三种携带GFP慢病毒转染兔骨髓间充质干细胞的效果

发布时间：2018-01-11 17:42

本文关键词：三种携带GFP慢病毒转染兔骨髓间充质干细胞的效果　出处：《中国组织工程研究》2017年29期 　论文类型：期刊论文

【摘要】：背景:研究不同慢病毒对同一骨髓来源间充质干细胞的转染效果,筛选合适的细胞转染工具。目的:探究3种慢病毒对兔骨髓间充质干细胞转染效果的差异。方法:选择2周龄日本大耳白兔,髂骨穿刺抽取骨髓液,采用密度梯度离心法提取骨髓间充质干细胞,流式细胞仪检测细胞表面标志物表达。分别用p Tomo-GFP慢病毒、Ad-GFP慢病毒、lenti-GFP慢病毒转染骨髓间充质干细胞,流式细胞仪检测3种慢病毒的转染率。CCK-8法检测转染前后细胞生长活力变化。结果与结论:①培养出的细胞CD44阳性率为75.64%,CD34、CD45阳性率分别为6.14%、9.48%;②pTomo-GFP慢病毒转染后GFP表达率为2.64%、Ad-GFP慢病毒转染后GFP表达率为16.72%、lenti-GFP慢病毒转染后GFP表达率为45.24%;③各慢病毒对细胞生长存在一定影响。p Tomo-GFP、Ad-GFP在第1-8天对细胞生长潜伏期和对数生长期存在影响,可能对前期细胞活性造成一定干扰。短期内lenti-GFP对细胞生长影响不大,但第12天时出现了细胞生长情况变化,考虑对细胞远期活力存在影响;④选用lenti-GFP慢病毒转染兔骨髓间充质干细胞能够在保证细胞正常生长的情况下提供最大的转染效率,适用于兔骨髓间充质干细胞的示踪及基因转染等相关研究。
[Abstract]:Background: to study the transfection effect of different lentiviruses on mesenchymal stem cells from the same bone marrow source. Objective: to investigate the transfection effects of three lentiviruses on rabbit bone marrow mesenchymal stem cells (BMSCs). Methods: two week-old Japanese large ear white rabbits were selected and bone marrow fluid was extracted by iliac puncture. Bone marrow mesenchymal stem cells (BMSCs) were extracted by density gradient centrifugation, and the expression of cell surface markers was detected by flow cytometry. Lenti-GFP lentivirus was transfected into bone marrow mesenchymal stem cells. Flow cytometry was used to detect the transfection rate of three lentiviruses. CCK-8 method was used to detect the changes of cell growth activity before and after transfection. Results and conclusion the CD44 positive rate of the cells cultured at 1: 1 was 75.64%. The positive rate of CD34 + CD45 was 6.14% and 9.48%, respectively. 2the GFP expression rate of pTomo-GFP lentivirus was 2.64% and the GFP expression rate of Ad-GFP lentivirus transfection was 16.72%. The expression rate of GFP was 45.24% after lenti-GFP lentivirus transfection. (3) Lentivirus had a certain effect on cell growth. P Tomo-GFP Ad-GFP had an effect on cell growth latency and logarithmic growth period on the 1st to 8th day. Lenti-GFP had little effect on cell growth in the short term, but the change of cell growth appeared on the 12th day. (4) transfection of rabbit bone marrow mesenchymal stem cells with lenti-GFP lentivirus could provide the maximum transfection efficiency under the condition of ensuring the normal growth of the cells. It is suitable for tracing and gene transfection of rabbit bone marrow mesenchymal stem cells.
【作者单位】：昆明医科大学第一附属医院运动医学科;
【基金】：国家自然科学基金(81460340) 云南省骨关节疾病诊疗省创新团队项目(2014HC1018) 医疗卫生内设机构研究项目(2014NS161)~~
【分类号】：R329.2
【正文快照】： 文章快速阅读:文题释义:绿色荧光蛋白:发现于维多利亚水母,是由238个氨基酸组成,经紫外光激发后可发出峰值为509 nm的绿色荧光,广泛用于基因工程中的基因表达检测、细胞示踪等。经过改良,研制出了增强型绿色荧光蛋白及其他颜色的荧光蛋白变体。慢病毒:属反转录病毒,具有长期潜

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