慢病毒介导RNAi干扰β-catenin对小鼠骨髓间充质干细胞生物学行为的影响

Effect of Interfering the β-catenin by Lentiviral Vector-mediated RNAi on the Biological Behavior of Mouse Bone Marrow Mesenchymal Stem Cells

本研究通过构建β-连环蛋白(β-catenin)特异的RNAi慢病毒表达载体,探讨阻断Wnt/β-catenin信号通路对骨髓间充质干细胞(MSC)生物学行为的影响。设计3对针对β-catenin mRNA不同位点的shRNA编码序列,分别连接到慢病毒载体质粒PLB中,构建PLB-β-catenin/shRNA1,PLB-β-catenin/shRNA2和PLB-β-catenin/shRNA3;将重组质粒与慢病毒包装质粒及包膜蛋白质粒共转染293FT细胞,收获病毒颗粒,浓缩后测定病毒滴度,感染MSC细胞,并应用流式细胞术分选GFP阳性细胞,Western blot和RT-PCR验证其对细胞内β-catenin基因表达的抑制效果,筛选干扰效率最高质粒;CCK-8法检测细胞增殖,Annexin-V/7-AAD法检测干扰后细胞凋亡情况,细胞划痕实验及Transwell实验检测MSC迁移能力。结果表明:构建的特异性慢病毒siRNA干扰组(PLB-β-catenin/shRNA2)能有效抑制β-catenin的mRNA和蛋白表达,并抑制细胞的增殖;而空载体对照组(PLB group)和正常对照组(control group)细胞增殖无明显变化,两组之间无显著的统计学差异(P>0.05);流式细胞术检测结果显示,采用血清饥饿法处理后,干扰组细胞的凋亡率明显增加,但两对照组之间无统计学差异(P>0.05);细胞划痕和Transwell实验显示,干扰组MSC的迁移能力明显降低,对照组细胞迁移能力无明显变化。结论:构建的特异RNAi慢病毒能够有效抑制β-catenin基因的表达,减少细胞内目的基因mRNA和蛋白的水平,从而对MSC细胞的增殖、凋亡和迁移能力产生重要影响。

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 plasimds and lentiviral packaging plasimds were co-transfected into the packaging cells 293FF, 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 fl-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 capalbility 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（inteference 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 inteference 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 inteference group dicreased significantly, while the migration capability of MSC in control group was not changed obvi-ously. 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.