CAJ | 학술논문

目的：建立表达microRNA（miRNA）tough decoy（TuD）的慢病毒载体构建方法，并检测其对细胞内miRNA表达水平及细胞表型的影响。方法在慢病毒表达质粒pSIH1-H1-copGFP中通过两步克隆，首先构建miRNA TuD通用骨架质粒，进一步构建特异性针对大鼠miR-203的TuD表达载体。在293T细胞中包装成慢病毒后，感染大鼠骨髓间充质干细胞（BM-MSCs），同时用pSIH1-H1-copGFP空质粒包装慢病毒，感染BM-MSCs作为对照。定量RT-PCR检测细胞中miR-203的水平变化，并用CCK-8法和Annexin V-PI双标记法分别检测BM-MSCs的活性和凋亡。结果成功构建了基于pSIH1-H1-copGFP质粒的miR-203 TuD表达载体，并对其序列进行了验证。用表达miR-203 TuD的重组慢病毒感染BM-MSCs后第3、6、9天检测细胞内源性miR-203表达水平降低，同时细胞活性增高，凋亡比例降低，与对照组相比差异有统计学意义。结论两步克隆法构建miRNA TuD表达载体是一种简便高效的方法，其表达产物能够有效抑制细胞内源性miRNA水平，同时影响细胞表型。
목적：건립표체microRNA（miRNA）tough decoy（TuD）적만병독재체구건방법，병검측기대세포내miRNA표체수평급세포표형적영향。방법재만병독표체질립pSIH1-H1-copGFP중통과량보극륭，수선구건miRNA TuD통용골가질립，진일보구건특이성침대대서miR-203적TuD표체재체。재293T세포중포장성만병독후，감염대서골수간충질간세포（BM-MSCs），동시용pSIH1-H1-copGFP공질립포장만병독，감염BM-MSCs작위대조。정량RT-PCR검측세포중miR-203적수평변화，병용CCK-8법화Annexin V-PI쌍표기법분별검측BM-MSCs적활성화조망。결과성공구건료기우pSIH1-H1-copGFP질립적miR-203 TuD표체재체，병대기서렬진행료험증。용표체miR-203 TuD적중조만병독감염BM-MSCs후제3、6、9천검측세포내원성miR-203표체수평강저，동시세포활성증고，조망비례강저，여대조조상비차이유통계학의의。결론량보극륭법구건miRNA TuD표체재체시일충간편고효적방법，기표체산물능구유효억제세포내원성miRNA수평，동시영향세포표형。
Objective To establish method of constructing lentiviral vectors to express microRNA (miRNA) ''tough decoy''(TuD)and to detect the effects of the TuD on cellular endogenous miRNA level and cellular phenotypes. Methods Two-step cloning strategy was utilized to first generate a universal miRNA TuD frame vector,followed by con-structing the TuD expression vector specially targeting miR-203. The package of the recombinant lentivirus was per-formed in 293T cells. Then the rat bone marrow mesenchymal stem cells(BM-MSCs)were infected by the miR-203 TuD expression lentivirus. The pSIH1-H1-copGFP vector was also packaged and the BM-MSCs infected by this lentivirus were served as control. Endogenous miR-203 level in BM-MSCs was measured by quantitative RT-PCR,and cellular vi-ability and apoptosis were detected by CCK-8 test and Annexin V-PI staining respectively. Results The miR-203 TuD expression vector was successfully constructed and inserted sequence was validated. At the 3rd,6th and 9th days after in-fected by the miR-203 TuD expression lentivirus,rat BM-MSCs exhibited a repressed endogenous miR-203 level. The miR-203 TuD also promoted viability and inhibited apoptosis of BM-MSCs. All these differences between miR-203 TuD group and control group were statistically significant. Conclusion The two-step cloning method for the construction of miRNA TuD expression vector is simple and efficient. The miRNA TuD can effectively suppress the level of the target miRNA and affect cellular phenotypes.