Bi Yang, Gong Min, He Yun, Zhang Yun, Chen Jie, Li Tingyu
Key Laboratory of Developmental Diseases in Childhood of Ministry of Education, Children's Nutritional Research Center, the Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
Sheng Wu Gong Cheng Xue Bao. 2012 May;28(5):632-42.
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.
构建表达大鼠视黄酸受体β（RARβ）基因特异性小干扰RNA（siRNA）的重组腺病毒载体，并检测其对全反式维甲酸（ATRA）处理的间充质干细胞（MSCs）中RARβ表达及神经元分化的影响。首先，我们设计了四对针对大鼠RARβ基因的siRNA序列，并在体外对互补寡核苷酸进行退火，然后将双链DNA克隆到含有U6/H1双启动子的pSES-HUS载体中，并与骨架载体重组以构建pAd-siRARβ质粒。我们使用在HEK293细胞系中包装的腺病毒Ad-siRARβ感染MSCs，然后进行实时定量PCR、蛋白质免疫印迹和免疫荧光检测RARβ的表达。我们使用ATRA和MNM联合诱导MSCs向神经样细胞分化，然后进行实时定量PCR和免疫荧光检测诱导神经细胞的神经元特异性标志物。通过PCR、内切酶切割和基因测序，我们证实靶基因正确克隆到腺病毒载体中。腺病毒感染后24小时，我们可以观察到超过60%的MSCs呈红色荧光蛋白（RFP）阳性。在ATRA处理的MSCs中，RARβ的表达显著增加至16.5±2.34倍（P<0.05），且定位于细胞核。四对siRNA中的三对可有效抑制RARβ的表达，抑制率分别为（66.26±9.12）%、（48.70±5.78）%、（64.09±0.53）%（P<0.05），尤其是siRNA-pool组，抑制率为（78.09±4.24）%（P<0.01）。ATRA和MNM联合诱导MSCs分化为表达神经元特异性标志物巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、微管相关蛋白2（MAP-2）和微管蛋白（Tau）的神经样细胞。免疫荧光结果显示，约50%-88%的细胞对Nestin、NSE、Tuj1呈阳性，然而，腺病毒介导的siRARβ表达可有效抑制神经特异性蛋白的表达水平及阳性染色细胞的比例（P<0.05）。因此，我们成功构建了含有大鼠RARP基因siRNA的重组腺病毒载体，腺病毒可有效感染MSCs并抑制ATRA处理MSCs中诱导的RARβ表达，进而抑制MSCs的神经元分化。
