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

构建携带针对大鼠维甲酸受体β(Retinoic acid receptorβ,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在BJ5183细菌中同源重组,并在HEK293细胞中包装获得重组腺病毒Ad-siRARβ。腺病毒感染大鼠MSCs后经ATRA处理24 h,Real-time、Western blotting及免疫荧光检测RARβ的表达情况。改良神经诱导培养基(Modified neuronal induction medium,MNM)诱导MSCs神经分化,Real-time PCR及免疫荧光检测神经相关蛋白表达。PCR、酶切及测序鉴定均证实siRNA正确克隆至腺病毒质粒中,腺病毒感染大鼠MSCs后可观察到60%以上的细胞有红色荧光蛋白(Red fluorescent protein,RFP)表达。经ATRA处理24 h,Real-time、Westernblotting及免疫荧光检测发现RARβ表达定位于细胞核,ATRA作用后MSCs中RARβ表达增高16.5±2.34倍(P<0.05),有3组siRNA能有效抑制ATRA诱导的RARβ表达增强,抑制率分别为(66.26±9.12)%、(48.70±5.78)%、(64.09±0.53)%(P<0.05),且以pool组效果最强,抑制率为(78.09±4.24)%(P<0.01)。ATRA联合MNM诱导MSCs成神经样细胞,表达相关神经特异蛋白Nestin、NSE、MAP-2、Tau,免疫荧光结果显示神经标志蛋白Nestin、NSE、Tju1表达阳性细胞率为(50-88)%,而腺病毒介导的siRARβ能有效抑制MSCs的神经标志物表达水平及阳性细胞率(P<0.05)。成功构建了携带针对大鼠RARβ基因的siRNA重组腺病毒,能有效感染MSCs并显著抑制ATRA诱导的RARβ表达增强和MSCs的神经分化。

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

To construct the recombinant adenovirus vector expressing specific siRNA for rat retinoic acid receptor-beta (RARbeta) gene, and to detect its effect on RARbeta 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 RARbeta 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-siRARbeta plasmid. We infected MSCs by using adenovirus Ad-siRARbeta which was packaged in HEK293 cell line, then performed Real-time, Western blotting and immunoflourencence to detect the expression of RARbeta. 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 RARbeta 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 RARbeta 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, Tjul, however, adenovirus medicated expression of siRARbeta 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 RARP gene, adenovirus could effectively infect MSCs and inhibit the expression of induced RARbeta in ATRA treated MSCs, then inhibit neuronal differentiation of MSCs.