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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。
In this study, β-catenin-specific RNAi lentivirus expression vector was constructed to investigate the effect of blocking Wnt / β-catenin signaling pathway on the biological behavior of bone marrow mesenchymal stem cells (MSC). Three pairs of shRNA encoding sequences targeting at different sites of β-catenin mRNA were designed and ligated into lentiviral plasmid PLB respectively to construct PLB-β-catenin / shRNA1, PLB-β-catenin / shRNA2 and PLB-β-catenin / shRNA3 293FT cells were co-transfected with the lentivirus packaging plasmid and the envelope protein plasmid, and the virus particles were harvested. The virus titer was determined after concentration, and the MSCs were infected. The GFP positive cells were sorted by flow cytometry and Western blot The inhibitory effect of β-catenin on the expression of β-catenin was detected by RT-PCR and the highest efficiency plasmid was screened. Cell proliferation was detected by CCK-8 assay. Cell apoptosis was detected by Annexin-V / 7-AAD assay. Experiments and Transwell experiments were performed to detect MSC migration ability. The results showed that PLB-β-catenin / shRNA2 could effectively inhibit the mRNA and protein expression of β-catenin and inhibit the proliferation of cells. However, PLB group and normal There was no significant difference in cell proliferation in control group (P> 0.05). The results of flow cytometry showed that the apoptosis rate of cells in control group But there was no significant difference between the two control groups (P> 0.05). Scratches and Transwell experiments showed that the migration ability of MSC in interference group was significantly decreased, while the migration ability of control group showed no significant change. CONCLUSION: The constructed specific RNAi lentivirus can effectively inhibit the expression of β-catenin gene and decrease the mRNA and protein levels of target genes in the cells, and thus have an important effect on the proliferation, apoptosis and migration of MSC cells.