.06±0.15)μmol/L降至(0.57±0.01)μmol/L、(4.04±017)μmol/L降至(1.56±0.05)μmol/L。提示shRNA干扰表达载体pSilencer 2.1-U6 neo-MDR1能够稳定、持久地抑制MDR1基因表达,并能有效增强HT9细胞对三尖杉酯碱、阿霉素的敏感性。
In this study, MDR1 gene expression in HT9-resistant acute promyelocytic leukemia cells was silenced by using small hairpin RNA (shRNA) expression vector to enhance the sensitivity of cells to harringtonine and doxorubicin. By designing a DNA template sequence encoding shRNA, we cloned into pSilencer 2.1-U6 neo plasmid successfully and constructed a shRNA expression vector with P-gp protein gene successfully. The shRNA was stably transfected into HT9 cells and the expression of MDR1 mRNA was analyzed by real-time fluorescence quantitative PCR The expression of P-gp protein was detected by Western blot, the efflux pump function of P-gp protein was detected by flow cytometry, and the drug sensitivity of cells was detected by MTT assay. The results showed that the shRNA expression vector pSilencer 2.1-U6 neo-MDR1 was successfully constructed and transfected into HT9 cells. The recombinant plasmids were integrated into the genomic DNA of HT9 / sh-2.1-1 cells for stable inheritance. HT9 / sh-2.1- 1 cells decreased 78.84% (P <0.01), the expression of P-gp protein decreased 48.27% (P <0.05), the relative fluorescence intensity of Rho123 increased from (10.8 ± 0.58)% to (73.56 ± 1.37) ). The sensitivity of the transfected cells to harringtonine and doxorubicin was significantly increased. The IC50 values were significantly decreased from (2.06 ± 0.15) μmol / L to (0.57 ± 0.01) μmol / L and (4.04 ± 0.17) μmol / L to (1.56 ± 0.05) μmol / L. The shRNA interference expression vector pSilencer 2.1-U6 neo-MDR1 could stably and durably suppress MDR1 gene expression and enhance the sensitivity of HT9 cells to harringtonine and doxorubicin.