Jmjd3基因RNAi慢病毒载体构建及转染BMSCs效应的实验研究

目的 构建针对SD大鼠Jmjd3基因RNAi慢病毒载体,转染大鼠骨髓间充质细胞(bone marrow stromal cells,BMSCs)后观察其对Jmjd3基因的抑制作用并筛选最有效干扰序列。方法 设计4对Jmjd3基因shRNA寡核苷酸序列,插入pGCSIL-GFP载体形成重组载体。重组载体经过测序鉴定后转染293T细胞生产病毒液,病毒液转染大鼠BMSCs,Western blot分析转染前后Jmjd3表达情况。通过计算Jmjd3灰度值/ACTIN灰度值,筛选出最有效的干扰序列。结果 测序结果证实Jmjd3基因shRNA寡核苷酸序列正确插入载体中,成功构建4个大鼠Jmjd3基因RNAi表达载体。Western blot检测显示转染后Jmjd3蛋白表达显著下调,半定量分析显示相对于未转染组,shRNA-1组、shRNA-2组、shRNA-3组、shRNA-4组分别下调79.5%(P<0.001)、90.7%(P<0.001)、73.7%(P<0.001)、56.5%(P<0.001),而GFP组下调11.5%(P>0.05),shRNA-2组为最佳干扰序列。结论 针对大鼠Jmjd3基因RNAi慢病毒载体构建成功,并能有效抑制BMSCs中Jmjd3基因的表达。

Construction of lentivirus vectors targeting Jmjd3 gene and their effects on BMSCs

Objective To construct specific RNAi expression vectors targeting rat Jmjd3 gene and study the inhibition effect of Jmjd3-RNAi on expression of Jmjd3 in bone marrow stromal cells(BMSCs) and select the vectors of optimal suppression efficiency.Methods Four pairs of specific shRNA single stranded DNA oligos for Jmjd3 were synthesized and cloned into pGCSIL-GFP vectors.The plasmid was identified by DNA sequencing.The recombined plasmids were transfected into 293 cell line.Viruses induced by 293 cell were transfected into rat BMSCs.Expression of Jmjd3 was detected by Western bolt.Results The shRNA oligo of Jmjd3 were correctly cloned into the pGCSIL-GFP plasmids and confirmed by DNA sequencing.Four RNAi vectors targeting rat Jmjd3 gene were constructed successfully.The results of Western blot showed that sequence-specific RNAi targeting Jmjd3 significantly down regulated the expression of Jmjd3 in rat BMSCs.Semi-quantitive analysis of Jmjd3 showed that decreased expression of Jmjd3 in the four different transfected cells was shRNA-1 79.5%(P<0.001),shRNA-2 90.7%(P<0.001),shRNA-3 73.7%(P<0.001),shRNA-4 56.5%(P<0.001) and that in the GFP group was 11.5%(P>0.05) compared with the untransfected BMSCs.Therefore,shRNA-2 was the most efficient interfering sequence.Conclusions Specific RNAi vectors targeting rat Jmjd3 gene was constructed successfully and they efficiently down regulated the expression of Jmdj3 in rat BMSCs.