慢病毒介导shRNA LINGO-1质粒转染促进骨髓间充质干细胞向神经细胞转化的研究

Lentivirus-Mediated shRNA LINGO-1 Plasmid Transfection Promotes Transformation of Bone Marrow Mesenchymal Stem Cells into Neurons

目的:探究慢病毒介导shRNA LINGO-1质粒转染促进骨髓间充质干细胞(BMSCs)向神经细胞转化的研究。方法:利用shRNA基因干扰技术,构建shRNA LINGO-1干扰载体,慢病毒转染BMSCs,根据病毒转染情况分成空白对照组(未转染慢病毒的BMSCs)、空载体病毒组(转染不含shRNA LINGO-1干扰载体慢病毒的BMSCs)、干扰载体病毒组(转染shRNA LINGO-1干扰载体慢病毒的BMSCs)。利用流式细胞仪检测BMSCs的表面蛋白,利用RT-PCR和Western-Blot检测胶质纤维酸性蛋白(GFAP)、神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)、巢蛋白的表达。结果:CD44和CD29在第2代BMSCs的表达为60.2%和58.3%;CD34和CD45在第2代BMSCs的表达为3.4%和2.6%。慢病毒转染效率为90%以上。空白对照组、空载体病毒组和干扰载体病毒组的GFAP、NSE、NF和巢蛋白m RNA的相对表达量比较差异有统计学意义(P<0.05),其中空载体病毒组和空白对照组的比较差异无统计学意义(P>0.05),干扰载体病毒组和空载体病毒组的差异有统计学意义(P<0.05)。Western-blot检测蛋白表达量也具有同样的表达特点。结论:慢病毒介导shRNA LINGO-1干扰载体转染促进BMSCs可提高BMSCs向神经细胞转化的效率。

Objective: To investigate the effect of lentivirus-mediated shRNA LINGO-1 plasmid transfection on the transformation of bone marrow mesenchymal stem cells (BMSCs) into neural cells. Methods: The shRNA LINGO-1 interfering vector was constructed by shRNA gene interference technique. Lentivirus was transfected into BMSCs. According to outcome of transfection, BMSCs were assigned to the blank control group (not transfected), empty vector virus group (transfected with lentivirus not containing the shRNA LINGO-1 interfering vector), and interfering vector virus group (transfected with lentivirus containing the shRNA LINGO-1 interfering vector). The surface proteins on BMSCs was detected by flow cytometry. The expression of glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), neurofilament protein (NF), and Nestin was detected by RT-PCR and Western-Blot. Results: In second generation BMSCs, the expression of CD44 and CD29 was 60.2% and 58.3%, respectively, and the expression of CD34 and CD45 was 3.4% and 2.6%, respectively. Lentivirus transfection efficiency was over 90%. The transfection efficiency was high. The relative expression of GFAP, NSE, NF, and Nestin mRNA in the blank control, empty vector virus, and interfering vector virus groups was significantly different (P<0.05), but there was no significant difference between the empty vector virus group and blank control group (P>0.05). There was a statistically significant difference between the interference vector virus group and the empty vector virus group (P<0.05). The protein expression level detected by Western-blot also showed the same expression characteristics. Conclusion: Lentivirus-mediated shRNA LINGO-1 interfering vector transfection promotes the transformation of BMSCs into neural cells.