mirRNA质粒和RAN干扰技术下调人脐血干细胞基因的研究

Efficient gene knockdown in human umbilical cord blood CD34^+ cells by microRNA-based shRNAs

目的探讨microRNA(miR)30为基础的shRNA干扰技术能否有效下调人脐血干细胞的基因。方法应用不同的第2代慢病毒包装质粒在293T细胞中包装病毒,检测病毒滴度和人脐血CD34+细胞转染效率。结果第2代慢病毒包装质粒可在人293T细胞中产生高滴度病毒(2×106TU/mL以上)。与包装质粒pCMV-dR8.91(6.8%~17.4%,平均14.2%)相比,pCMV-dR8.74(psPAX2)(12.3%~31.8%,平均26.5%,P<0.05)产生的病毒转染脐血干细胞的效率更高。结论应用shRNAmir和RNA干扰技术可有效下调人脐血CD34+细胞的基因。

Objective To evaluate the gene knockdown effect of microRNA-Based short hairpin RNAs （shRNAmir） on human cord blood CD34 cells. Methods shRNAmir plasmid, the second generation lentivirus package plasmid and the envelope plasmid were mixed together and transfected 293T cells with the help of lipofectamin 2000. The titre of virus soup produced by 293T cells was detected. Human cord blood CD34 cells were transduced with 48 h and 72 h virus soup. Results 293T cells can produce high titre virus （ above 2 x 106 TU/mL）. Compared with pCMV-dR8.91 （6. 8% - 17.4% ,average 14. 2% ） ,the transduction efficiency of pCMV-dRS. 74（psPAX2） （ 12.3% - 31.8% ,average 26. 5% ,P 〈 0. 05 ） was better in human cord blood cells. Conclusion Our results indicate that shRNAmir based RNA interference can successfully transduce genes expressiong in human cord blood CD34 ＋ cells.