大熊猫白血病抑制因子RNAi干扰载体的构建及应用

Construction and application of giant panda LIF RNAi interference vector

旨在构建大熊猫白血病抑制因子(LIF)RNAi重组质粒,以期进一步研究LIF对大熊猫骨髓间充质干细胞(PDBM-MSCs)的调控作用.试验设计3组大熊猫LIF shRNA慢病毒pLKO.1 puro-LIF-down干扰载体,通过慢病毒的方式感染PDBM-MSCs,经q-PCR鉴定重组质粒pLKO.1 puro-LIF-down-1抑制效果最显著.pLKO.1 puro-LIF-down-1实验组和空质粒对照组比较发现,LIF mRNA表达水平抑制后能够显著下调细胞周期调控基因CDK2、CCNB2、CCND3的mRNA表达水平(P<0.05);显著上调凋亡基因P53、P16、P27、caspase3的mRNA表达水平(P<0.05);显著下调干性基因KLF4、SOX2的mRNA表达水平(P<0.05).以上结果表明,此次研究成功构建出大熊猫LIF RNAi干扰载体,LIF对PDBM-MSCs的生长和干性维持具有重要的调控作用,为大熊猫干细胞的体外培养及干性机制的研究奠定基础.

The aim was to construct a giant panda leukemia inhibitory factor(LIF)RNAi recombinant plasmid to study the reg-ulatory effect of LIF on giant panda bone marrow mesenchymal stem cells(PDBM-MSCs).The experiment was designed with three groups of giant panda LIF shRNA lentiviral pLKO.1 puro-LIF-down interfering vectors,which infected PDBM-MSCs by lentiviral means.q-PCR identified the most significant inhibitory effect of the recombinant plasmid pLKO.1 puro-LIF-down-1.Comparison of pLKO.1 puro-LIF-down-1 experimental group and the empty plasmid control group revealed that the inhibition of LIF mRNA expression level was able to significantly down-regulate the mRNA expression level of the cell cycle regulatory genes CDK2,CCNB2,CCND3(P<0.05),significantly up-regulate the mRNA expression level of the apoptotic genes P53,P16,P27,caspase3(P<0.05),and significantly down-regulate the stemness genes KLF4,SOX2 mRNA expression level(P<0.05).The above results indicated that the giant pandas LIF RNAi interference vector was successfully constructed in this study,and LIF had an important regulatory role in the growth and stemness maintenance of PDBM-MSCs,which lay the foundation for the in vitro culture of pandas stem cells and the study of stemness mechanism.