CAJ | 학술논문

目的构建shRNA/mrp1的质粒表达载体并验证其体外表达效率.方法根据业已筛选出的mrp1基因RNAi靶序列,按照pSUPER的设计要求合成64 bp的寡核苷酸序列,将其退火后形成双链并用双酶切法克隆到pSUPER得到质粒pSUPER-shRNA/mrp1,转染感受态大肠杆菌,筛选阳性克隆,经测序证实后扩增培养并以去内毒素试剂盒提取,然后转染HepG2/mrp1细胞,阴性载体为对照组,Real-time PCR、流式细胞术检测mrp1基因mRNA、MRP1表达、细胞耐药性等功能变化.结果成功构建质粒载体pSUPER-shRNA/mrp1,经基因测序证实靶序列插入正确;HepG_2/mrp1组Ct值较GAPDH增加5.61,HepG_2/mrp1-si组Ct值比GAPDH升高11.35,mrp1基因的表达水平是耐药株HepG_2/mrp1的179分之一;实验组HepG_2/mrp1细胞和阴性对照组HepG_2/mrp1细胞的MRP表达率分别为11.2%和97.6%,差别有统计学意义(P<0.05);药物敏感试验显示HepG_2/mrp1细胞耐药倍数从45.0下降到1.2,相对逆转效率99.62%;细胞内DNR累积量也明显增加,与对照组比较(78.58%vs 38.44%,P<0.05).结论成功构建质粒载体pSUPER-shRNA/mrp1,在HepG2/mrp1内稳定表达,逆转其耐药性.
목적 구건shRNA/mrp1적질립표체재체병험증기체외표체효솔.방법 근거업이사선출적mrp1기인RNAi파서렬,안조pSUPER적설계요구합성64 bp적과핵감산서렬,장기퇴화후형성쌍련병용쌍매절법극륭도pSUPER득도질립pSUPER-shRNA/mrp1,전염감수태대장간균,사선양성극륭,경측서증실후확증배양병이거내독소시제합제취,연후전염HepG2/mrp1세포,음성재체위대조조,Real-time PCR、류식세포술검측mrp1기인mRNA、MRP1표체、세포내약성등공능변화.결과 성공구건질립재체pSUPER-shRNA/mrp1,경기인측서증실파서렬삽입정학;HepG_2/mrp1조Ct치교GAPDH증가5.61,HepG_2/mrp1-si조Ct치비GAPDH승고11.35,mrp1기인적표체수평시내약주HepG_2/mrp1적179분지일;실험조HepG_2/mrp1세포화음성대조조HepG_2/mrp1세포적MRP표체솔분별위11.2%화97.6%,차별유통계학의의(P<0.05);약물민감시험현시HepG_2/mrp1세포내약배수종45.0하강도1.2,상대역전효솔99.62%;세포내DNR루적량야명현증가,여대조조비교(78.58%vs 38.44%,P<0.05).결론 성공구건질립재체pSUPER-shRNA/mrp1,재HepG2/mrp1내은정표체,역전기내약성.
Objective To construct the expressing vector of shRNA/mrp1 and study its expression in vitro. Methods 64bp oligonucleotides of pSUPER and the targeted senquence of siRNA/mrp1 were synthesized and annealed to form duplex strand, then were cloned into pSUPER to construct pSUPER-shRNA/mrp1 vector. Competenced Ecoli was transfected by vector of pSUPER-shRNA/ mrp1 to screen the positive clones for sequencing and extracting plasmid. The plasmids extracted were used to transfected HepG2/mrp1 cells with a control groups by negative vectors. The expression of mrp1 mRNA and MRP1 was measured by real-time PCR and resistance of HepG2/mrp1 by flowcytometry. Results pSUPER-shRNA/mrp1 was established successfully and was sequenced to test its accuracy. Expression of mrp1 mRNA in HepG2/mrp1-si was lower than that in HepG2/mrp1 (1-fold vs 179.76-fold, P<0.001). Compared to HepG2/mrp1, the expression of MRP1 in HepG2/mrp1-si was lower (11.2% vs 97.6%, P<0. 05). The sensitivity of HepG2/mrp1-si to adiramycin was higher than that of HepG2/mrp1(45.0-fold vs 1.2-fold, P<0.01). Meanwhile, the accumulation of DNR in HepG2/mrp1-si increased significantly as compared with the control (78.58 % vs 38.44%,P<0.05).Conclusion Vector of pSUPER-shRNA/mrp1 can be constructed by the technique of enzymatic incision. The multidrug resistance of HepG2/mrp1 can be reversed by RNA interference.