共表达人血管内皮生长因子165和人骨形态发生蛋白7基因的重组腺相关病毒载体基因表达的时间效应研究

[Study on time effect of gene expression of recombinant adeno-associated virus vector co-expressing human vascular endothelial growth factor 165 and human bone morphogenetic protein 7 genes].

作者信息

Zhang Chen, Qiang Hui, Dang Xiaoqian, Wang Kunzheng

机构信息

Department of Orthopedics, the Second Affiliated Hospital of Medicine College, Xi'an Jiaotong University, Xi'an Shaanxi, 710004, PR China.

出版信息

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Sep;24(9):1121-7.

PMID:20939488

Abstract

OBJECTIVE

To study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothelial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis.

METHODS

The best multiplicity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 x 10(6) cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sliced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo.

RESULTS

The best MOI of BMSCs transfected with rAAV was 5 x 10(4) v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the strong level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and strong expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 +/- 75.58) pg/mL and (4.80 +/- 0.61) ng/mL respectively in experimental group 1, and were (32.28 +/- 8.42) pg/mL and (0.64 +/- 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P < 0.05).

CONCLUSION

The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.

摘要

目的

研究共表达人血管内皮生长因子165（hVEGF165）和人骨形态发生蛋白7（hBMP - 7）基因的重组腺相关病毒（rAAV）载体基因表达的时间效应，为骨坏死的基因治疗奠定理论基础。

方法

通过荧光细胞计数法检测rAAV转染骨髓间充质干细胞（BMSCs）的最佳感染复数（MOI）。将第3代兔骨髓间充质干细胞分别用rAAV - hVEGF165 - 内部核糖体进入位点（IRES） - hBMP - 7（实验组）和绿色荧光蛋白（GFP）标记的rAAV - IRES - GFP（对照组）转染。用倒置荧光显微镜观察GFP的表达。体外通过RT - PCR法和蛋白质印迹法评估hVEGF165和hBMP - 7的表达。将两组转染后的细胞制成浓度为5×10⁶个细胞/mL的悬液，分别注入实验组1（n = 9）和对照组1（n = 9）的兔大腿肌肉中。采用冰冻切片法对注射rAAV - IRES - GFP的肌肉进行切片，用倒置荧光显微镜观察GFP蛋白的表达。体内通过蛋白质印迹法和ELISA法评估hVEGF165和hBMP - 7的表达。

结果

rAAV转染BMSCs的最佳MOI为5×10⁴ v.g/细胞。体外，GFP、hVEGF165和hBMP - 7基因的表达在转染后1天开始，转染后14天表达明显增加，转染后28天表达维持在较高水平。体内，注射后2周可检测到GFP、hVEGF165和hBMP - 7基因的表达，注射后6至8周表达较强。实验组1中hVEGF165和hBMP - 7的值分别为（248.67±75.58）pg/mL和（4.80±0.61）ng/mL，对照组1中分别为（32.28±8.42）pg/mL和（0.64±0.42）ng/mL；两组间差异有统计学意义（P < 0.05）。