摘要
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。
ObjectiveTo build the lentiviral vectors of pigment epithelial derived factor （PEDF） gene, and investigate their expression in human umbilical cord mesenchymal stem cells （hUCMSCs）.MethodsThe 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.ResultsThe 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）.ConclusionsWe successfully built a lentivirus vector carrying PEDF gene and obtained hUCMSCs with overexpressed PEDF.
出处
《中华眼底病杂志》
CSCD
北大核心
2017年第6期621-625,共5页
Chinese Journal of Ocular Fundus Diseases
基金
天津市科技计划项目（13ZCZDSY01500）
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
Vascular endothelial growth factors
Mesenchymal stem cells
Lentivirus infections
Cells, cultured
