红色荧光基因慢病毒转染骨髓间充质干细胞:表达21d对细胞活性无影响

发布时间：2018-08-12 18:43

【摘要】：背景:掌握转染的最佳感染复数和产生较强荧光强度的时间,可为后期观察人骨髓间充质干细胞在动物模型体内的示踪奠定基础。目的:探讨HIV-1来源的慢病毒携带增强型红色荧光蛋白转染人骨髓间充质干细胞的可行性。方法:将第4代人骨髓间充质干细胞分成空白组和感染复数为2,3,4组,以细胞数5.0×105个接种于12孔培养皿,添加含有体积分数为1%胎牛血清的成人骨髓间质干细胞完全培养基1 m L。调整慢病毒携带增强型红色荧光蛋白感染滴度为1.0×1011TU/L,加入各组培养皿中病毒液体积分别为10,15,20μL,感染复数分别为2,3,4,空白组加入10μL PBS。转染后24,72 h荧光倒置显微镜观察细胞红色荧光表达情况并计算出转染率。结果与结论:增强型红色荧光蛋白在骨髓间充质干细胞中稳定表达,转染后24 h倒置荧光显微镜下可见红色荧光,72 h荧光达到最强,细胞转染率与感染复数值呈线性增长关系。转染后21 d内,各转染实验组的人骨髓间充质干细胞数量与空白组比较差异无显著性意义(P0.05),以上结果提示采用HIV-1来源的慢病毒载体介导增强型红色荧光蛋白转染标记人骨髓间充质干细胞是可行的,当感染复数为4时转染效率高并可持续表达至少21 d,且标记后对其增殖活性无影响。
[Abstract]:Background: mastering the optimal number of infections and the time of producing strong fluorescence intensity can lay a foundation for the later observation of human bone marrow mesenchymal stem cells (BMSCs) tracer in animal models. Aim: to investigate the feasibility of transfection of human bone marrow mesenchymal stem cells (BMSCs) with lentivirus derived from HIV-1 and enhanced red fluorescent protein. Methods: the fourth generation of human bone marrow mesenchymal stem cells (BMSCs) were divided into blank group and infected plural group (2). The cells were inoculated in 12 well culture dish with 5. 0 脳 10 ~ 5 cells. The adult bone marrow mesenchymal stem cells containing 1% fetal bovine serum were added to the complete culture medium of 1 mL of adult bone marrow mesenchymal stem cells. The titer of lentivirus carrying enhanced red fluorescent protein infection was adjusted to 1.0 脳 1011TU / L, the volume of virus solution in the culture dish was 10 ~ 1520 渭 L, the complex number of infection was 2 ~ 3 ~ 3 ~ (4), and the blank group was added 10 渭 L PBS. The expression of red fluorescence was observed by fluorescence inverted microscope at 24 ~ 72 h after transfection and the transfection rate was calculated. Results and conclusion: the enhanced red fluorescent protein was stably expressed in bone marrow mesenchymal stem cells. The fluorescence intensity of 72 h was the strongest under the inverted fluorescence microscope 24 h after transfection, and the cell transfection rate was linearly increased with the infection complex value. Within 21 days after transfection, There was no significant difference in the number of human bone marrow mesenchymal stem cells between each transfection group and the blank group (P0.05). The above results suggested that the lentivirus vector derived from HIV-1 was used to mediate the transfection of enhanced red fluorescent protein to label human bone marrow mesenchymal cells (BMSCs). Stem cells are feasible. When the complex number of infection is 4, the transfection efficiency is high and the expression can be sustained for at least 21 days, and the labeling has no effect on its proliferation activity.
【作者单位】：广州医科大学附属第三医院骨科;
【基金】：国家青年科学基金项目(31200726) 广州市应用基础研究项目(2013J4100101)~~
【分类号】：R329.2;R373

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