摘要
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。
The study investigated the effects of RNAi silencing MDR1 gene, increase the sensitivity of multidrug resistant actue promyelocytic leukemia cells HT9 to harringtonine and doxorubicin. One short hairpin RNA （small hairpin RNA, shRNA） was designed and constructed into pSilencer2, lU6 neo plasmid. MDR1 shRNA expression plasmid pSilencer 2. lU6 neoMDR1 was constructed and introduced into HT9 cells. MDR1 mRNA was assayed by realtime fluorescent quantitative PCR. The Pgp protein was assayed by Western blot. The pump function of Pgp was assayed by FCM. The sensitivity of cells to drugs were assayed by MTT. The results suggested that pSilencer 2. lU6 neoMDR1 expression plasmid was constructed successfully. The results of PCR showed that the shR NA recombinant plasmid had integrated into genome. In HT9/sh2.11 cells, MDR1 mRNA were decreased by 78.84% （P〈0.01）, and Pgp protein were decreased by 48.27% （P〈0.05）; The Rho123 were increased from （10.80±0.58）% to （73.564±1.37）%; The sensitivity of transfected cells to harringtonine and doxorubicin were increased significantly, ICs0 were decreased from （2.06±0.15） gmol/L to （0.574±0.01） gmol/L, （4.04±0.17） gmol/L to （1.56±0.05） μmol/L, respectively. So shRNA expression plasmid pSilencer 2. lU6 neoMDR1 can permanently inhibit the expression of MDR1 gene stability, and increase the sensitivity of HT9 cells to harringtonine and doxorubicin.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2012年第10期983-987,共5页
Chinese Journal of Cell Biology
基金
黑龙江省自然科学基金(No.C200624)
黑龙江省教育厅科学技术项目(No.11511447
No.12511611)资助项目~~
