携带CXCR4基因慢病毒载体转染大鼠骨髓间充质干细胞的体外实验

发布时间：2018-01-30 14:13

本文关键词： 骨髓间质干细胞慢病毒属受体 CXCR 干细胞骨髓干细胞慢病毒骨髓间充质干细胞 CXCR 细胞增殖免疫调节因子辽宁省自然科学基金　出处：《中国组织工程研究》2014年41期 　论文类型：期刊论文

【摘要】：背景:间充质干细胞在体内归巢主要调控因子是CXCR4,如何提高干细胞自身CXCR4的表达量是调节干细胞体内靶器官归巢能力的关键。目的:构建CXCR4或GFP基因慢病毒表达载体,并观察其对骨髓间充质干细胞自身生长特性及分泌功能的影响。方法:骨髓间充质干细胞应用慢病毒载体转染系统转染CXCR4基因达到过表达,同时单独转GFP基因作为对照。通过荧光显微镜、RT-PCR、免疫蛋白印迹方法验证病毒转染效率;流式细胞技术测定骨髓间充质干细胞的增殖、凋亡情况;Western Blot法测定间充质干细胞培养基上清中分泌蛋白及细胞因子变化。结果与结论:慢病毒转染系统可以安全、有效地转染CXCR4基因或GFP基因到骨髓间充质干细胞。流式细胞仪检测显示过表达CXCR4的骨髓间充质干细胞增殖能力、生存能力加强,凋亡细胞减少(P0.05);细胞上清蛋白分析提示过表达CXCR4后,骨髓间充质干细胞培养上清内出现很多的蛋白因子升高(P0.05),其中大多数对细胞自身复制及免疫调节有益。结果表明慢病毒转染是一种安全高效的基因修饰手段,可以通过CXCR4基因修饰的方法提高骨髓间充质干细胞的生存能力、降低凋亡率,调节细胞保护性因子及免疫调节因子的分泌功能。
[Abstract]:Background: the main regulatory factor for homing of mesenchymal stem cells in vivo is CXCR4. How to improve the expression of stem cells CXCR4 is the key to regulate the homing ability of target organs in stem cells. Objective: to construct the lentivirus expression vector of CXCR4 or GFP gene. Methods: bone marrow mesenchymal stem cells (BMSCs) were transfected with lentivirus vector transfection system to achieve overexpression of CXCR4 gene. At the same time, the GFP gene was transferred alone as control. The transfection efficiency was verified by fluorescence microscope and Western blot. The proliferation and apoptosis of bone marrow mesenchymal stem cells were measured by flow cytometry. The secretory protein and cytokines in the supernatant of mesenchymal stem cell medium were determined by Western Blot. Results & conclusion: lentivirus transfection system is safe. CXCR4 gene or GFP gene was effectively transfected into bone marrow mesenchymal stem cells. Flow cytometry showed the proliferation ability and survival ability of bone marrow mesenchymal stem cells expressing CXCR4. Apoptotic cells decreased P0.05A; The analysis of superalbumin showed that after overexpression of CXCR4, there were a lot of protein factors in the supernatant of bone marrow mesenchymal stem cells (P0.05). The results showed that lentivirus transfection was a safe and efficient method for gene modification. CXCR4 gene modification can improve the viability of bone marrow mesenchymal stem cells, reduce apoptosis rate and regulate the secretion of protective and immunomodulatory factors.
【作者单位】：解放军沈阳军区总医院泌尿男科;
【基金】：辽宁省自然科学基金项目(2013020199)~~
【分类号】：R329.2
【正文快照】： 文章亮点:1细胞上清分泌蛋白是干细胞在体内修复组织损伤、调节免疫功能的主要手段。研究提示转染CXCR4的骨髓间充质干细胞可以分泌更多的免疫调节蛋白,对治疗免疫性疾病有重要意义。2转染CXCR4的骨髓间充质干细胞凋亡率下降、细胞多处于增殖期,提示过表达CXCR4的间充质干细胞

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