| 陈 娟,李福军,方 堃,李松洋,叶 英.慢病毒介导的GFP转染对大鼠骨髓间充质干细胞增殖、细胞表型及脑脊液诱导后分化能力的影响[J].现代生物医学进展英文版,2021,(19):3617-3621. |
| 慢病毒介导的GFP转染对大鼠骨髓间充质干细胞增殖、细胞表型及脑脊液诱导后分化能力的影响 |
| Effects of Lentivirus-GFP Transfection System on Proliferation, Phenotype and Differentiation Ability of Rat Bone Marrow Mesenchymal Stem Cells Induced by Cerebrospinal Fluid |
| Received:January 27, 2021 Revised:February 23, 2021 |
| DOI：10.13241/j.cnki.pmb.2021.19.004 |
| 中文关键词: 髓间充质干细胞 慢病毒 绿色荧光蛋白 脑脊液 |
| 英文关键词: BMSCs Lentiviral vector Green fluorescence protein gene Human cerebrospinal fluid |
| 基金项目:江苏省高校自然科学研究重大项目（12KJA320002）；江苏省青蓝工程资助 |
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| 中文摘要: |
| 摘要 目的：研究慢病毒（Lentivirus）介导的绿色荧光蛋白（Lentivirus-GFP）转染大鼠骨髓间充质干细胞（BMSCs）的可行性及稳定性，以及对人脑脊液诱导转染后BMSCs（BMSCs-GFP）成神经分化能力的影响。方法：全骨髓贴壁法培养BMSCs，Lentivirus-GFP以5、10、30、50的感染复数（MOI）转染BMSCs，96h后倒置显微镜下观察GFP转染效率和表达情况，筛选最适MOI；流式细胞术检测细胞表型；CCK8法检测细胞活力。人脑脊液诱导BMSCs-GFP向神经细胞分化，蛋白印迹法检测神经细胞表面标记物MAP-2和Nestin表达。结果：全骨髓贴壁法分离培养的BMSCs生长旺盛。MOI值为5、10、30、50的转染效率分别为56.2%、87.3%、94.7%和95.1%，当MOI为30时，Lentivirus-GFP转染BMSCs效率较高，且对BMSCs生长状态无显著性影响。BMSCs-GFP表达CD29、CD90，较少表达CD45、CD54，符合干细胞特性。BMSCs-GFP增殖活性与未转染GFP基因的BMSCs相比，差异无统计学意义（P>0.05）。人脑脊液诱导BMSCs-GFP成神经细胞分化后表达神经元标记物MAP-2和Nestin。结论：Lentivirus-GFP能高效稳定转染大鼠BMSCs（最适MOI值为30），同时不影响其生物学特性，人脑脊液能诱导BMSCs-GFP成神经细胞分化。 |
| 英文摘要: |
| ABSTRACT Objective: The object of this study is to evaluate the transfection efficiency and stability of lentiviral-GFP into rat bone marrow mesenchymal stem cells (BMSCs) and detect the differentiation ability of BMSCs carrying GFP (BMSCs-GFP) induced by Human cerebrospinal fluid. Methods: BMSCs derived from rat bone marrow were isolated and cultivated in vitro. Cells were transfected with lentiviral-GFP at different multiplicity of infection (MOI=5, 10, 30, 50). The GFP expression was observed under inverted fluorescence microscopy after 96h. Flow cytometry was used to evaluate the transfection efficiency at proper MOI and CD phenotype on the surface of BMSCs-GFP. Growth curve was acquired by CCK8 to compare the difference of the growth rate between BMSCs and BMSCs-GFP. BMSCs-GFP were induced by Human cerebrospinal fluid and the expression of neuron specific protein MAP-2 and Nestin were detected by western blot method. Results: Under the inverted fluorescence microscope, green fluorescent was found partly after 96h. The transfection rate were 56.2%, 87.3%, 94.7% and 95.1%, while the transfect MOI were 5, 10, 30 and 50, respectively. At transfect MOI 30, the transfection efficiency of BMSCs was the best, and there was no obvious effect on cell activity observed. The results of flow cytometry analysis are shown BMSCs-GFP expressed CD29 and CD90 and rarely express CD45 and CD54. The result of CCK8 measured growth curve showed that there was no significant difference in the proliferative capability between the BMSCs and BMSCs-GFP （P>0.05）. BMSCs-GFP was induced to differentiate into neuron like cells by Human cerebrospinal fluid and could express nerve cell markers MAP-2 and Nestin. Conclusion: The most proper MOI is 30 in this experiment. The lentiviral-GFP could infect BMSCs efficiently and expressed stably, and BMSCs-GFP could be induced by Human cerebrospinal fluid to differentiate into neuron like cells. |
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