腺病毒介导siRNA抑制全反式维甲酸诱导的骨髓间充质干细胞RARβ表达

Recombinant adenovirus expressing siRNA is generated to inhibit the expression of RARβ in rat mesenchymal stem cells treated by all-trans retinoic acid

摘要:

构建携带针对大鼠维甲酸受体β (Retinoic acid receptor β，RARβ)基因的siRNA重组腺病毒，并感染全反式维甲酸 (All-trans retinoic acid, ATRA)处理的骨髓间充质干细胞 (Mesenchymal stem cells, MSCs)，检测其对RARβ的表达及MSCs成神经分化的影响。设计针对大鼠RARβ的4对siRNA的DNA序列，体外退火形成双链，定向克隆至含有U6/H1双启动子的腺病毒穿梭质粒pSES-HUS，随后与腺病毒骨架质粒pAd-Easy1

Abstract:

To construct the recombinant adenovirus vector expressing specific siRNA for rat retinoic acid receptor-β (RARβ) gene, and to detect its effect on RARβ expression and neuronal differentiation of all-trans retinoic acid (ATRA) treated mesenchymal stem cells (MSCs). First, we designed four pairs of siRNA sequence for rat RARβ gene and annealed complementary oligonucleotides in vitro, then cloned double-stranded DNA in pSES-HUS vector containing U6/H1 double-promoter and recombinated with the backbone vector to construct pAd-siRARβ plasmid. We infected MSCs by using adenovirus Ad-siRARβ which was packaged in HEK293 cell line, then performed Real-time, Western blotting and immunoflourencence to detect the expression of RARβ. We used combination of ATRA and MNM to induce MSCs into neural-like cells, then performed Real-time PCR and immunoflourencence to detect neuronal specific markers of induced neural cells. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in adenovirus vector. We could observe more than 60% RFP-positive MSCs at 24 h after adenovirus infection. The expression of RARβ was significantly increased to 16.5±2.34 fold in ATRA treated MSCs (P<0.05) and located in nucleus. Three of four pairs siRNA could effectively inhibit the expression of RARβ with inhibition efficacy of (66.26±9.12)%, (48.70±5.78)%, (64.09±0.53)% (P<0.05), especially siRNA-pool group with inhibition efficacy of (78.09±4.24)% (P<0.01). Combination of ATRA and MNM induced MSCs into neural-like cells which expressed neuronal specific markers, Nestin, NSE, MAP-2, and Tau. Immunoflourencence result showed that about 50~88 present of cells were positive for Nestin, NSE, Tju1, however, adenovirus medicated expression of siRARβ could effectively inhibit the expression level of neural specific proteins and the ratio of positive stained cells (P<0.05). Therefore, we successfully constructed the recombinant adenovirus vector containing siRNA for rat RARβ gene, adenovirus could effectively infect MSCs and inhibit the expression of induced RARβ in ATRA treated MSCs, then inhibit neuronal differentiation of MSCs.