中华眼底病杂志

中华眼底病杂志

慢病毒载体介导色素上皮衍生因子基因转染人脐带间充质干细胞研究

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目的 构建携带色素上皮衍生因子(PEDF)基因的慢病毒(LV)载体并转染人脐带间充质干细胞(hUCMSCs),初步探讨基因修饰PEDF-间充质干细胞的可行性。 方法 采用基因重组方法构建LV-PEDF载体,测定其LV滴度,以感染复数(MOI)值为10、30、50 的LV体外转染hUCMSCs,并据此分为相应MOI值实验组;以不含PEDF基因的相同MOI值的重组LV载体[LV-绿色荧光蛋白(GFP)]转染hUCMSCs,并据此分为 相应MOI值对照组。荧光显微镜观察两组细胞转染效率并计算转染率。采用细胞免疫荧光法、免疫细胞化学法、实时定量聚合酶链反应(RT-PCR)检测50MOI实验组、50MOI对照组细胞中PEDF、血管内皮生长因子(VEGF) 表达水平。 结果 经酶切以及基因测序鉴定证明LV载体构建成功。转染96 h后,50MOI实验组细胞可见大量GFP表达,转染效率为72.1%。荧光显微镜观察发现,转染后96 h,50MOI实验组细胞胞浆PEDF、VEGF表达增强。光学显微镜观察发现,转染后96 h,50MOI实验组细胞胞浆PEDF表达较低,而VEGF表达较强。RT-PCR检测结果发现,50MOI实验组、50MOI对照组细胞中PEDF mRNA相对表达量分别为0.170±0.028、0.015±0.007;VEGF mRNA相对表达量分别为0.265±0.022、0.285±0.049。两组细胞中PEDF mRNA相对表达量比较,差异有统计学意义(F=70.29,P<0.001);VEGF mRNA相对表达量比较,差异无统计学意义(F=9.57,P>0.05)。 结论 成功构建携带PEDF基因LV表达载体,并在hUCMSCs中过表达PEDF基因。

Objective To build the lentiviral vectors of pigment epithelial derived factor (PEDF) gene, and investigate their expression in human umbilical cord mesenchymal stem cells (hUCMSCs). Methods The PEDF lentiviral vectors (LV-PEDF) were built by DNA recombination and confirmed by DNA sequencing. hUCMSCs were transfected by LV-PEDF with MOI 10, 30, 50, respectively. The transfection efficiency was observed under fluorescence microscope. Cell immunofluorescence, immunocytochemistry and real-time PCR methods were used for detecting the expression of PEDF and VEGF. Results The PEDF cDNA was sub-cloned into pCDH-CMV-MCS-EF1-copGFP vector successfully. DNA sequencing analysis confirmed that PEDF gene sequence was exactly the same with that reported in GenBank. pCDH-PEDF infected cells could show green fluorescence under fluorescence microscope. The transfection efficiency was 72.1% in PEDF-MSCs. Immunofluorescence and immunochemical staining confirmed that PEDF protein was overexpressed in hUCMSCs. The relative expression of PEDF mRNA in experimental group and control group was (0.170±0.028) and (0.015±0.007) respectively by RT-PCR, the difference was statistically significant (P<0.001). The relative expression levels of VEGF mRNA in the two groups were (0.265±0.022) and (0.285±0.049), respectively, with no significant difference (P>0.05). Conclusions We successfully built a lentivirus vector carrying PEDF gene and obtained hUCMSCs with overexpressed PEDF.

关键词: 血管内皮生长因子类; 间充质干细胞; 慢病毒感染; 细胞,培养的

Key words: Vascular endothelial growth factors; Mesenchymal stem cells; Lentivirus infections; Cells, cultured

引用本文: 段红涛, 陈松, 董蒙, 王月欣, 孔佳慧, 宋建, 李泽东, 王继明. 慢病毒载体介导色素上皮衍生因子基因转染人脐带间充质干细胞研究. 中华眼底病杂志, 2017, 33(6): 621-625. doi: 10.3760/cma.j.issn.1005-1015.2017.06.016 复制

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