Fu Cheng-Juan, Li Zhen-Yu, Li De-Peng, Yan Zhi-Ling, Chen Wei, Chen Chong, Wu Qing-Yun, Pan Xiu-Ying, Xu Kai-Lin
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Laboratory of Transplantation Immunology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China.
Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Laboratory of Transplantation Immunology, Xuzhou Medical College, Xuzhou 221002, Jiangsu Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Dec;21(6):1546-51. doi: 10.7534/j.issn.1009-2137.2013.06.034.
This study was aimed to investigate the effect of Wnt/β-catenin signaling pathway on the biologic behavior of mouse bone marrow mesenchymal stem cells (mBM-MSC) by constructing a RNAi lentiviral vector specific to β-catenin. Three pairs of shRNA coding sequences directed against different sites of β-catenin mRNA were designed and were linked into lentiviral vector plasmid PLB for constructing the PLB-β-catenin/shRNA1, PLB-β-catenin/shRNA2 and PLB-β-catenin/ shRNA3. Those plasmids and lentiviral packaging plasmids were co-transfected into the packaging cells 293FT, then the virus particles were collected and the viral titer was assayed after concentration, and these viral particles were infected to MSC. The flow cytometry was used to sort GFP (+) cells, Western blot and RT-PCR were used to verify the inhibitory effect of those cells on expression of β-catenin gene in cells, CCK-8 method was used to detect the cell proliferation level, Annexin-V/7-AAD was used to determine the cell apoptosis after interference, the cell scratch and transwell tests were used to detect the migration capability of MSC. The results showed that the efficient inhibition of β-catenin mRNA and protein expression, and the suppression of MSC proliferation were observed in group of PLB-β-catenin/shRNA2 (interference group), while there was no significant changes of MSC proliferation between negative group (PLB group) and the normal group (control group). The flow cytometric detection indicated that after induced by serum starvation for 72 h, the apoptosis of MSC increased in interference group, but there was no difference between PLB and control groups (P > 0.05). The cell scratch and transwell tests demonstrated that the migration capability of MSC in interference group decreased significantly, while the migration capability of MSC in control group was not changed obviously. It is concluded that the constructed specific RNAi lentivirus can effectively inhibit the expression of β-catenin gene, decrease expression level of β-catenin mRNA and protein. The Wnt/β-catenin signaling pathway plays an important role in biological behavior of BM-MSC.
本研究旨在通过构建针对β-连环蛋白的RNAi慢病毒载体，探讨Wnt/β-连环蛋白信号通路对小鼠骨髓间充质干细胞（mBM-MSC）生物学行为的影响。设计了三对针对β-连环蛋白mRNA不同位点的shRNA编码序列，并将其连接到慢病毒载体质粒PLB中，构建PLB-β-连环蛋白/shRNA1、PLB-β-连环蛋白/shRNA2和PLB-β-连环蛋白/shRNA3。将这些质粒与慢病毒包装质粒共转染至包装细胞293FT，然后收集病毒颗粒，浓缩后测定病毒滴度，并将这些病毒颗粒感染MSC。采用流式细胞术分选GFP(+)细胞，运用蛋白质免疫印迹法和RT-PCR验证这些细胞对细胞中β-连环蛋白基因表达的抑制作用，采用CCK-8法检测细胞增殖水平，运用Annexin-V/7-AAD法测定干扰后细胞凋亡情况，采用细胞划痕试验和transwell试验检测MSC的迁移能力。结果显示，PLB-β-连环蛋白/shRNA2组（干扰组）可有效抑制β-连环蛋白mRNA和蛋白表达，抑制MSC增殖，而阴性组（PLB组）与正常组（对照组）之间MSC增殖无明显变化。流式细胞术检测表明，血清饥饿诱导72 h后，干扰组MSC凋亡增加，但PLB组与对照组之间无差异（P>0.05）。细胞划痕试验和transwell试验表明，干扰组MSC迁移能力显著降低，而对照组MSC迁移能力无明显变化。结论：构建的特异性RNAi慢病毒可有效抑制β-连环蛋白基因表达，降低β-连环蛋白mRNA和蛋白表达水平。Wnt/β-连环蛋白信号通路在BM-MSC生物学行为中起重要作用。
